Targeting immunoproteasome and glutamine supplementation prevent intestinal hyperpermeability.

PubMed

Ghouzali, Ibtissem; Lemaitre, Caroline; Bahlouli, Wafa; Azhar, Saïda; Bôle-Feysot, Christine; Meleine, Mathieu; Ducrotté, Philippe; Déchelotte, Pierre; Coëffier, Moïse

2017-01-01

Intestinal hyperpermeability has been reported in several intestinal and non-intestinal disorders. We aimed to investigate the role of the ubiquitin proteasome system in gut barrier regulation in two mice models: the water avoidance stress model (WAS) and a post-inflammatory model (post-TNBS). Both models were applied in C57BL/6 male mice (n=7-8/group); Proteasome was targeted by injection of a selective proteasome inhibitor or by using knock-out mice for β2i proteasome subunit. Finally, glutamine supplementation was evaluated. In both models (WAS at day 10, post-TNBS at day 28), we observed an increase in proteasome trypsin-like activity and in inducible β2/constitutive β2 subunit protein expression ratio, associated with an increase in intestinal permeability. Moreover, intestinal hyperpermeability was blunted by intraperitoneal injection of selective proteasome inhibitor in WAS and post-TNBS mice. Of note, knock-out mice for the β2i subunit exhibited a significant decrease in intestinal permeability and fecal pellet output during WAS. Glutamine supplementation also improved colonic permeability in both models. In conclusion, the proteasome system is altered in the colonic mucosa of WAS and post-TNBS mice with increased trypsin-like activity. Associated intestinal hyperpermeability was blunted by immunoproteasome inhibition. Copyright © 2016 Elsevier B.V. All rights reserved.

Proteasome function is not impaired in healthy aging of the lung.

PubMed

Caniard, Anne; Ballweg, Korbinian; Lukas, Christina; Yildirim, Ali Ö; Eickelberg, Oliver; Meiners, Silke

2015-10-01

Aging is the progressive loss of cellular function which inevitably leads to death. Failure of proteostasis including the decrease in proteasome function is one hallmark of aging. In the lung, proteasome activity was shown to be impaired in age-related diseases such as chronic obstructive pulmonary disease. However, little is known on proteasome function during healthy aging. Here, we comprehensively analyzed healthy lung aging and proteasome function in wildtype, proteasome reporter and immunoproteasome knockout mice. Wildtype mice spontaneously developed senile lung emphysema while expression and activity of proteasome complexes and turnover of ubiquitinated substrates was not grossly altered in lungs of aged mice. Immunoproteasome subunits were specifically upregulated in the aged lung and the caspase-like proteasome activity concomitantly decreased. Aged knockout mice for the LMP2 or LMP7 immunoproteasome subunits showed no alteration in proteasome activities but exhibited typical lung aging phenotypes suggesting that immunoproteasome function is dispensable for physiological lung aging in mice. Our results indicate that healthy aging of the lung does not involve impairment of proteasome function. Apparently, the reserve capacity of the proteostasis systems in the lung is sufficient to avoid severe proteostasis imbalance during healthy aging.

Iron Loading Selectively Increases Hippocampal Levels of Ubiquitinated Proteins and Impairs Hippocampus-Dependent Memory.

PubMed

Figueiredo, Luciana Silva; de Freitas, Betânia Souza; Garcia, Vanessa Athaíde; Dargél, Vinícius Ayub; Köbe, Luiza Machado; Kist, Luiza Wilges; Bogo, Maurício Reis; Schröder, Nadja

2016-11-01

Alterations of brain iron levels have been observed in a number of neurodegenerative disorders. We have previously demonstrated that iron overload in the neonatal period results in severe and persistent memory deficits in the adulthood. Protein degradation mediated by the ubiquitin-proteasome system (UPS) plays a central regulatory role in several cellular processes. Impairment of the UPS has been implicated in the pathogenesis of neurodegenerative disorders. Here, we examined the effects of iron exposure in the neonatal period (12th-14th day of postnatal life) on the expression of proteasome β-1, β-2, and β-5 subunits, and ubiquitinated proteins in brains of 15-day-old rats, to evaluate the immediate effect of the treatment, and in adulthood to assess long-lasting effects. Two different memory types, emotionally motivated conditioning and object recognition were assessed in adult animals. We found that iron administered in the neonatal period impairs both emotionally motivated and recognition memory. Polyubiquitinated protein levels were increased in the hippocampus, but not in the cortex, of adult animals treated with iron. Gene expression of subunits β1 and β5 was affected by age, being higher in the early stages of development in the hippocampus, accompanied by an age-related increase in polyubiquitinated protein levels in adults. In the cortex, gene expression of the three proteasome subunits was significantly higher in adulthood than in the neonatal period. These findings suggest that expression of proteasome subunits and activity are age-dependently regulated. Iron exposure in the neonatal period produces long-lasting harmful effects on the UPS functioning, which may be related with iron-induced memory impairment.

Inhibition of the Host Proteasome Facilitates Papaya Ringspot Virus Accumulation and Proteosomal Catalytic Activity Is Modulated by Viral Factor HcPro

PubMed Central

Sahana, Nandita; Kaur, Harpreet; Basavaraj; Tena, Fatima; Jain, Rakesh Kumar; Palukaitis, Peter; Canto, Tomas; Praveen, Shelly

2012-01-01

The ubiquitin/26S proteasome system plays an essential role not only in maintaining protein turnover, but also in regulating many other plant responses, including plant–pathogen interactions. Previous studies highlighted different roles of the 20S proteasome in plant defense during virus infection, either indirectly through viral suppressor-mediated degradation of Argonaute proteins, affecting the RNA interference pathway, or directly through modulation of the proteolytic and RNase activity of the 20S proteasome, a component of the 20S proteasome, by viral proteins, affecting the levels of viral proteins and RNAs. Here we show that MG132, a cell permeable proteasomal inhibitor, caused an increase in papaya ringspot virus (PRSV) accumulation in its natural host papaya (Carica papaya). We also show that the PRSV HcPro interacts with the papaya homologue of the Arabidopsis PAA (α1 subunit of the 20S proteasome), but not with the papaya homologue of Arabidopsis PAE (α5 subunit of the 20S proteasome), associated with the RNase activity, although the two 20S proteasome subunits interacted with each other. Mutated forms of PRSV HcPro showed that the conserved KITC54 motif in the N-terminal domain of HcPro was necessary for its binding to PAA. Co-agroinfiltration assays demonstrated that HcPro expression mimicked the action of MG132, and facilitated the accumulation of bothtotal ubiquitinated proteins and viral/non-viral exogenous RNA in Nicotiana benthamiana leaves. These effects were not observed by using an HcPro mutant (KITS54), which impaired the HcPro – PAA interaction. Thus, the PRSV HcPro interacts with a proteasomal subunit, inhibiting the action of the 20S proteasome, suggesting that HcPro might be crucial for modulating its catalytic activities in support of virus accumulation. PMID:23300704

Inhibition of the host proteasome facilitates papaya ringspot virus accumulation and proteosomal catalytic activity is modulated by viral factor HcPro.

PubMed

Sahana, Nandita; Kaur, Harpreet; Basavaraj; Tena, Fatima; Jain, Rakesh Kumar; Palukaitis, Peter; Canto, Tomas; Praveen, Shelly

2012-01-01

The ubiquitin/26S proteasome system plays an essential role not only in maintaining protein turnover, but also in regulating many other plant responses, including plant-pathogen interactions. Previous studies highlighted different roles of the 20S proteasome in plant defense during virus infection, either indirectly through viral suppressor-mediated degradation of Argonaute proteins, affecting the RNA interference pathway, or directly through modulation of the proteolytic and RNase activity of the 20S proteasome, a component of the 20S proteasome, by viral proteins, affecting the levels of viral proteins and RNAs. Here we show that MG132, a cell permeable proteasomal inhibitor, caused an increase in papaya ringspot virus (PRSV) accumulation in its natural host papaya (Carica papaya). We also show that the PRSV HcPro interacts with the papaya homologue of the Arabidopsis PAA (α1 subunit of the 20S proteasome), but not with the papaya homologue of Arabidopsis PAE (α5 subunit of the 20S proteasome), associated with the RNase activity, although the two 20S proteasome subunits interacted with each other. Mutated forms of PRSV HcPro showed that the conserved KITC54 motif in the N-terminal domain of HcPro was necessary for its binding to PAA. Co-agroinfiltration assays demonstrated that HcPro expression mimicked the action of MG132, and facilitated the accumulation of bothtotal ubiquitinated proteins and viral/non-viral exogenous RNA in Nicotiana benthamiana leaves. These effects were not observed by using an HcPro mutant (KITS54), which impaired the HcPro - PAA interaction. Thus, the PRSV HcPro interacts with a proteasomal subunit, inhibiting the action of the 20S proteasome, suggesting that HcPro might be crucial for modulating its catalytic activities in support of virus accumulation.

Sulforaphane attenuates postnatal proteasome inhibition and improves spatial learning in adult mice.

PubMed

Sunkaria, Aditya; Bhardwaj, Supriya; Yadav, Aarti; Halder, Avishek; Sandhir, Rajat

2018-01-01

Proteasomes are known to degrade proteins involved in various processes like metabolism, signal transduction, cell-cycle regulation, inflammation, and apoptosis. Evidence showed that protein degradation has a strong influence on developing neurons as well as synaptic plasticity. Here, we have shown that sulforaphane (SFN) could prevent the deleterious effects of postnatal proteasomal inhibition on spatial reference and working memory of adult mice. One day old Balb/c mice received intracerebroventricular injections of MG132 and SFN. Sham received an equal volume of aCSF. We observed that SFN pre-administration could attenuate MG132 mediated decrease in proteasome and calpain activities. In vitro findings revealed that SFN could induce proteasomal activity by enhancing the expression of catalytic subunit-β5. SFN pre-administration prevented the hippocampus based spatial memory impairments during adulthood, mediated by postnatal MG132 exposure. Histological examination showed deleterious effects of MG132 on pyramidal neurons and granule cell neurons in DG and CA3 sub-regions respectively. Furthermore, SFN pre-administration has shown to attenuate the effect of MG132 on proteasome subunit-β5 expression and also induce the Nrf2 nuclear translocation. In addition, SFN pre-administered mice have also shown to induce expression of pCaMKII, pCreb, and mature/pro-Bdnf, molecules which play a crucial role in spatial learning and memory consolidation. Our findings have shown that proteasomes play an important role in hippocampal synaptic plasticity during the early postnatal period and SFN pre-administration could enhance the proteasomal activity as well as improve spatial learning and memory consolidation. Copyright © 2017 Elsevier Inc. All rights reserved.

A monoclonal antibody that distinguishes latent and active forms of the proteasome (multicatalytic proteinase complex)

NASA Technical Reports Server (NTRS)

Weitman, D.; Etlinger, J. D.

1992-01-01

Monoclonal antibodies (mAbs) were generated to proteasome purified from human erythrocytes. Five of six proteasome-specific mAbs reacted with three subunits in the molecular mass range of 25-28 kDa, indicating a common epitope. The other mAb (AP5C10) exhibited a more restricted reactivity, recognizing a 32-kDa subunit of the proteasome purified in its latent state. However, when the proteasome is isolated in its active state, AP5C10 reacts with a 28-kDa subunit, evidence for processing of the proteasome subunits during purification. Purified proteasome preparations which exhibited partial latency have both AP5C10 reactive subunits. Although the 32-kDa subunit appears required for latency, loss of this component and generation of the 28-kDa component are not obligatory for activation. The 32- and 28-kDa subunits can each be further resolved into three components by isoelectric focusing. The apparent loss of 4 kDa during the conversion of the 32- to 28-kDa subunit is accompanied by a shift to a more basic pI for each polypeptide. Western blots of the early steps of proteasome purification reveal an AP5C10-reactive protein at 41 kDa. This protein was separated from proteasomes by sizing chromatography and may represent a pool of precursor subunits. Since the 32-kDa subunit appears necessary for latency, it is speculated to play a regulatory role in ATP-dependent proteolytic activity.

Modulation of the Proteasome Pathway by Nano-Curcumin and Curcumin in Retinal Pigment Epithelial Cells.

PubMed

Ramos de Carvalho, J Emanuel; Verwoert, Milan T; Vogels, Ilse M C; Schipper-Krom, Sabine; Van Noorden, Cornelis J F; Reits, Eric A; Klaassen, Ingeborg; Schlingemann, Reinier O

2018-01-01

Curcumin has multiple biological effects including the modulation of protein homeostasis by the ubiquitin-proteasome system. The purpose of this study was to assess the in vitro cytotoxic and oxidative effects of nano-curcumin and standard curcumin and characterize their effects on proteasome regulation in retinal pigment epithelial (RPE) cells. Viability, cell cycle progression, and reactive oxygen species (ROS) production were determined after treatment with nano-curcumin or curcumin. Subsequently, the effects of nano-curcumin and curcumin on proteasome activity and the gene and protein expression of proteasome subunits PA28α, α7, β5, and β5i were assessed. Nano-curcumin (5-100 μM) did not show significant cytotoxicity or anti-oxidative effects against H2O2-induced oxidative stress, whereas curcumin (≥10 μM) was cytotoxic and a potent inducer of ROS production. Both nano-curcumin and curcumin induced changes in proteasome-mediated proteolytic activity characterized by increased activity of the proteasome subunits β2 and β5i/β1 and reduced activity of β5/β1i. Likewise, nano-curcumin and curcumin affected mRNA and protein levels of household and immunoproteasome subunits. Nano-curcumin is less toxic to RPE cells and less prone to induce ROS production than curcumin. Both nano-curcumin and curcumin increase proteasome-mediated proteolytic activity. These results suggest that nano-curcumin may be regarded as a proteasome-modulating agent of limited cytotoxicity for RPE cells. The Author(s). Published by S. Karger AG, Basel.

Global Proteome Analysis Identifies Active Immunoproteasome Subunits in Human Platelets*

PubMed Central

Klockenbusch, Cordula; Walsh, Geraldine M.; Brown, Lyda M.; Hoffman, Michael D.; Ignatchenko, Vladimir; Kislinger, Thomas; Kast, Juergen

2014-01-01

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets. PMID:25146974

Involvement of the Nrf2-proteasome pathway in the endoplasmic reticulum stress response in pancreatic β-cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Sanghwan; Hur, Eu-gene; Ryoo, In-geun

2012-11-01

The ubiquitin-proteasome system plays a central role in protein quality control through endoplasmic reticulum (ER)-associated degradation (ERAD) of unfolded and misfolded proteins. NF-E2‐related factor 2 (Nrf2) is a transcription factor that controls the expression of an array of phase II detoxification and antioxidant genes. Nrf2 signaling has additionally been shown to upregulate the expression of the proteasome catalytic subunits in several cell types. Here, we investigated the role of Nrf2 in tunicamycin-induced ER stress using a murine insulinoma β-cell line, βTC-6. shRNA-mediated silencing of Nrf2 expression in βTC-6 cells significantly increased tunicamycin-induced cytotoxicity, elevated the expression of the pro-apoptotic ERmore » stress marker Chop10, and inhibited tunicamycin-inducible expression of the proteasomal catalytic subunits Psmb5 and Psmb6. The effects of 3H-1,2-dithiole-3-thione (D3T), a small molecule Nrf2 activator, on ER stress were also examined in βTC-6 cells. D3T pretreatment reduced tunicamycin cytotoxicity and attenuated the tunicamycin-inducible Chop10 and protein kinase RNA-activated‐like ER kinase (Perk). The protective effect of D3T was shown to be associated with increased ERAD. D3T increased the expression of Psmb5 and Psmb6 and elevated chymotrypsin-like peptidase activity; proteasome inhibitor treatment blocked D3T effects on tunicamycin cytotoxicity and ER stress marker changes. Similarly, silencing of Nrf2 abolished the protective effect of D3T against ER stress. These results indicate that the Nrf2 pathway contributes to the ER stress response in pancreatic β-cells by enhancing proteasome-mediated ERAD. -- Highlights: ► Nrf2 silencing in pancreatic β-cells enhanced tunicamycin-mediated ER stress. ► Expression of the proteasome was inducible by Nrf2 signaling. ► Nrf2 activator D3T protected β-cells from tunicamycin-mediated ER stress. ► Protective effect of D3T was associated with Nrf2-dependent proteasome induction.« less

FAT10 knock out mice livers fail to develop Mallory-Denk bodies in the DDC mouse model.

PubMed

French, S W; French, B A; Oliva, J; Li, J; Bardag-Gorce, F; Tillman, B; Canaan, A

2012-12-01

Mallory-Denk bodies (MDBs) are aggresomes composed of undigested ubiqutinated short lived proteins which have accumulated because of a decrease in the rate of their degradation by the 26s proteasome. The decrease in the activity of the proteasome is due to a shift in the activity of the 26s proteasome to the immunoproteasome triggered by an increase in expression of the catalytic subunits of the immunoproteasome which replaces the catalytic subunits of the 26s proteasome. This switch in the type of proteasome in liver cells is triggered by the binding of IFNγ to the IFNγ sequence response element (ISRE) located on the FAT10 promoter. To determine if either FAT10 or IFNγ are essential for the formation of MDBs we fed both IFNγ and FAT10 knock out (KO) mice DDC added to the control diet for 10weeks in order to induce MDBs. Mice fed the control diet and Wild type mice fed the DDC or control diet were compared. MDBs were located by immunofluorescent double stains using antibodies to ubiquitin to stain MDBs and FAT10 to localize the increased expression of FAT10 in MDB forming hepatocytes. We found that MDB formation occurred in the IFNγ KO mice but not in the FAT10 KO mice. Western blots showed an increase in the ubiquitin smears and decreases β 5 (chymotrypsin-like 26S proteasome subunit) in the Wild type mice fed DDC but not in the FAT10 KO mice fed DDC. To conclude, we have demonstrated that FAT10 is essential to the induction of MDB formation in the DDC fed mice. Copyright © 2012 Elsevier Inc. All rights reserved.

Possible roles of the transcription factor Nrf1 (NFE2L1) in neural homeostasis by regulating the gene expression of deubiquitinating enzymes.

PubMed

Taniguchi, Hiroaki; Okamuro, Shota; Koji, Misaki; Waku, Tsuyoshi; Kubo, Kaori; Hatanaka, Atsushi; Sun, Yimeng; Chowdhury, A M Masudul Azad; Fukamizu, Akiyoshi; Kobayashi, Akira

2017-02-26

The transcription factor Nrf1 (NFE2L1) maintains protein homeostasis (proteostasis) by regulating the gene expression of proteasome subunits in response to proteasome inhibition. The deletion of the Nrf1 gene in neural stem/progenitor cells causes severe neurodegeneration due to the accumulation of ubiquitinated proteins in Purkinje cells and motor neurons (Nrf1 NKO mice). However, the molecular mechanisms governing this neurodegenerative process remain unclear. We demonstrate herein that the loss of Nrf1 leads to the reduced gene expression of the deubiquitinating enzymes (DUBs) but not proteasome subunits in Nrf1 NKO mice between P7 and P18. First, we show that K48-linked polyubiquitinated proteins accumulate in Nrf1-deficient Purkinje cells and cerebral cortex neurons. Nevertheless, loss of Nrf1 does not alter the expression and proteolytic activity of proteasome. A significantly reduced expression of deubiquitinating enzymes was also demonstrated in Nrf1-deficient cerebellar tissue using microarray analysis. The genome database further reveals species-conserved ARE, a Nrf1 recognition element, in the regulatory region of certain DUB genes. Furthermore, we show that Nrf1 can activate Usp9x gene expression related to neurodegeneration. Altogether these findings suggest that neurodegeneration in Nrf1 NKO mice may stem from the dysfunction of the ubiquitin-mediated regulation of neuronal proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Global proteome analysis identifies active immunoproteasome subunits in human platelets.

PubMed

Klockenbusch, Cordula; Walsh, Geraldine M; Brown, Lyda M; Hoffman, Michael D; Ignatchenko, Vladimir; Kislinger, Thomas; Kast, Juergen

2014-12-01

The discovery of new functions for platelets, particularly in inflammation and immunity, has expanded the role of these anucleate cell fragments beyond their primary hemostatic function. Here, four in-depth human platelet proteomic data sets were generated to explore potential new functions for platelets based on their protein content and this led to the identification of 2559 high confidence proteins. During a more detailed analysis, consistently high expression of the proteasome was discovered, and the composition and function of this complex, whose role in platelets has not been thoroughly investigated, was examined. Data set mining resulted in identification of nearly all members of the 26S proteasome in one or more data sets, except the β5 subunit. However, β5i, a component of the immunoproteasome, was identified. Biochemical analyses confirmed the presence of all catalytically active subunits of the standard 20S proteasome and immunoproteasome in human platelets, including β5, which was predominantly found in its precursor form. It was demonstrated that these components were assembled into the proteasome complex and that standard proteasome as well as immunoproteasome subunits were constitutively active in platelets. These findings suggest potential new roles for platelets in the immune system. For example, the immunoproteasome may be involved in major histocompatibility complex I (MHC I) peptide generation, as the MHC I machinery was also identified in our data sets. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels.

PubMed

Fu, Ssu-Ju; Jeng, Chung-Jiuan; Ma, Chia-Hao; Peng, Yi-Jheng; Lee, Chi-Ming; Fang, Ya-Ching; Lee, Yi-Ching; Tang, Sung-Chun; Hu, Meng-Chun; Tang, Chih-Yung

2017-03-01

Voltage-gated Ca V 2.1 channels comprise a pore-forming α 1A subunit with auxiliary α 2 δ and β subunits. Ca V 2.1 channels play an essential role in regulating synaptic signaling. Mutations in the human gene encoding the Ca V 2.1 subunit are associated with the cerebellar disease episodic ataxia type 2 (EA2). Several EA2-causing mutants exhibit impaired protein stability and exert dominant-negative suppression of Ca V 2.1 wild-type (WT) protein expression via aberrant proteasomal degradation. Here, we set out to delineate the protein degradation mechanism of human Ca V 2.1 subunit by identifying RNF138, an E3 ubiquitin ligase, as a novel Ca V 2.1-binding partner. In neurons, RNF138 and Ca V 2.1 coexist in the same protein complex and display notable subcellular colocalization at presynaptic and postsynaptic regions. Overexpression of RNF138 promotes polyubiquitination and accelerates protein turnover of Ca V 2.1. Disrupting endogenous RNF138 function with a mutant (RNF138-H36E) or shRNA infection significantly upregulates the Ca V 2.1 protein level and enhances Ca V 2.1 protein stability. Disrupting endogenous RNF138 function also effectively rescues the defective protein expression of EA2 mutants, as well as fully reversing EA2 mutant-induced excessive proteasomal degradation of Ca V 2.1 WT subunits. RNF138-H36E coexpression only partially restores the dominant-negative effect of EA2 mutants on Ca V 2.1 WT functional expression, which can be attributed to defective membrane trafficking of Ca V 2.1 WT in the presence of EA2 mutants. We propose that RNF138 plays a critical role in the homeostatic regulation of Ca V 2.1 protein level and functional expression and that RNF138 serves as the primary E3 ubiquitin ligase promoting EA2-associated aberrant degradation of human Ca V 2.1 subunits. SIGNIFICANCE STATEMENT Loss-of-function mutations in the human Ca V 2.1 subunit are linked to episodic ataxia type 2 (EA2), a dominantly inherited disease characterized by paroxysmal attacks of ataxia and nystagmus. EA2-causing mutants may exert dominant-negative effects on the Ca V 2.1 wild-type subunit via aberrant proteasomal degradation. The molecular nature of the Ca V 2.1 ubiquitin-proteasome degradation pathway is currently unknown. The present study reports the first identification of an E3 ubiquitin ligase for Ca V 2.1, RNF138. Ca V 2.1 protein stability is dynamically regulated by RNF138 and auxiliary α 2 δ and β subunits. We provide a proof of concept that protecting the human Ca V 2.1 subunit from excessive proteasomal degradation with specific interruption of endogenous RNF138 function may partially contribute to the future development of a novel therapeutic strategy for EA2 patients. Copyright © 2017 the authors 0270-6474/17/372485-19$15.00/0.

Cannabinoids synergize with carfilzomib, reducing multiple myeloma cells viability and migration.

PubMed

Nabissi, Massimo; Morelli, Maria Beatrice; Offidani, Massimo; Amantini, Consuelo; Gentili, Silvia; Soriani, Alessandra; Cardinali, Claudio; Leoni, Pietro; Santoni, Giorgio

2016-11-22

Several studies showed a potential anti-tumor role for cannabinoids, by modulating cell signaling pathways involved in cancer cell proliferation, chemo-resistance and migration. Cannabidiol (CBD) was previously noted in multiple myeloma (MM), both alone and in synergy with the proteasome inhibitor bortezomib, to induce cell death. In other type of human cancers, the combination of CBD with Δ9-tetrahydrocannabinol (THC) was found to act synergistically with other chemotherapeutic drugs suggesting their use in combination therapy. In the current study, we evaluated the effects of THC alone and in combination with CBD in MM cell lines. We found that CBD and THC, mainly in combination, were able to reduce cell viability by inducing autophagic-dependent necrosis. Moreover, we showed that the CBD-THC combination was able to reduce MM cells migration by down-regulating expression of the chemokine receptor CXCR4 and of the CD147 plasma membrane glycoprotein. Furthermore, since the immuno-proteasome is considered a new target in MM and also since carfilzomib (CFZ) is a new promising immuno-proteasome inhibitor that creates irreversible adducts with the β5i subunit of immuno-proteasome, we evaluated the effect of CBD and THC in regulating the expression of the β5i subunit and their effect in combination with CFZ. Herein, we also found that the CBD and THC combination is able to reduce expression of the β5i subunit as well as to act in synergy with CFZ to increase MM cell death and inhibits cell migration. In summary, these results proved that this combination exerts strong anti-myeloma activities.

Proteomics of the 26S proteasome in Spodoptera frugiperda cells infected with the nucleopolyhedrovirus, AcMNPV.

PubMed

Lyupina, Yulia V; Zatsepina, Olga G; Serebryakova, Marina V; Erokhov, Pavel A; Abaturova, Svetlana B; Kravchuk, Oksana I; Orlova, Olga V; Beljelarskaya, Svetlana N; Lavrov, Andrey I; Sokolova, Olga S; Mikhailov, Victor S

2016-06-01

Baculoviruses are large DNA viruses that infect insect species such as Lepidoptera and are used in biotechnology for protein production and in agriculture as insecticides against crop pests. Baculoviruses require activity of host proteasomes for efficient reproduction, but how they control the cellular proteome and interact with the ubiquitin proteasome system (UPS) of infected cells remains unknown. In this report, we analyzed possible changes in the subunit composition of 26S proteasomes of the fall armyworm, Spodoptera frugiperda (Sf9), cells in the course of infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 26S proteasomes were purified from Sf9 cells by an immune affinity method and subjected to 2D gel electrophoresis followed by MALDI-TOF mass spectrometry and Mascot search in bioinformatics databases. A total of 34 homologues of 26S proteasome subunits of eukaryotic species were identified including 14 subunits of the 20S core particle (7 α and 7 β subunits) and 20 subunits of the 19S regulatory particle (RP). The RP contained homologues of 11 of RPN-type and 6 of RPT-type subunits, 2 deubiquitinating enzymes (UCH-14/UBP6 and UCH-L5/UCH37), and thioredoxin. Similar 2D-gel maps of 26S proteasomes purified from uninfected and AcMNPV-infected cells at 48hpi confirmed the structural integrity of the 26S proteasome in insect cells during baculovirus infection. However, subtle changes in minor forms of some proteasome subunits were detected. A portion of the α5(zeta) cellular pool that presumably was not associated with the proteasome underwent partial proteolysis at a late stage in infection. Copyright © 2016 Elsevier B.V. All rights reserved.

Novel Aspects of Degradation of T Cell Receptor Subunits from the Endoplasmic Reticulum (ER) in T Cells: Importance of Oligosaccharide Processing, Ubiquitination, and Proteasome-dependent Removal from ER Membranes

PubMed Central

Yang, Mei; Omura, Satoshi; Bonifacino, Juan S.; Weissman, Allan M.

1998-01-01

Expression of the T cell antigen receptor (TCR) on the surface of thymocytes and mature T cells is dependent on the assembly of receptor subunits into TCRs in the endoplasmic reticulum (ER) and their successful traversal of the secretory pathway to the plasma membrane. TCR subunits that fail to exit the ER for the Golgi complex are degraded by nonlysosomal processes that have been referred to as “ER degradation”. The molecular basis for the loss of the TCR CD3-δ and TCR-α subunits from the ER was investigated in lymphocytes. For CD3-δ, we describe a process leading to its degradation that includes trimming of mannose residues from asparagine-linked (N-linked) oligosaccharides, generation of ubiquitinated membrane-bound intermediates, and proteasome-dependent removal from the ER membrane. When either mannosidase activity or the catalytic activity of proteasomes was inhibited, loss of CD3-δ was markedly curtailed and CD3-δ remained membrane bound in a complex with CD3-ε. TCR-α was also found to be degraded in a proteasome-dependent manner with ubiquitinated intermediates. However, no evidence of a role for mannosidases was found for TCR-α, and significant retrograde movement through the ER membrane took place even when proteasome function was inhibited. These findings provide new insights into mechanisms employed to regulate levels of TCRs, and underscore that cells use multiple mechanisms to target proteins from the ER to the cytosol for degradation. PMID:9500786

Bacterial self-resistance to the natural proteasome inhibitor salinosporamide A

PubMed Central

Kale, Andrew J.; McGlinchey, Ryan P.; Lechner, Anna; Moore, Bradley S.

2011-01-01

Proteasome inhibitors have recently emerged as a therapeutic strategy in cancer chemotherapy but susceptibility to drug resistance limits their efficacy. The marine actinobacterium Salinispora tropica produces salinosporamide A (NPI-0052, marizomib), a potent proteasome inhibitor and promising clinical agent in the treatment of multiple myeloma. Actinobacteria also possess 20S proteasome machinery, raising the question of self-resistance. We identified a redundant proteasome β-subunit, SalI, encoded within the salinosporamide biosynthetic gene cluster and biochemically characterized the SalI proteasome complex. The SalI β-subunit has an altered substrate specificity profile, 30-fold resistance to salinosporamide A, and cross-resistance to the FDA-approved proteasome inhibitor bortezomib. An A49V mutation in SalI correlates to clinical bortezomib resistance from a human proteasome β 5-subunit A49T mutation, suggesting that intrinsic resistance to natural proteasome inhibitors may predict clinical outcomes. PMID:21882868

[Expression of proteasome subunits PSMB5 and PSMB9 mRNA in hippocampal neurons in experimental diabetes mellitus: link with apoptosis and necrosis].

PubMed

Lebid', Iu V; Dosenko, V Ie; Skybo, H H

2010-01-01

There is a huge body of evidence showing that long-termed diabetes mellitus is followed with hippocampal dysfunction. The goal of this work was to investigate the expression of proteasome subunits PSMB5 and PSMB9 mRNA in CA1, CA2 and CA3 areas of hippocampus in parallel with processes of cell death (apoptosis and necrosis) in development dynamics of streptozotocine-induced diabetes. We have studied hippocampal neurons using chromatine dye Hoechst-33342 and immunohistochemical detection of apoptotic cell death marker caspase-3. At day 3 and 7 after injection of streptozotocine we have performed visualization of caspase-3-immunopositive neurons showing signs of neurodegeneration in hippocampal sections using confocal microscope Olympus FV1000. The rate of proteasome subunits PSMB5 and PSMB9 mRNA expression was determined with RT-PCR. The results indicated elevation of PSMB9 mRNA content (from 4807 +/- 0.392 arbU up to 20,023 +/- 4949 arbU on day 3 and up to 20,253 +/- 5141 arbU on day 7). A maximal number of cells with signs of chromatin condensation was observed at day 3 and day 7 in CA2 and CA3 area (11.51% and 12.49% respectively). That indicates an intensification of proapoptotic processes. Summarizing the results presented above we can conclude that during the first week of diabetes mellitus development, hippocampal cells undergo the process of impairment and degeneration.

Assessing subunit dependency of the Plasmodium proteasome using small molecule inhibitors and active site probes.

PubMed

Li, Hao; van der Linden, Wouter A; Verdoes, Martijn; Florea, Bogdan I; McAllister, Fiona E; Govindaswamy, Kavitha; Elias, Joshua E; Bhanot, Purnima; Overkleeft, Herman S; Bogyo, Matthew

2014-08-15

The ubiquitin-proteasome system (UPS) is a potential pathway for therapeutic intervention for pathogens such as Plasmodium, the causative agent of malaria. However, due to the essential nature of this proteolytic pathway, proteasome inhibitors must avoid inhibition of the host enzyme complex to prevent toxic side effects. The Plasmodium proteasome is poorly characterized, making rational design of inhibitors that induce selective parasite killing difficult. In this study, we developed a chemical probe that labels all catalytic sites of the Plasmodium proteasome. Using this probe, we identified several subunit selective small molecule inhibitors of the parasite enzyme complex. Treatment with an inhibitor that is specific for the β5 subunit during blood stage schizogony led to a dramatic decrease in parasite replication while short-term inhibition of the β2 subunit did not affect viability. Interestingly, coinhibition of both the β2 and β5 catalytic subunits resulted in enhanced parasite killing at all stages of the blood stage life cycle and reduced parasite levels in vivo to barely detectable levels. Parasite killing was achieved with overall low host toxicity, something that has not been possible with existing proteasome inhibitors. Our results highlight differences in the subunit dependency of the parasite and human proteasome, thus providing a strategy for development of potent antimalarial drugs with overall low host toxicity.

The RPN5 subunit of the 26s proteasome is essential for gametogenesis, sporophyte development, and complex assembly in Arabidopsis.

PubMed

Book, Adam J; Smalle, Jan; Lee, Kwang-Hee; Yang, Peizhen; Walker, Joseph M; Casper, Sarah; Holmes, James H; Russo, Laura A; Buzzinotti, Zachri W; Jenik, Pablo D; Vierstra, Richard D

2009-02-01

The 26S proteasome is an essential multicatalytic protease complex that degrades a wide range of intracellular proteins, especially those modified with ubiquitin. Arabidopsis thaliana and other plants use pairs of genes to encode most of the core subunits, with both of the isoforms often incorporated into the mature complex. Here, we show that the gene pair encoding the regulatory particle non-ATPase subunit (RPN5) has a unique role in proteasome function and Arabidopsis development. Homozygous rpn5a rpn5b mutants could not be generated due to a defect in male gametogenesis. While single rpn5b mutants appear wild-type, single rpn5a mutants display a host of morphogenic defects, including abnormal embryogenesis, partially deetiolated development in the dark, a severely dwarfed phenotype when grown in the light, and infertility. Proteasome complexes missing RPN5a are less stable in vitro, suggesting that some of the rpn5a defects are caused by altered complex integrity. The rpn5a phenotype could be rescued by expression of either RPN5a or RPN5b, indicating functional redundancy. However, abnormal phenotypes generated by overexpression implied that paralog-specific functions also exist. Collectively, the data point to a specific role for RPN5 in the plant 26S proteasome and suggest that its two paralogous genes in Arabidopsis have both redundant and unique roles in development.

Selective inhibitors of the osteoblast proteasome stimulate bone formation in vivo and in vitro

PubMed Central

Garrett, I.R.; Chen, D.; Gutierrez, G.; Zhao, M.; Escobedo, A.; Rossini, G.; Harris, S.E.; Gallwitz, W.; Kim, K.B.; Hu, S.; Crews, C.M.; Mundy, G.R.

2003-01-01

We have found that the ubiquitin-proteasome pathway exerts exquisite control of osteoblast differentiation and bone formation in vitro and in vivo in rodents. Structurally different inhibitors that bind to specific catalytic β subunits of the 20S proteasome stimulated bone formation in bone organ cultures in concentrations as low as 10 nM. When administered systemically to mice, the proteasome inhibitors epoxomicin and proteasome inhibitor–1 increased bone volume and bone formation rates over 70% after only 5 days of treatment. Since the ubiquitin-proteasome pathway has been shown to modulate expression of the Drosophila homologue of the bone morphogenetic protein-2 and -4 (BMP-2 and BMP-4) genes, we examined the effects of noggin, an endogenous inhibitor of BMP-2 and BMP-4 on bone formation stimulated by these compounds and found that it was abrogated. These compounds increased BMP-2 but not BMP-4 or BMP-6 mRNA expression in osteoblastic cells, suggesting that BMP-2 was responsible for the observed bone formation that was inhibited by noggin. We show proteasome inhibitors regulate BMP-2 gene expression at least in part through inhibiting the proteolytic processing of Gli3 protein. Our results suggest that the ubiquitin-proteasome machinery regulates osteoblast differentiation and bone formation and that inhibition of specific components of this system may be useful therapeutically in common diseases of bone loss. PMID:12782679

Host Gene Expression Analysis in Sri Lankan Melioidosis Patients

DTIC Science & Technology

2017-06-19

response genes and epigenetic regulators during melioidosis infection. Methods Patient enrollment Nationwide active surveillance for melioidosis... activator complex subunit 2 TR-17-140 Distribution Statement A: Approved for public release; distribution is unlimited. 13 PSMA5 Proteasome subunit...B-cell activation and T-cell proliferation, thus acting as a key regulator of humoral and adaptive immunity. Its role as an anti-inflammatory

Different effects of the nonsteroidal anti-inflammatory drugs meclofenamate sodium and naproxen sodium on proteasome activity in cardiac cells.

PubMed

Ghosh, Rajeshwary; Hwang, Soyun M; Cui, Ziyou; Gilda, Jennifer E; Gomes, Aldrin V

2016-05-01

The use of nonsteroidal anti-inflammatory drugs (NSAIDs) like meclofenamate sodium (MS), used to reduce pain, has been associated with an increased risk of cardiovascular disease (CVD). Naproxen (NAP), another NSAID, is not associated with increased risk of CVD. The molecular mechanism(s) by which NSAIDs induce CVD is unknown. We investigated the effects of MS and NAP on protein homeostasis and cardiotoxicity in rat cardiac H9c2 cells and murine neonatal cardiomyocytes. MS, but not NAP, significantly inhibited proteasome activity and reduced cardiac cell viability at pharmacological levels found in humans. Although proteasome subunit gene and protein expression were unaffected by NSAIDs, MS treated cell lysates showed higher 20S proteasome content, while purified proteasomes from MS treated cells had lower proteasome activity and higher levels of oxidized subunits than proteasomes from control cells. Addition of exogenous proteasome to MS treated cells improved cell viability. Both MS and NAP increased ROS production, but the rate of ROS production was greater in MS than in NAP treated cells. The ROS production is likely from mitochondria, as MS inhibited mitochondrial Complexes I and III, major sources of ROS, while NAP inhibited Complex I. MS also impaired mitochondrial membrane potential while NAP did not. Antioxidants were able to prevent the reduced cell viability caused by MS treatment. These results suggest that NSAIDs induce cardiotoxicity by a ROS dependent mechanism involving mitochondrial and proteasome dysfunction and may explain why some NSAIDs should not be given to patients for long periods. Copyright © 2016 Elsevier Ltd. All rights reserved.

Targeting the 19S proteasomal subunit, Rpt4, for the treatment of colon cancer.

PubMed

Boland, Karen; Flanagan, Lorna; McCawley, Niamh; Pabari, Ritesh; Kay, Elaine W; McNamara, Deborah A; Murray, Frank; Byrne, Annette T; Ramtoola, Zebunnissa; Concannon, Caoimhín G; Prehn, Jochen H M

2016-06-05

Deregulation of the ubiquitin-proteasome pathway has been frequently observed in a number of malignancies. Using quantitative Western blotting of normal and matched tumour tissue, we here identified a significant increase in the 19S proteasome subunit Rpt4 in response to chemoradiation in locally advanced rectal cancer patients with unfavourable outcome. We therefore explored the potential of Rpt4 reduction as a therapeutic strategy in colorectal cancer (CRC). Utilizing siRNA to down regulate Rpt4 expression, we show that silencing of Rpt4 reduced proteasomal activity and induced endoplasmic reticulum stress. Gene silencing of Rpt4 also inhibited cell proliferation, reduced clonogenic survival and induced apoptosis in HCT-116 colon cancer cells. We next developed a cell penetrating peptide-based nanoparticle delivery system to achieve in vivo gene silencing of Rpt4. Administration of Rpt4 siRNA nanoparticles reduced tumour growth and improved survival in a HCT-116 colon cancer xenograft tumour model in vivo. Collectively, our data suggest that inhibition of Rpt4 represents a novel strategy for the treatment of CRC. Copyright © 2016 Elsevier B.V. All rights reserved.

CRISPR Genome-Wide Screening Identifies Dependence on the Proteasome Subunit PSMC6 for Bortezomib Sensitivity in Multiple Myeloma.

PubMed

Shi, Chang-Xin; Kortüm, K Martin; Zhu, Yuan Xiao; Bruins, Laura A; Jedlowski, Patrick; Votruba, Patrick G; Luo, Moulun; Stewart, Robert A; Ahmann, Jonathan; Braggio, Esteban; Stewart, A Keith

2017-12-01

Bortezomib is highly effective in the treatment of multiple myeloma; however, emergent drug resistance is common. Consequently, we employed CRISPR targeting 19,052 human genes to identify unbiased targets that contribute to bortezomib resistance. Specifically, we engineered an RPMI8226 multiple myeloma cell line to express Cas9 infected by lentiviral vector CRISPR library and cultured derived cells in doses of bortezomib lethal to parental cells. Sequencing was performed on surviving cells to identify inactivated genes responsible for drug resistance. From two independent whole-genome screens, we selected 31 candidate genes and constructed a second CRISPR sgRNA library, specifically targeting each of these 31 genes with four sgRNAs. After secondary screening for bortezomib resistance, the top 20 "resistance" genes were selected for individual validation. Of these 20 targets, the proteasome regulatory subunit PSMC6 was the only gene validated to reproducibly confer bortezomib resistance. We confirmed that inhibition of chymotrypsin-like proteasome activity by bortezomib was significantly reduced in cells lacking PSMC6. We individually investigated other members of the PSMC group (PSMC1 to 5) and found that deficiency in each of those subunits also imparts bortezomib resistance. We found 36 mutations in 19S proteasome subunits out of 895 patients in the IA10 release of the CoMMpass study (https://themmrf.org). Our findings demonstrate that the PSMC6 subunit is the most prominent target required for bortezomib sensitivity in multiple myeloma cells and should be examined in drug-refractory populations. Mol Cancer Ther; 16(12); 2862-70. ©2017 AACR . ©2017 American Association for Cancer Research.

Downregulation of 26S proteasome catalytic activity promotes epithelial-mesenchymal transition

PubMed Central

van Baarsel, Eric D.; Metz, Patrick J.; Fisch, Kathleen; Widjaja, Christella E.; Kim, Stephanie H.; Lopez, Justine; Chang, Aaron N.; Geurink, Paul P.; Florea, Bogdan I.; Overkleeft, Hermen S.; Ovaa, Huib; Bui, Jack D.; Yang, Jing; Chang, John T.

2016-01-01

The epithelial-mesenchymal transition (EMT) endows carcinoma cells with phenotypic plasticity that can facilitate the formation of cancer stem cells (CSCs) and contribute to the metastatic cascade. While there is substantial support for the role of EMT in driving cancer cell dissemination, less is known about the intracellular molecular mechanisms that govern formation of CSCs via EMT. Here we show that β2 and β5 proteasome subunit activity is downregulated during EMT in immortalized human mammary epithelial cells. Moreover, selective proteasome inhibition enabled mammary epithelial cells to acquire certain morphologic and functional characteristics reminiscent of cancer stem cells, including CD44 expression, self-renewal, and tumor formation. Transcriptomic analyses suggested that proteasome-inhibited cells share gene expression signatures with cells that have undergone EMT, in part, through modulation of the TGF-β signaling pathway. These findings suggest that selective downregulation of proteasome activity in mammary epithelial cells can initiate the EMT program and acquisition of a cancer stem cell-like phenotype. As proteasome inhibitors become increasingly used in cancer treatment, our findings highlight a potential risk of these therapeutic strategies and suggest a possible mechanism by which carcinoma cells may escape from proteasome inhibitor-based therapy. PMID:26930717

Dual function of Rpn5 in two PCI complexes, the 26S proteasome and COP9 signalosome.

PubMed

Yu, Zanlin; Kleifeld, Oded; Lande-Atir, Avigail; Bsoul, Maisa; Kleiman, Maya; Krutauz, Daria; Book, Adam; Vierstra, Richard D; Hofmann, Kay; Reis, Noa; Glickman, Michael H; Pick, Elah

2011-04-01

Subunit composition and architectural structure of the 26S proteasome lid is strictly conserved between all eukaryotes. This eight-subunit complex bears high similarity to the eukaryotic translation initiation factor 3 and to the COP9 signalosome (CSN), which together define the proteasome CSN/COP9/initiation factor (PCI) troika. In some unicellular eukaryotes, the latter two complexes lack key subunits, encouraging questions about the conservation of their structural design. Here we demonstrate that, in Saccharomyces cerevisiae, Rpn5 plays dual roles by stabilizing proteasome and CSN structures independently. Proteasome and CSN complexes are easily dissected, with Rpn5 the only subunit in common. Together with Rpn5, we identified a total of six bona fide subunits at roughly stoichiometric ratios in isolated, affinity-purified CSN. Moreover, the copy of Rpn5 associated with the CSN is required for enzymatic hydrolysis of Rub1/Nedd8 conjugated to cullins. We propose that multitasking by a single subunit, Rpn5 in this case, allows it to function in different complexes simultaneously. These observations demonstrate that functional substitution of subunits by paralogues is feasible, implying that the canonical composition of the three PCI complexes in S. cerevisiae is more robust than hitherto appreciated.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Osna, Natalia A., E-mail: nosna@UNMC.edu; Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68105; White, Ronda L.

The proteasome is a multi-catalytic protein degradation enzyme that is regulated by ethanol-induced oxidative stress; such suppression is attributed to CYP2E1-generated metabolites. However, under certain conditions, it appears that in addition to oxidative stress, other mechanisms are also involved in proteasome regulation. This study investigated whether impaired protein methylation that occurs during exposure of liver cells to ethanol, may contribute to suppression of proteasome activity. We measured the chymotrypsin-like proteasome activity in Huh7CYP cells, hepatocytes, liver cytosols and nuclear extracts or purified 20S proteasome under conditions that maintain or prevent protein methylation. Reduction of proteasome activity of hepatoma cell andmore » hepatocytes by ethanol or tubercidin was prevented by simultaneous treatment with S-adenosylmethionine (SAM). Moreover, the tubercidin-induced decline in proteasome activity occurred in both nuclear and cytosolic fractions. In vitro exposure of cell cytosolic fractions or highly purified 20S proteasome to low SAM:S-adenosylhomocysteine (SAH) ratios in the buffer also suppressed proteasome function, indicating that one or more methyltransferase(s) may be associated with proteasomal subunits. Immunoblotting a purified 20S rabbit red cell proteasome preparation using methyl lysine-specific antibodies revealed a 25 kDa proteasome subunit that showed positive reactivity with anti-methyl lysine. This reactivity was modified when 20S proteasome was exposed to differential SAM:SAH ratios. We conclude that impaired methylation of proteasome subunits suppressed proteasome activity in liver cells indicating an additional, yet novel mechanism of proteasome activity regulation by ethanol.« less

Nucleotide-dependent switch in proteasome assembly mediated by the Nas6 chaperone

PubMed Central

Li, Frances; Tian, Geng; Langager, Deanna; Sokolova, Vladyslava; Finley, Daniel; Park, Soyeon

2017-01-01

The proteasome is assembled via the nine-subunit lid, nine-subunit base, and 28-subunit core particle (CP). Previous work has shown that the chaperones Rpn14, Nas6, Hsm3, and Nas2 each bind a specific ATPase subunit of the base and antagonize base–CP interaction. Here, we show that the Nas6 chaperone also obstructs base–lid association. Nas6 alternates between these two inhibitory modes according to the nucleotide state of the base. When ATP cannot be hydrolyzed, Nas6 interferes with base–lid, but not base–CP, association. In contrast, under conditions of ATP hydrolysis, Nas6 obstructs base–CP, but not base–lid, association. Modeling of Nas6 into cryoelectron microscopy structures of the proteasome suggests that Nas6 controls both base–lid affinity and base–CP affinity through steric hindrance; Nas6 clashes with the lid in the ATP-hydrolysis–blocked proteasome, but clashes instead with the CP in the ATP-hydrolysis–competent proteasome. Thus, Nas6 provides a dual mechanism to control assembly at both major interfaces of the proteasome. PMID:28137839

Impaired proteasome function in sporadic amyotrophic lateral sclerosis.

PubMed

Kabashi, Edor; Agar, Jeffrey N; Strong, Michael J; Durham, Heather D

2012-06-01

Abstract The ubiquitin-proteasome system, important for maintaining protein quality control, is compromised in experimental models of familial ALS. The objective of this study was to determine if proteasome function is impaired in sporadic ALS. Proteasomal activities and subunit composition were evaluated in homogenates of spinal cord samples obtained at autopsy from sporadic ALS and non-neurological control cases, compared to cerebellum as a clinically spared tissue. The level of 20S α structural proteasome subunits was assessed in motor neurons by immunohistochemistry. Catalysis of peptide substrates of the three major proteasomal activities was substantially reduced in ALS thoracic spinal cord, but not in cerebellum, accompanied by alterations in the constitutive proteasome machinery. Chymotrypsin-like activity was decreased to 60% and 65% of control in ventral and dorsal spinal cord, respectively, concomitant with reduction in the β5 subunit with this catalytic activity. Caspase- and trypsin-like activities were reduced to a similar extent (46% - 68% of control). Proteasome levels, although generally maintained, appeared reduced specifically in motor neurons by immunolabelling. In conclusion, there are commonalities of findings in sporadic ALS patients and presymptomatic SOD1-G93A transgenic mice and these implicate inadequate proteasome function in the pathogenesis of both familial and sporadic ALS.

Basic leucine zipper protein Cnc-C is a substrate and transcriptional regulator of the Drosophila 26S proteasome.

PubMed

Grimberg, Kristian Björk; Beskow, Anne; Lundin, Daniel; Davis, Monica M; Young, Patrick

2011-02-01

While the 26S proteasome is a key proteolytic complex, little is known about how proteasome levels are maintained in higher eukaryotic cells. Here we describe an RNA interference (RNAi) screen of Drosophila melanogaster that was used to identify transcription factors that may play a role in maintaining levels of the 26S proteasome. We used an RNAi library against 993 Drosophila transcription factor genes to identify genes whose suppression in Schneider 2 cells stabilized a ubiquitin-green fluorescent protein reporter protein. This screen identified Cnc (cap 'n' collar [CNC]; basic region leucine zipper) as a candidate transcriptional regulator of proteasome component expression. In fact, 20S proteasome activity was reduced in cells depleted of cnc. Immunoblot assays against proteasome components revealed a general decline in both 19S regulatory complex and 20S proteasome subunits after RNAi depletion of this transcription factor. Transcript-specific silencing revealed that the longest of the seven transcripts for the cnc gene, cnc-C, was needed for proteasome and p97 ATPase production. Quantitative reverse transcription-PCR confirmed the role of Cnc-C in activation of transcription of genes encoding proteasome components. Expression of a V5-His-tagged form of Cnc-C revealed that the transcription factor is itself a proteasome substrate that is stabilized when the proteasome is inhibited. We propose that this single cnc gene in Drosophila resembles the ancestral gene family of mammalian nuclear factor erythroid-derived 2-related transcription factors, which are essential in regulating oxidative stress and proteolysis.

Homopiperazine Derivatives as a Novel Class of Proteasome Inhibitors with a Unique Mode of Proteasome Binding

PubMed Central

Kikuchi, Jiro; Shibayama, Naoya; Yamada, Satoshi; Wada, Taeko; Nobuyoshi, Masaharu; Izumi, Tohru; Akutsu, Miyuki; Kano, Yasuhiko; Sugiyama, Kanako; Ohki, Mio; Park, Sam-Yong; Furukawa, Yusuke

2013-01-01

The proteasome is a proteolytic machinery that executes the degradation of polyubiquitinated proteins to maintain cellular homeostasis. Proteasome inhibition is a unique and effective way to kill cancer cells because they are sensitive to proteotoxic stress. Indeed, the proteasome inhibitor bortezomib is now indispensable for the treatment of multiple myeloma and other intractable malignancies, but is associated with patient inconvenience due to intravenous injection and emerging drug resistance. To resolve these problems, we attempted to develop orally bioavailable proteasome inhibitors with distinct mechanisms of action and identified homopiperazine derivatives (HPDs) as promising candidates. Biochemical and crystallographic studies revealed that some HPDs inhibit all three catalytic subunits (ß 1, ß 2 and ß 5) of the proteasome by direct binding, whereas bortezomib and other proteasome inhibitors mainly act on the ß5 subunit. Proteasome-inhibitory HPDs exhibited cytotoxic effects on cell lines from various hematological malignancies including myeloma. Furthermore, K-7174, one of the HPDs, was able to inhibit the growth of bortezomib-resistant myeloma cells carrying a ß5-subunit mutation. Finally, K-7174 had additive effects with bortezomib on proteasome inhibition and apoptosis induction in myeloma cells. Taken together, HPDs could be a new class of proteasome inhibitors, which compensate for the weak points of conventional ones and overcome the resistance to bortezomib. PMID:23593271

Androgen receptor polyglutamine expansion drives age-dependent quality control defects and muscle dysfunction.

PubMed

Nath, Samir R; Yu, Zhigang; Gipson, Theresa A; Marsh, Gregory B; Yoshidome, Eriko; Robins, Diane M; Todi, Sokol V; Housman, David E; Lieberman, Andrew P

2018-05-29

Skeletal muscle has emerged as a critical, disease-relevant target tissue in spinal and bulbar muscular atrophy, a degenerative disorder of the neuromuscular system caused by a CAG/polyglutamine (polyQ) expansion in the androgen receptor (AR) gene. Here, we used RNA-Seq to identify pathways that are disrupted in diseased muscle using AR113Q knock-in mice. This analysis unexpectedly identified significantly diminished expression of numerous ubiquitin-proteasome pathway genes in AR113Q muscle, encoding approximately 30% of proteasome subunits and 20% of E2 ubiquitin conjugases. These changes were age-, hormone- and glutamine length-dependent and arose due to a toxic gain-of-function conferred by the mutation. Moreover, altered gene expression was associated with decreased level of the proteasome transcription factor NRF1 and its activator DDI2 and resulted in diminished proteasome activity. Ubiquitinated ADRM1 was detected in AR113Q muscle, indicating the occurrence of stalled proteasomes in mutant mice. Finally, diminished expression of Drosophila orthologues of NRF1 or ADRM1 promoted the accumulation of polyQ AR protein and increased toxicity. Collectively, these data indicate that AR113Q muscle develops progressive proteasome dysfunction that leads to the impairment of quality control and the accumulation of polyQ AR protein, key features that contribute to the age-dependent onset and progression of this disorder.

Apigenin manipulates the ubiquitin-proteasome system to rescue estrogen receptor-β from degradation and induce apoptosis in prostate cancer cells.

PubMed

Singh, Vishal; Sharma, Vikas; Verma, Vikas; Pandey, Deepti; Yadav, Santosh K; Maikhuri, Jagdamba P; Gupta, Gopal

2015-12-01

To investigate apigenin (5,7,4-trihydroxyflavone), a dietary flavonoid with proteasome-inhibitory activity (desired for the management of multiple types of cancers), against FDA-approved anticancer proteasome inhibitor bortezomib in context to its effects on the tumor suppressor estrogen receptor-beta (ER-β) in prostate cancer cells. Prostate cancer (PC-3) cells were treated with either apigenin or bortezomib, and proliferation inhibition was correlated with proteasomal biochemistry, ER-degradation and cell apoptosis. Apigenin specifically inhibited only chymotrypsin-like activity of proteasome without affecting trypsin and caspase-like activities, which was in contrast to the non-specific inhibition of all the three activities by bortezomib. Apigenin selectively increased the protein levels of ER-β at 1.8 and 10.0 µM (without affecting mRNA levels) and preferentially accumulated ubiquitinated ER-β over ER-α in PC-3. Apigenin-treated cells exhibited increased ER-β interactions with ubiquitin-protein ligase E6AP, downregulated PSMA5 (α-5 subunit for assembly of 20S proteasome) without affecting PSMB1 (β-1 subunit), PSMB2 (β-2 subunit) and PSMB5 (β-5 subunit, whose overexpression by bortezomib causes drug resistance) of proteasome at mRNA levels. Caspase-3 activation in PC-3 by apigenin was dependent on caspase-8 activity but independent of mitochondrial membrane depolarization. The deubiquitinase USP14 activity, which antagonizes degradation of proteins via proteasome, was significantly increased by apigenin treatment. Apigenin selectively inhibits proteasomal degradation of tumor suppressor ER-β by specifically inhibiting chymotrypsin-like activity of proteasome, preventing its assembly via PSMA5 and inhibiting USP14 enzyme activity in prostate cancer cells, resulting in cancer cell apoptosis. Unlike bortezomib, apigenin's actions are subtle, precise, mechanistically distinct and capable of abstaining drug resistance.

Proteasome, but Not Autophagy, Disruption Results in Severe Eye and Wing Dysmorphia: A Subunit- and Regulator-Dependent Process in Drosophila

PubMed Central

Pantazi, Asimina D.; Mpakou, Vassiliki E.; Zervas, Christos G.; Papassideri, Issidora S.; Stravopodis, Dimitrios J.

2013-01-01

Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, we herein examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}BxMS1096 genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, we proved that proteasomal integrity and ubiquitination proficiency essentially control fly’s eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, α5, β5 and β6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing’s, but not eye’s, morphogenetic organization and architecture. However, Atg9 proved indispensable for the maintenance of structural integrity of adult wings in aged flies. In toto, our findings clearly demonstrate the gene-specific fundamental contribution of proteasome, but not autophagy, in invertebrate eye and wing organ development. PMID:24282550

Proteasome, but not autophagy, disruption results in severe eye and wing dysmorphia: a subunit- and regulator-dependent process in Drosophila.

PubMed

Velentzas, Panagiotis D; Velentzas, Athanassios D; Pantazi, Asimina D; Mpakou, Vassiliki E; Zervas, Christos G; Papassideri, Issidora S; Stravopodis, Dimitrios J

2013-01-01

Proteasome-dependent and autophagy-mediated degradation of eukaryotic cellular proteins represent the two major proteostatic mechanisms that are critically implicated in a number of signaling pathways and cellular processes. Deregulation of functions engaged in protein elimination frequently leads to development of morbid states and diseases. In this context, and through the utilization of GAL4/UAS genetic tool, we herein examined the in vivo contribution of proteasome and autophagy systems in Drosophila eye and wing morphogenesis. By exploiting the ability of GAL4-ninaE. GMR and P{GawB}Bx(MS1096) genetic drivers to be strongly and preferentially expressed in the eye and wing discs, respectively, we proved that proteasomal integrity and ubiquitination proficiency essentially control fly's eye and wing development. Indeed, subunit- and regulator-specific patterns of severe organ dysmorphia were obtained after the RNAi-induced downregulation of critical proteasome components (Rpn1, Rpn2, α5, β5 and β6) or distinct protein-ubiquitin conjugators (UbcD6, but not UbcD1 and UbcD4). Proteasome deficient eyes presented with either rough phenotypes or strongly dysmorphic shapes, while transgenic mutant wings were severely folded and carried blistered structures together with loss of vein differentiation. Moreover, transgenic fly eyes overexpressing the UBP2-yeast deubiquitinase enzyme were characterized by an eyeless-like phenotype. Therefore, the proteasome/ubiquitin proteolytic activities are undoubtedly required for the normal course of eye and wing development. In contrast, the RNAi-mediated downregulation of critical Atg (1, 4, 7, 9 and 18) autophagic proteins revealed their non-essential, or redundant, functional roles in Drosophila eye and wing formation under physiological growth conditions, since their reduced expression levels could only marginally disturb wing's, but not eye's, morphogenetic organization and architecture. However, Atg9 proved indispensable for the maintenance of structural integrity of adult wings in aged flies. In toto, our findings clearly demonstrate the gene-specific fundamental contribution of proteasome, but not autophagy, in invertebrate eye and wing organ development.

Proteasome inhibition induces DNA damage and reorganizes nuclear architecture and protein synthesis machinery in sensory ganglion neurons.

PubMed

Palanca, Ana; Casafont, Iñigo; Berciano, María T; Lafarga, Miguel

2014-05-01

Bortezomib is a reversible proteasome inhibitor used as an anticancer drug. However, its clinical use is limited since it causes peripheral neurotoxicity. We have used Sprague-Dawley rats as an animal model to investigate the cellular mechanisms affected by both short-term and chronic bortezomib treatments in sensory ganglia neurons. Proteasome inhibition induces dose-dependent alterations in the architecture, positioning, shape and polarity of the neuronal nucleus. It also produces DNA damage without affecting neuronal survival, and severe disruption of the protein synthesis machinery at the central cytoplasm accompanied by decreased expression of the brain-derived neurotrophic factor. As a compensatory or adaptive survival response against proteotoxic stress caused by bortezomib treatment, sensory neurons preserve basal levels of transcriptional activity, up-regulate the expression of proteasome subunit genes, and generate a new cytoplasmic perinuclear domain for protein synthesis. We propose that proteasome activity is crucial for controlling nuclear architecture, DNA repair and the organization of the protein synthesis machinery in sensory neurons. These neurons are primary targets of bortezomib neurotoxicity, for which reason their dysfunction may contribute to the pathogenesis of the bortezomib-induced peripheral neuropathy in treated patients.

Molecular modeling on porphyrin derivatives as β5 subunit inhibitor of 20S proteasome.

PubMed

Arba, Muhammad; Nur-Hidayat, Andry; Ruslin; Yusuf, Muhammad; Sumarlin; Hertadi, Rukman; Wahyudi, Setyanto Tri; Surantaadmaja, Slamet Ibrahim; Tjahjono, Daryono H

2018-06-01

The ubiquitin-proteasome system plays an important role in protein quality control. Currently, inhibition of the proteasome has been validated as a promising approach in anticancer therapy. The 20S core particle of the proteasome harbors β5 subunit which is a crucial active site in proteolysis. Targeting proteasome β5 subunit which is responsible for the chymotrypsin-like activity of small molecules has been regarded as an important way for achieving therapeutics target. In the present study, a series of porphyrin derivatives bearing either pyridine or pyrazole rings as meso-substituents were designed and evaluated as an inhibitor for the β5 subunit of the proteasome by employing molecular docking and dynamics simulations. The molecular docking was performed with the help of AutoDock 4.2, while molecular dynamics simulation was done using AMBER 14. All compounds bound to the proteasome with similar binding modes, and each porphyrin-proteasome complex was stable during 30 ns MD simulation as indicated by root-mean-square-deviation (RMSD) value. An analysis on protein residue fluctuation of porphyrin binding demonstrates that in all complexes, porphyrin binding produces minor fluctuation on amino acid residues. The molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) free energy calculation shows that the binding affinities of mono-H 2 PyP, bis-H 2 PzP, and tetra-H 2 PyP were comparable with that of the potential inhibitor, HU10. It is noted that the electrostatic interaction increases with the number of meso-substituents, which was favourable for porphyrin binding. The present study shows that both electrostatic and van der Waals interaction are the main force which controls the interaction of porphyrin compounds with the proteasome. Copyright © 2018 Elsevier Ltd. All rights reserved.

Myostatin Activates the Ubiquitin-Proteasome and Autophagy-Lysosome Systems Contributing to Muscle Wasting in Chronic Kidney Disease

PubMed Central

Wang, Dong-Tao; Yang, Ya-Jun; Huang, Ren-Hua; Zhang, Zhi-Hua; Lin, Xin

2015-01-01

Our evidence demonstrated that CKD upregulated the expression of myostatin, TNF-α, and p-IkBa and downregulated the phosphorylation of PI3K, Akt, and FoxO3a, which were also associated with protein degradation and muscle atrophy. The autophagosome formation and protein expression of autophagy-related genes were increased in muscle of CKD rats. The mRNA level and protein expression of MAFbx and MuRF-1 were also upregulated in CKD rats, as well as proteasome activity of 26S. Moreover, activation of myostatin elicited by TNF-α induces C2C12 myotube atrophy via upregulating the expression of autophagy-related genes, including MAFbx and MuRF1 and proteasome subunits. Inactivation of FoxO3a triggered by PI3K inhibitor LY294002 prevented the myostatin-induced increase of expression of MuRF1, MAFbx, and LC3-II protein in C2C12 myotubes. The findings were further consolidated by using siRNA interference and overexpression of myostatin. Additionally, expression of myostatin was activated by TNF-α via a NF-κB dependent pathway in C2C12 myotubes, while inhibition of NF-κB activity suppressed myostatin and improved myotube atrophy. Collectively, myostatin mediated CKD-induced muscle catabolism via coordinate activation of the autophagy and the ubiquitin-proteasome systems. PMID:26448817

Myostatin Activates the Ubiquitin-Proteasome and Autophagy-Lysosome Systems Contributing to Muscle Wasting in Chronic Kidney Disease.

PubMed

Wang, Dong-Tao; Yang, Ya-Jun; Huang, Ren-Hua; Zhang, Zhi-Hua; Lin, Xin

2015-01-01

Our evidence demonstrated that CKD upregulated the expression of myostatin, TNF-α, and p-IkBa and downregulated the phosphorylation of PI3K, Akt, and FoxO3a, which were also associated with protein degradation and muscle atrophy. The autophagosome formation and protein expression of autophagy-related genes were increased in muscle of CKD rats. The mRNA level and protein expression of MAFbx and MuRF-1 were also upregulated in CKD rats, as well as proteasome activity of 26S. Moreover, activation of myostatin elicited by TNF-α induces C2C12 myotube atrophy via upregulating the expression of autophagy-related genes, including MAFbx and MuRF1 and proteasome subunits. Inactivation of FoxO3a triggered by PI3K inhibitor LY294002 prevented the myostatin-induced increase of expression of MuRF1, MAFbx, and LC3-II protein in C2C12 myotubes. The findings were further consolidated by using siRNA interference and overexpression of myostatin. Additionally, expression of myostatin was activated by TNF-α via a NF-κB dependent pathway in C2C12 myotubes, while inhibition of NF-κB activity suppressed myostatin and improved myotube atrophy. Collectively, myostatin mediated CKD-induced muscle catabolism via coordinate activation of the autophagy and the ubiquitin-proteasome systems.

Chronic aspirin via dose-dependent and selective inhibition of cardiac proteasome possibly contributed a potential risk to the ischemic heart.

PubMed

Tan, Chunjiang; Chen, Wenlie; Wu, Yanbin; Lin, Jiumao; Lin, Ruhui; Tan, Xuerui; Chen, Songming

2013-08-01

Impaired cardiac proteasome has been reported in ischemic heart and heart failure. Recent data highlighted aspirin as an inhibitor of the ubiquitin-proteasome system, however, it's unclear whether it affects cardiac proteasome functions. Myocardial infarction (MI), sham or normal male SD rats were injected intraperitoneally with high (300 mg/kg), low (5 mg/kg) aspirin or saline (control) once a day for seven weeks. Parallel experiments were performed in the hypoxia/reoxygenated human ventricular myocytes. Dose-related increases in heart and ventricular weight, and impaired cardiac functions, were found more exacerbated in the aspirin-treated MI rat hearts than the saline-treated MI counterparts. The activity of 26S, 20S and 19S declined by about 30%, or the 20S proteasome subunits β5, β2 and β1 decreased by 40%, 20% and 30%, respectively, in the MI rats compared with the non-MI rats (P<0.05). Compared with the saline-treated MI rats, 26S and 20S in high or low dose aspirin-treated MI rats further decreased by 30% and 20%, β5 by 30% and 12%, and β1 by 40% and 30%, respectively, and the lost activity was correlated with the compromised cardiac functions or the decreased cell viability. The dose-related and selective inhibition of 26S and 20S proteasome, or the 20S proteasome subunits β5 and β1 by aspirin was comparable to their protein expressions in the MI rats and in the cultured cells. The impaired cardiac proteasome, enhanced by chronic aspirin treatment, attenuated the removal of oxidative and ubiquitinated proteins, and chronic aspirin treatment via selective and dose-dependent inhibition of cardiac proteasome possibly constituted a potential risk to ischemic heart. Copyright © 2013 Elsevier Inc. All rights reserved.

A novel crosstalk between two major protein degradation systems: regulation of proteasomal activity by autophagy.

PubMed

Wang, Xiao J; Yu, Jun; Wong, Sunny H; Cheng, Alfred S L; Chan, Francis K L; Ng, Simon S M; Cho, Chi H; Sung, Joseph J Y; Wu, William K K

2013-10-01

Eukaryotes have two major intracellular protein degradation pathways, namely the ubiquitin-proteasome system (UPS) and autophagy. Inhibition of proteasomal activities has been previously shown to induce autophagy, indicating a coordinated and complementary relationship between these two systems. However, little is known about the regulation of the UPS by autophagy. In this study, we showed for the first time that proteasomes were activated in response to pharmacological inhibition of autophagy as well as disruption of autophagy-related genes by RNA interference under nutrient-deficient conditions in cultured human colon cancer cells. The induction was evidenced by the increased proteasomal activities and the upregulation of proteasomal subunits, including the proteasome β5 subunit, PSMB5. Co-inhibition of the proteasome and autophagy also synergistically increased the accumulation of polyubiquitinated proteins. Collectively, our findings suggest that proteasomes are activated in a compensatory manner for protein degradation upon autophagy inhibition. Our studies unveiled a novel regulatory mechanism between the two protein degradation pathways.

Autophagic degradation of the 26S proteasome is mediated by the dual ATG8/ubiquitin receptor RPN10 in Arabidopsis

DOE PAGES

Marshall, Richard S.; Li, Faqiang; Gemperline, David C.; ...

2015-05-21

Autophagic turnover of intracellular constituents is critical for cellular housekeeping, nutrient recycling, and various aspects of growth and development in eukaryotes. In this paper, we show that autophagy impacts the other major degradative route involving the ubiquitin-proteasome system by eliminating 26S proteasomes, a process we termed proteaphagy. Using Arabidopsis proteasomes tagged with GFP, we observed their deposition into vacuoles via a route requiring components of the autophagy machinery. This transport can be initiated separately by nitrogen starvation and chemical or genetic inhibition of the proteasome, implying distinct induction mechanisms. Proteasome inhibition stimulates comprehensive ubiquitylation of the complex, with the ensuingmore » proteaphagy requiring the proteasome subunit RPN10, which can simultaneously bind both ATG8 and ubiquitin. Finally and collectively, we propose that Arabidopsis RPN10 acts as a selective autophagy receptor that targets inactive 26S proteasomes by concurrent interactions with ubiquitylated proteasome subunits/targets and lipidated ATG8 lining the enveloping autophagic membranes.« less

Developmentally regulated, alternative splicing of the Rpn10 gene generates multiple forms of 26S proteasomes

PubMed Central

Kawahara, Hiroyuki; Kasahara, Masanori; Nishiyama, Atsuya; Ohsumi, Keita; Goto, Tetsuya; Kishimoto, Takeo; Saeki, Yasushi; Yokosawa, Hideyoshi; Shimbara, Naoki; Murata, Shigeo; Chiba, Tomoki; Suzuki, Koichi; Tanaka, Keiji

2000-01-01

The 26S proteasome is a multisubunit protein- destroying machinery that degrades ubiquitin-tagged proteins. To date only a single species of Rpn10, which possibly functions as a multiubiquitin chain-binding subunit, has been identified in various organisms. Here we report that mouse Rpn10 mRNAs occur in at least five distinct forms, named Rpn10a to Rpn10e, and that they are generated from a single gene by developmentally regulated, alternative splicing. Rpn10a is ubiquitously expressed, whereas Rpn10e is expressed only in embryos, with the highest levels of expression in the brain. Both forms of Rpn10 are components of the 26S proteasome, with an apparently similar affinity for multiubiquitylated [125I]lysozyme in vitro. However, they exert markedly divergent effects on the destruction of B-type cyclin in Xenopus egg extracts. Thus, the 26S proteasome occurs in at least two functionally distinct forms: one containing a ubiquitously expressed Rpn10a and the other a newly identified, embryo-specific Rpn10e. While the former is thought to perform proteolysis constitutively in a wide variety of cells, the latter may play a specialized role in early embryonic development. PMID:10921894

Proteasomes remain intact, but show early focal alteration in their composition in a mouse model of amyotrophic lateral sclerosis.

PubMed

Kabashi, Edor; Agar, Jeffrey N; Hong, Yu; Taylor, David M; Minotti, Sandra; Figlewicz, Denise A; Durham, Heather D

2008-06-01

In amyotrophic lateral sclerosis caused by mutations in Cu/Zn-superoxide dismutase (SOD1), altered solubility and aggregation of the mutant protein implicates failure of pathways for detecting and catabolizing misfolded proteins. Our previous studies demonstrated early reduction of proteasome-mediated proteolytic activity in lumbar spinal cord of SOD1(G93A) transgenic mice, tissue particularly vulnerable to disease. The purpose of this study was to identify any underlying abnormalities in proteasomal structure. In lumbar spinal cord of pre-symptomatic mice [postnatal day 45 (P45) and P75], normal levels of structural 20S alpha subunits were incorporated into 20S/26S proteasomes; however, proteasomal complexes separated by native gel electrophoresis showed decreased immunoreactivity with antibodies to beta3, a structural subunit of the 20S proteasome core, and beta5, the subunit with chymotrypsin-like activity. This occurred prior to increase in beta5i immunoproteasomal subunit. mRNA levels were maintained and no association of mutant SOD1 with proteasomes was identified, implicating post-transcriptional mechanisms. mRNAs also were maintained in laser captured motor neurons at a later stage of disease (P100) in which multiple 20S proteins are reduced relative to the surrounding neuropil. Increase in detergent-insoluble, ubiquitinated proteins at P75 provided further evidence of stress on mechanisms of protein quality control in multiple cell types prior to significant motor neuron death.

The Role of Epibionts of Bacteria of the Genus Pseudoalteromonas and Cellular Proteasomes in the Adaptive Plasticity of Marine Cold-Water Sponges.

PubMed

Kravchuk, O I; Lavrov, A I; Finoshin, A D; Gornostaev, N G; Georgiev, A A; Abaturova, S B; Mikhailov, V S; Lyupina, Yu V

2018-03-01

It was found that cells of different color morphs of the cold-water marine sponges Halichondria panicea (Pallas, 1766) of the class Demospongiae differ in the content of epibionts of bacteria of the genus Pseudoalteromonas. The sponge cells with elevated levels of epibionts of bacteria of the genus Pseudoalteromonas showed an increased expression of Hsp70 proteins but had a reduced level of the proteasomal catalytic beta 5 subunit, which was accompanied by a change in their activity. Probably, epibionts of bacteria of the genus Pseudoalteromonas may affect the ubiquitin-proteasome system in the cells of cold-water marine sponges and, thereby, ensure their adaptive plasticity.

Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bai, Lin; Hu, Kuan; Wang, Tong

Here, the human pathogen Mycobacterium tuberculosis ( Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb. Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly,more » the truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria.« less

Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis

DOE PAGES

Bai, Lin; Hu, Kuan; Wang, Tong; ...

2016-03-21

Here, the human pathogen Mycobacterium tuberculosis ( Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb. Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly,more » the truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria.« less

UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis.

PubMed

Xie, Youming; Varshavsky, Alexander

2002-12-01

The ubiquitin system recognizes degradation signals of protein substrates through E3-E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4(DeltaN), which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.

Affinity purification of the Arabidopsis 26 S proteasome reveals a diverse array of plant proteolytic complexes.

PubMed

Book, Adam J; Gladman, Nicholas P; Lee, Sang-Sook; Scalf, Mark; Smith, Lloyd M; Vierstra, Richard D

2010-08-13

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis.

Affinity Purification of the Arabidopsis 26 S Proteasome Reveals a Diverse Array of Plant Proteolytic Complexes*

PubMed Central

Book, Adam J.; Gladman, Nicholas P.; Lee, Sang-Sook; Scalf, Mark; Smith, Lloyd M.; Vierstra, Richard D.

2010-01-01

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis. PMID:20516081

Derlin-1 promotes ubiquitylation and degradation of the epithelial Na+ channel, ENaC.

PubMed

You, Hui; Ge, Yamei; Zhang, Jian; Cao, Yizhi; Xing, Jing; Su, Dongming; Huang, Yujie; Li, Min; Qu, Shen; Sun, Fei; Liang, Xiubin

2017-03-15

Ubiquitylation of the epithelial Na + channel (ENaC) plays a critical role in cellular functions, including transmembrane transport of Na + , Na + and water balance, and blood pressure stabilization. Published studies have suggested that ENaC subunits are targets of ER-related degradation (ERAD) in yeast systems. However, the molecular mechanism underlying proteasome-mediated degradation of ENaC subunits remains to be established. Derlin-1, an E3 ligase mediator, links recognized target proteins to ubiquitin-mediated proteasomal degradation in the cytosol. In the present study, we found that derlin-1 suppressed the expression of ENaC at the protein level and that the subunit α-ENaC (also known as SCNN1A) physically interacted with derlin-1 at the membrane-anchored domains or the loop regions, and that derlin-1 initiated α-ENaC retrotranslocation. In addition, HUWE1, an endoplasmic reticulum (ER)-resident E3 ubiquitin ligase, was recruited and promoted K11-linked polyubiquitylation of α-ENaC and, hence, formation of an α-ENaC ubiquitin-mediated degradation complex. These findings suggest that derlin-1 promotes ENaC ubiquitylation and enhances ENaC ubiquitin- mediated proteasome degradation. The derlin-1 pathway therefore may represent a significant early checkpoint in the recognition and degradation of ENaC in mammalian cells. © 2017. Published by The Company of Biologists Ltd.

Hepatic expression of proteasome subunit alpha type-6 is upregulated during viral hepatitis and putatively regulates the expression of ISG15 ubiquitin-like modifier, a proviral host gene in hepatitis C virus infection.

PubMed

Broering, R; Trippler, M; Werner, M; Real, C I; Megger, D A; Bracht, T; Schweinsberg, V; Sitek, B; Eisenacher, M; Meyer, H E; Baba, H A; Weber, F; Hoffmann, A-C; Gerken, G; Schlaak, J F

2016-05-01

The interferon-stimulated gene 15 (ISG15) plays an important role in the pathogenesis of hepatitis C virus (HCV) infection. ISG15-regulated proteins have previously been identified that putatively affect this proviral interaction. The present observational study aimed to elucidate the relation between ISG15 and these host factors during HCV infection. Transcriptomic and proteomic analyses were performed using liver samples of HCV-infected (n = 54) and uninfected (n = 10) or HBV-infected controls (n = 23). Primary human hepatocytes (PHH) were treated with Toll-like receptor ligands, interferons and kinase inhibitors. Expression of ISG15 and proteasome subunit alpha type-6 (PSMA6) was suppressed in subgenomic HCV replicon cell lines using specific siRNAs. Comparison of hepatic expression patterns revealed significantly increased signals for ISG15, IFIT1, HNRNPK and PSMA6 on the protein level as well as ISG15, IFIT1 and PSMA6 on the mRNA level in HCV-infected patients. In contrast to interferon-stimulated genes, PSMA6 expression occurred independent of HCV load and genotype. In PHH, the expression of ISG15 and PSMA6 was distinctly induced by poly(I:C), depending on IRF3 activation or PI3K/AKT signalling, respectively. Suppression of PSMA6 in HCV replicon cells led to significant induction of ISG15 expression, thus combined knock-down of both genes abrogated the antiviral effect induced by the separate suppression of ISG15. These data indicate that hepatic expression of PSMA6, which is upregulated during viral hepatitis, likely depends on TLR3 activation. PSMA6 affects the expression of immunoregulatory ISG15, a proviral factor in the pathogenesis of HCV infection. Therefore, the proteasome might be involved in the enigmatic interaction between ISG15 and HCV. © 2016 John Wiley & Sons Ltd.

Comparative Proteomic Analysis of Methanothermobacter themautotrophicus ΔH in Pure Culture and in Co-Culture with a Butyrate-Oxidizing Bacterium

PubMed Central

Enoki, Miho; Shinzato, Naoya; Sato, Hiroaki; Nakamura, Kohei; Kamagata, Yoichi

2011-01-01

To understand the physiological basis of methanogenic archaea living on interspecies H2 transfer, the protein expression of a hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain ΔH, was investigated in both pure culture and syntrophic coculture with an anaerobic butyrate oxidizer Syntrophothermus lipocalidus strain TGB-C1 as an H2 supplier. Comparative proteomic analysis showed that global protein expression of methanogen cells in the model coculture was substantially different from that of pure cultured cells. In brief, in syntrophic coculture, although methanogenesis-driven energy generation appeared to be maintained by shifting the pathway to the alternative methyl coenzyme M reductase isozyme I and cofactor F420-dependent process, the machinery proteins involved in carbon fixation, amino acid synthesis, and RNA/DNA metabolisms tended to be down-regulated, indicating restrained cell growth rather than vigorous proliferation. In addition, our proteome analysis revealed that α subunits of proteasome were differentially acetylated between the two culture conditions. Since the relevant modification has been suspected to regulate proteolytic activity of the proteasome, the global protein turnover rate could be controlled under syntrophic growth conditions. To our knowledge, the present study is the first report on N-acetylation of proteasome subunits in methanogenic archaea. These results clearly indicated that physiological adaptation of hydrogenotrophic methanogens to syntrophic growth is more complicated than that of hitherto proposed. PMID:21904627

Proteasome expression and activity in cancer and cancer stem cells.

PubMed

Voutsadakis, Ioannis A

2017-03-01

Proteasome is a multi-protein organelle that participates in cellular proteostasis by destroying damaged or short-lived proteins in an organized manner guided by the ubiquitination signal. By being in a central place in the cellular protein complement homeostasis, proteasome is involved in virtually all cell processes including decisions on cell survival or death, cell cycle, and differentiation. These processes are important also in cancer, and thus, the proteasome is an important regulator of carcinogenesis. Cancers include a variety of cells which, according to the cancer stem cell theory, descend from a small percentage of cancer stem cells, alternatively termed tumor-initiating cells. These cells constitute the subsets that have the ability to propagate the whole variety of cancer and repopulate tumors after cytostatic therapies. Proteasome plays a role in cellular processes in cancer stem cells, but it has been found to have a decreased function in them compared to the rest of cancer cells. This article will discuss the transcriptional regulation of proteasome sub-unit proteins in cancer and in particular cancer stem cells and the relationship of the proteasome with the pluripotency that is the defining characteristic of stem cells. Therapeutic opportunities that present from the understanding of the proteasome role will also be discussed.

Data for a proteomic analysis of p53-independent induction of apoptosis by bortezomib

PubMed Central

Yerlikaya, Azmi; Okur, Emrah; Tarık Baykal, Ahmet; Acılan, Ceyda; Boyacı, İhsan; Ulukaya, Engin

2014-01-01

This data article contains data related to the research article entitled, “A proteomic analysis of p53-independent induction of apoptosis by bortezomib in 4T1 breast cancer cell line” by Yerlikaya et al. [1]. The research article presented 2-DE and nLC-MS/MS based proteomic analysis of proteasome inhibitor bortezomib-induced changes in the expression of cellular proteins. The report showed that GRP78 and TCEB2 were over-expressed in response to treatment with bortezomib for 24 h. In addition, the report demonstrated that Hsp70, the 26S proteasome non-ATPase regulatory subunit 14 and sequestosome 1 were increased at least 2 fold in p53-deficient 4T1 cells. The data here show for the first time the increased expressions of Card10, Dffb, Traf3 and Trp53bp2 in response to inhibition of the 26S proteasome. The information presented here also shows that both Traf1 and Xiap (a member of IAPs) are also downregulated simultaneously upon proteasomal inhibition. The increases in the level of Card10 and Trp53bp2 proteins were verified by Western blot analysis in response to varying concentrations of bortezomib for 24 h. PMID:26217687

Structural Defects in the Regulatory Particle-Core Particle Interface of the Proteasome Induce a Novel Proteasome Stress Response*

PubMed Central

Park, Soyeon; Kim, Woong; Tian, Geng; Gygi, Steven P.; Finley, Daniel

2011-01-01

Proteasomes consist of a 19-subunit regulatory particle (RP) and 28-subunit core particle (CP), an α7β7β7α7 structure. The RP recognizes substrates and translocates them into the CP for degradation. At the RP-CP interface, a heterohexameric Rpt ring joins to a heteroheptameric CP α ring. Rpt C termini insert individually into the α ring pockets to form a salt bridge with a pocket lysine residue. We report that substitutions of α pocket lysine residues produce an unexpected block to CP assembly, arising from a late stage defect in β ring assembly. Substitutions α5K66A and α6K62A resulted in abundant incorporation of immature CP β subunits, associated with a complete β ring, into proteasome holoenzymes. Incorporation of immature CP into the proteasome depended on a proteasome-associated protein, Ecm29. Using ump1 mutants, we identified Ecm29 as a potent negative regulator of RP assembly and confirmed our previous findings that proper RP assembly requires the CP. Ecm29 was enriched on proteasomes of pocket lysine mutants, as well as those of rpt4-Δ1 and rpt6-Δ1 mutants, in which the C-terminal residue, thought to contact the pocket lysine, is deleted. In both rpt6-Δ1 and α6K62A proteasomes, Ecm29 suppressed opening of the CP substrate translocation channel, which is gated through interactions between Rpt C termini and the α pockets. The ubiquitin ligase Hul5 was recruited to these proteasomes together with Ecm29. Proteasome remodeling through the addition of Ecm29 and Hul5 suggests a new layer of the proteasome stress response and may be a common response to structurally aberrant proteasomes or deficient proteasome function. PMID:21878652

von Hippel–Lindau binding protein 1-mediated degradation of integrase affects HIV-1 gene expression at a postintegration step

PubMed Central

Mousnier, Aurélie; Kubat, Nicole; Massias-Simon, Aurélie; Ségéral, Emmanuel; Rain, Jean-Christophe; Benarous, Richard; Emiliani, Stéphane; Dargemont, Catherine

2007-01-01

HIV-1 integrase, the viral enzyme responsible for provirus integration into the host genome, can be actively degraded by the ubiquitin–proteasome pathway. Here, we identify von Hippel–Lindau binding protein 1(VBP1), a subunit of the prefoldin chaperone, as an integrase cellular binding protein that bridges interaction between integrase and the cullin2 (Cul2)-based von Hippel–Lindau (VHL) ubiquitin ligase. We demonstrate that VBP1 and Cul2/VHL are required for proper HIV-1 expression at a step between integrase-dependent proviral integration into the host genome and transcription of viral genes. Using both an siRNA approach and Cul2/VHL mutant cells, we show that VBP1 and the Cul2/VHL ligase cooperate in the efficient polyubiquitylation of integrase and its subsequent proteasome-mediated degradation. Results presented here support a role for integrase degradation by the prefoldin–VHL–proteasome pathway in the integration–transcription transition of the viral replication cycle. PMID:17698809

Aberrant Splicing Promotes Proteasomal Degradation of L-type CaV1.2 Calcium Channels by Competitive Binding for CaVβ Subunits in Cardiac Hypertrophy.

PubMed

Hu, Zhenyu; Wang, Jiong-Wei; Yu, Dejie; Soon, Jia Lin; de Kleijn, Dominique P V; Foo, Roger; Liao, Ping; Colecraft, Henry M; Soong, Tuck Wah

2016-10-12

Decreased expression and activity of Ca V 1.2 calcium channels has been reported in pressure overload-induced cardiac hypertrophy and heart failure. However, the underlying mechanisms remain unknown. Here we identified in rodents a splice variant of Ca V 1.2 channel, named Ca V 1.2 e21+22 , that contained the pair of mutually exclusive exons 21 and 22. This variant was highly expressed in neonatal hearts. The abundance of this variant was gradually increased by 12.5-folds within 14 days of transverse aortic banding that induced cardiac hypertrophy in adult mouse hearts and was also elevated in left ventricles from patients with dilated cardiomyopathy. Although this variant did not conduct Ca 2+ ions, it reduced the cell-surface expression of wild-type Ca V 1.2 channels and consequently decreased the whole-cell Ca 2+ influx via the Ca V 1.2 channels. In addition, the Ca V 1.2 e21+22 variant interacted with Ca V β subunits significantly more than wild-type Ca V 1.2 channels, and competition of Ca V β subunits by Ca V 1.2 e21+22 consequently enhanced ubiquitination and subsequent proteasomal degradation of the wild-type Ca V 1.2 channels. Our findings show that the resurgence of a specific neonatal splice variant of Ca V 1.2 channels in adult heart under stress may contribute to heart failure.

Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits*

PubMed Central

Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

2011-01-01

The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation. PMID:21478150

Rice Stripe Tenuivirus Nonstructural Protein 3 Hijacks the 26S Proteasome of the Small Brown Planthopper via Direct Interaction with Regulatory Particle Non-ATPase Subunit 3

PubMed Central

Xu, Yi; Wu, Jianxiang; Fu, Shuai; Li, Chenyang; Zhu, Zeng-Rong

2015-01-01

ABSTRACT The ubiquitin/26S proteasome system plays a vital role in regulating host defenses against pathogens. Previous studies have highlighted different roles for the ubiquitin/26S proteasome in defense during virus infection in both mammals and plants, but their role in the vectors that transmit those viruses is still unclear. In this study, we determined that the 26S proteasome is present in the small brown planthopper (SBPH) (Laodelphgax striatellus) and has components similar to those in plants and mammals. There was an increase in the accumulation of Rice stripe virus (RSV) in the transmitting vector SBPH after disrupting the 26S proteasome, indicating that the SBPH 26S proteasome plays a role in defense against RSV infection by regulating RSV accumulation. Yeast two-hybrid analysis determined that a subunit of the 26S proteasome, named RPN3, could interact with RSV NS3. Transient overexpression of RPN3 had no effect on the RNA silencing suppressor activity of RSV NS3. However, NS3 could inhibit the ability of SBPH rpn3 to complement an rpn3 mutation in yeast. Our findings also indicate that the direct interaction between RPN3 and NS3 was responsible for inhibiting the complementation ability of RPN3. In vivo, we found an accumulation of ubiquitinated protein in SBPH tissues where the RSV titer was high, and silencing of rpn3 resulted in malfunction of the SBPH proteasome-mediated proteolysis. Consequently, viruliferous SBPH in which RPN3 was repressed transmitted the virus more effectively as a result of higher accumulation of RSV. Our results suggest that the RSV NS3 protein is able to hijack the 26S proteasome in SBPH via a direct interaction with the RPN3 subunit to attenuate the host defense response. IMPORTANCE We show, for the first time, that the 26S proteasome components are present in the small brown planthopper and play a role in defense against its vectored plant virus (RSV). In turn, RSV encodes a protein that subverts the SBPH 26S proteasome via direct interaction with the 26S proteasome subunit RPN3. Our results imply that the molecular arms race observed in plant hosts can be extended to the insect vector that transmits those viruses. PMID:25653432

The Nrf1 CNC-bZIP protein is regulated by the proteasome and activated by hypoxia.

PubMed

Chepelev, Nikolai L; Bennitz, Joshua D; Huang, Ting; McBride, Skye; Willmore, William G

2011-01-01

Nrf1 (nuclear factor-erythroid 2 p45 subunit-related factor 1) is a transcription factor mediating cellular responses to xenobiotic and pro-oxidant stress. Nrf1 regulates the transcription of many stress-related genes through the electrophile response elements (EpREs) located in their promoter regions. Despite its potential importance in human health, the mechanisms controlling Nrf1 have not been addressed fully. We found that proteasomal inhibitors MG-132 and clasto-lactacystin-β-lactone stabilized the protein expression of full-length Nrf1 in both COS7 and WFF2002 cells. Concomitantly, proteasomal inhibition decreased the expression of a smaller, N-terminal Nrf1 fragment, with an approximate molecular weight of 23 kDa. The EpRE-luciferase reporter assays revealed that proteasomal inhibition markedly inhibited the Nrf1 transactivational activity. These results support earlier hypotheses that the 26 S proteasome processes Nrf1 into its active form by removing its inhibitory N-terminal domain anchoring Nrf1 to the endoplasmic reticulum. Immunoprecipitation demonstrated that Nrf1 is ubiquitinated and that proteasomal inhibition increased the degree of Nrf1 ubiquitination. Furthermore, Nrf1 protein had a half-life of approximately 5 hours in COS7 cells. In contrast, hypoxia (1% O(2)) significantly increased the luciferase reporter activity of exogenous Nrf1 protein, while decreasing the protein expression of p65, a shorter form of Nrf1, known to act as a repressor of EpRE-controlled gene expression. Finally, the protein phosphatase inhibitor okadaic acid activated Nrf1 reporter activity, while the latter was repressed by the PKC inhibitor staurosporine. Collectively, our data suggests that Nrf1 is controlled by several post-translational mechanisms, including ubiquitination, proteolytic processing and proteasomal-mediated degradation as well as by its phosphorylation status. © 2011 Chepelev et al.

Discovery of a potent and highly specific β2 proteasome inhibitor from a library of copper complexes.

PubMed

Zhou, Tongliang; Cai, Yuanbo; Liang, Lei; Yang, Lingfei; Xu, Fengrong; Niu, Yan; Wang, Chao; Zhang, Jun-Long; Xu, Ping

2016-12-01

We reported the synthesis, characterization and biological activity of several copper(II) Schiff base complexes, which exhibit high proteasome inhibitory activities with particular selectivity of β 2 subunit. Structure-activity relationships information obtained from complex Na 2 [Cu(a4s1)] demonstrated that distinct bonding modes in β 2 and β 5 subunits determines its selectivity and potent inhibition for β 2 subunit. Copyright © 2016 Elsevier Ltd. All rights reserved.

Selective Proteasomal Degradation of the B′β Subunit of Protein Phosphatase 2A by the E3 Ubiquitin Ligase Adaptor Kelch-like 15*

PubMed Central

Oberg, Elizabeth A.; Nifoussi, Shanna K.; Gingras, Anne-Claude; Strack, Stefan

2012-01-01

Protein phosphatase 2A (PP2A), a ubiquitous and pleiotropic regulator of intracellular signaling, is composed of a core dimer (AC) bound to a variable (B) regulatory subunit. PP2A is an enzyme family of dozens of heterotrimers with different subcellular locations and cellular substrates dictated by the B subunit. B′β is a brain-specific PP2A regulatory subunit that mediates dephosphorylation of Ca2+/calmodulin-dependent protein kinase II and tyrosine hydroxylase. Unbiased proteomic screens for B′β interactors identified Cullin3 (Cul3), a scaffolding component of E3 ubiquitin ligase complexes, and the previously uncharacterized Kelch-like 15 (KLHL15). KLHL15 is one of ∼40 Kelch-like proteins, many of which have been identified as adaptors for the recruitment of substrates to Cul3-based E3 ubiquitin ligases. Here, we report that KLHL15-Cul3 specifically targets B′β to promote turnover of the PP2A subunit by ubiquitylation and proteasomal degradation. Comparison of KLHL15 and B′β tissue expression profiles suggests that the E3 ligase adaptor contributes to selective expression of the PP2A/B′β holoenzyme in the brain. We mapped KLHL15 residues critical for homodimerization as well as interaction with Cul3 and B′β. Explaining PP2A subunit selectivity, the divergent N terminus of B′β was found necessary and sufficient for KLHL15-mediated degradation, with Tyr-52 having an obligatory role. Although KLHL15 can interact with the PP2A/B′β heterotrimer, it only degrades B′β, thus promoting exchange with other regulatory subunits. E3 ligase adaptor-mediated control of PP2A holoenzyme composition thereby adds another layer of regulation to cellular dephosphorylation events. PMID:23135275

Changes in the Expression and the Enzymic Properties of the 20S Proteasome in Sugar-Starved Maize Roots. Evidence for an in Vivo Oxidation of the Proteasome1

PubMed Central

Basset , Gilles; Raymond, Philippe; Malek, Lada; Brouquisse, Renaud

2002-01-01

The 20S proteasome (multicatalytic proteinase) was purified from maize (Zea mays L. cv DEA 1992) roots through a five-step procedure. After biochemical characterization, it was shown to be similar to most eukaryotic proteasomes. We investigated the involvement of the 20S proteasome in the response to carbon starvation in excised maize root tips. Using polyclonal antibodies, we showed that the amount of proteasome increased in 24-h-carbon-starved root tips compared with freshly excised tips, whereas the mRNA levels of α3 and β6 subunits of 20S proteasome decreased. Moreover, in carbon-starved tissues, chymotrypsin-like and caseinolytic activities of the 20S proteasome were found to increase, whereas trypsin-like activities decreased. The measurement of specific activities and kinetic parameters of 20S proteasome purified from 24-h-starved root tips suggested that it was subjected to posttranslational modifications. Using dinitrophenylhydrazine, a carbonyl-specific reagent, we observed an increase in carbonyl residues in 20S proteasome purified from starved root tips. This means that 20S proteasome was oxidized during starvation treatment. Moreover, an in vitro mild oxidative treatment of 20S proteasome from non-starved material resulted in the activation of chymotrypsin-like, peptidyl-glutamyl-peptide hydrolase and caseinolytic-specific activities and in the inhibition of trypsin-like specific activities, similar to that observed for proteasome from starved root tips. Our results provide the first evidence, to our knowledge, for an in vivo carbonylation of the 20S proteasome. They suggest that sugar deprivation induces an oxidative stress, and that oxidized 20S proteasome could be associated to the degradation of oxidatively damaged proteins in carbon starvation situations. PMID:11891269

Proteomics of a new esophageal cancer cell line established from Persian patient.

PubMed

Moghanibashi, Mehdi; Jazii, Ferdous Rastgar; Soheili, Zahra-Soheila; Zare, Maryam; Karkhane, Aliasghar; Parivar, Kazem; Mohamadynejad, Parisa

2012-05-25

Although the highest incidence of esophageal squamous cell carcinoma (ESCC) has repeatedly been reported from Persia (Iran), nevertheless the so far proteomic published reports were limited to one study on tissue specimens. Here we report the proteome of a newly established cell line from Persian ESCC patients and compare it with the normal primary cell proteome. Among polypeptides, whose expression was different in cell line sixteen polypeptides were identified by MALDI/TOF/TOF spectrometry. S100-A8 protein, annexin A1, annexin A2, regulatory subunit of calpain, subunit alpha type-3 of proteasome and glutamate dehydrogenase 1 were proteins down-regulated in cell line while peroxiredoxin-5, non-muscle myosin light polypeptide 6, keratin 1, annexin A4, keratin 8, tropomyosin 3, stress-induced-phosphoprotein 1 and albumin were found to be subject of up-regulation in cell line compared to the primary normal cells. The proteomic results were further verified by western blotting and RT-PCR on annexin A1 and keratin 8. In addition, among the aforementioned proteins, glutamate dehydrogenase 1, regulatory subunit of calpain, subunit alpha of type-3 proteasome and annexin A4 are proteins whose deregulation in ESCC is reported for the first time by this study. Copyright © 2012 Elsevier B.V. All rights reserved.

Nucleolar Proteome Analysis and Proteasomal Activity Assays Reveal a Link between Nucleolus and 26S Proteasome in A. thaliana

PubMed Central

Montacié, Charlotte; Durut, Nathalie; Opsomer, Alison; Palm, Denise; Comella, Pascale; Picart, Claire; Carpentier, Marie-Christine; Pontvianne, Frederic; Carapito, Christine; Schleiff, Enrico; Sáez-Vásquez, Julio

2017-01-01

In all eukaryotic cells, the nucleolus is functionally and structurally linked to rRNA synthesis and ribosome biogenesis. This compartment contains as well factors involved in other cellular activities, but the functional interconnection between non-ribosomal activities and the nucleolus (structure and function) still remains an open question. Here, we report a novel mass spectrometry analysis of isolated nucleoli from Arabidopsis thaliana plants using the FANoS (Fluorescence Assisted Nucleolus Sorting) strategy. We identified many ribosome biogenesis factors (RBF) and proteins non-related with ribosome biogenesis, in agreement with the recognized multi-functionality of the nucleolus. Interestingly, we found that 26S proteasome subunits localize in the nucleolus and demonstrated that proteasome activity and nucleolus organization are intimately linked to each other. Proteasome subunits form discrete foci in the disorganized nucleolus of nuc1.2 plants. Nuc1.2 protein extracts display reduced proteasome activity in vitro compared to WT protein extracts. Remarkably, proteasome activity in nuc1.2 is similar to proteasome activity in WT plants treated with proteasome inhibitors (MG132 or ALLN). Finally, we show that MG132 treatment induces disruption of nucleolar structures in WT but not in nuc1.2 plants. Altogether, our data suggest a functional interconnection between nucleolus structure and proteasome activity. PMID:29104584

Baicalin and scutellarin are proteasome inhibitors that specifically target chymotrypsin-like catalytic activity.

PubMed

Wu, Yi-Xin; Sato, Eiji; Kimura, Wataru; Miura, Naoyuki

2013-09-01

Baicalin and scutellarin are the major active principal flavonoids extracted from the Chinese herbal medicines Scutellaria baicalensis and Erigeron breviscapus (Vant.) Hand-Mazz. It has recently been reported that baicalin and scutellarin have antitumor activity. However, the mechanisms of action are unknown. We previously reported that some flavonoids have a specific role in the inhibition of the activity of proteasome subunits and induced apoptosis in tumor cells. To further investigate these pharmacological effects, we examined the inhibitory activity of baicalin and scutellarin on the extracted proteasomes from mice and cancer cells. Using fluorogenic substrates for proteasome catalytic subunits, we found that baicalin and scutellarin specifically inhibited chymotrypsin-like activity but did not inhibit trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities. These data suggested that baicalin and scutellarin specifically inhibit chymotrypsin-like catalytic activity in the proteasome. Copyright © 2012 John Wiley & Sons, Ltd.

Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators

PubMed Central

Liu, Yangfan P.; Tsai, I-Chun; Morleo, Manuela; Oh, Edwin C.; Leitch, Carmen C.; Massa, Filomena; Lee, Byung-Hoon; Parker, David S.; Finley, Daniel; Zaghloul, Norann A.; Franco, Brunella; Katsanis, Nicholas

2014-01-01

Cilia are critical mediators of paracrine signaling; however, it is unknown whether proteins that contribute to ciliopathies converge on multiple paracrine pathways through a common mechanism. Here, we show that loss of cilopathy-associated proteins Bardet-Biedl syndrome 4 (BBS4) or oral-facial-digital syndrome 1 (OFD1) results in the accumulation of signaling mediators normally targeted for proteasomal degradation. In WT cells, several BBS proteins and OFD1 interacted with proteasomal subunits, and loss of either BBS4 or OFD1 led to depletion of multiple subunits from the centrosomal proteasome. Furthermore, overexpression of proteasomal regulatory components or treatment with proteasomal activators sulforaphane (SFN) and mevalonolactone (MVA) ameliorated signaling defects in cells lacking BBS1, BBS4, and OFD1, in morphant zebrafish embryos, and in induced neurons from Ofd1-deficient mice. Finally, we tested the hypothesis that other proteasome-dependent pathways not known to be associated with ciliopathies are defective in the absence of ciliopathy proteins. We found that loss of BBS1, BBS4, or OFD1 led to decreased NF-κB activity and concomitant IκBβ accumulation and that these defects were ameliorated with SFN treatment. Taken together, our data indicate that basal body proteasomal regulation governs paracrine signaling pathways and suggest that augmenting proteasomal function might benefit ciliopathy patients. PMID:24691443

NOX4-mediated ROS production induces apoptotic cell death via down-regulation of c-FLIP and Mcl-1 expression in combined treatment with thioridazine and curcumin.

PubMed

Seo, Seung Un; Kim, Tae Hwan; Kim, Dong Eun; Min, Kyoung-Jin; Kwon, Taeg Kyu

2017-10-01

Thioridazine is known to have anti-tumor effects by inhibiting PI3K/Akt signaling, which is an important signaling pathway in cell survival. However, thioridazine alone does not induce apoptosis in head and neck squamous cell carcinoma (AMC-HN4), human breast carcinoma (MDA-MB231), and human glioma (U87MG) cells. Therefore, we investigated whether combined treatment with thioridazine and curcumin induces apoptosis. Combined treatment with thioridazine and curcumin markedly induced apoptosis in cancer cells without inducing apoptosis in human normal mesangial cells and human normal umbilical vein cells (EA.hy926). We found that combined treatment with thioridazine and curcumin had synergistic effects in AMC-HN4 cells. Among apoptosis-related proteins, thioridazine plus curcumin induced down-regulation of c-FLIP and Mcl-1 expression at the post-translational levels in a proteasome-dependent manner. Augmentation of proteasome activity was related to the up-regulation of proteasome subunit alpha 5 (PSMA5) expression in curcumin plus thioridazine-treated cells. Combined treatment with curcumin and thioridazine produced intracellular ROS in a NOX4-dependent manner, and ROS-mediated activation of Nrf2/ARE signaling played a critical role in the up-regulation of PSMA5 expression. Furthermore, ectopic expression of c-FLIP and Mcl-1 inhibited apoptosis in thioridazine and curcumin-treated cells. Therefore, we demonstrated that thioridazine plus curcumin induces proteasome activity by up-regulating PSMA5 expression via NOX4-mediated ROS production and that down-regulation of c-FLIP and Mcl-1 expression post-translationally is involved in apoptosis. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Inhibition of 19S proteasomal regulatory complex subunit PSMD8 increases polyspermy during porcine fertilization in vitro.

PubMed

Yi, Young-Joo; Manandhar, Gaurishankar; Sutovsky, Miriam; Jonáková, Vera; Park, Chang-Sik; Sutovsky, Peter

2010-03-01

The 26S proteoasome is a multi-subunit protease specific to ubiquitinated substrate proteins. It is composed of a 20S proteasomal core with substrate degradation activity, and a 19S regulatory complex that acts in substrate recognition, deubiquitination, priming and transport to the 20S core. Inhibition of proteolytic activities associated with the sperm acrosome-borne 20S core prevents fertilization in mammals, ascidians and echinoderms. Less is known about the function of the proteasomal 19S complex during fertilization. The present study examined the role of PSMD8, an essential non-ATPase subunit of the 19S complex, in sperm-ZP penetration during porcine fertilization in vitro (IVF). Immunofluorescence localized PSMD8 to the outer acrosomal membrane, acrosomal matrix and the inner acrosomal membrane. Colloidal gold transmission electron microscopy detected PSMD8 on the surface of vesicles in the acrosomal shroud, formed as a result of zona pellucida-induced acrosomal exocytosis. Contrary to the inhibition of fertilization by blocking of the 20S core activities, fertilization and polyspermy rates were increased by adding anti-PSMD8 antibody to fertilization medium. This observation is consistent with a possible role of PSMD8 in substrate deubiquitination, a process which when blocked, may actually accelerate substrate proteolysis by the 26S proteasome. Subunit PSMD8 co-immunoprecipitated with acrosomal surface-associated spermadhesin AQN1. This association indicates that the sperm acrosome-borne proteasomes become exposed onto the sperm surface following the acrosomal exocytosis. Since immunological blocking of subunit PSMD8 increases the rate of polyspermy during porcine fertilization, the activity of the 19S complex may be a rate-limiting factor contributing to anti-polyspermy defense during porcine fertilization. Copyright 2009. Published by Elsevier Ireland Ltd.

Phosphorylation of Rpt6 regulates synaptic strength in hippocampal neurons.

PubMed

Djakovic, Stevan N; Marquez-Lona, Esther M; Jakawich, Sonya K; Wright, Rebecca; Chu, Carissa; Sutton, Michael A; Patrick, Gentry N

2012-04-11

It has become increasingly evident that protein degradation via the ubiquitin proteasome system plays a fundamental role in the development, maintenance and remodeling of synaptic connections in the CNS. We and others have recently described the activity-dependent regulation of proteasome activity and recruitment of proteasomes into spine compartments involving the phosphorylation of the 19S ATPase subunit, Rpt6, by the plasticity kinase Ca(2+)/calmodulin-dependent protein kinase II α (CaMKIIα) (Bingol and Schuman, 2006; Djakovic et al., 2009; Bingol et al, 2010). Here, we investigated the role of Rpt6 phosphorylation on proteasome function and synaptic strength. Utilizing a phospho-specific antibody we verified that Rpt6 is phosphorylated at Serine 120 (S120) by CaMKIIα. In addition, we found that Rpt6 is phosphorylated by CaMKIIα in an activity-dependent manner. Furthermore, we showed that a serine 120 to aspartic acid phospho-mimetic mutant of Rpt6 (S120D) increases its resistance to detergent extraction in rat hippocampal dendrites, indicating phosphorylated Rpt6 may promote the tethering of proteasomes to scaffolds and cytoskeletal components. Expression of Rpt6 S120D decreased miniature EPSC (mEPSC) amplitude, while expression of a phospho-dead mutant (S120A) increased mEPSC amplitude. Surprisingly, homeostatic scaling of mEPSC amplitude produced by chronic application of bicuculline or tetrodotoxin is both mimicked and occluded by altered Rpt6 phosphorylation. Together, these data suggest that CaMKII-dependent phosphorylation of Rpt6 at S120 may be an important regulatory mechanism for proteasome-dependent control of synaptic remodeling in slow homeostatic plasticity.

Ameliorating replicative senescence of human bone marrow stromal cells by PSMB5 overexpression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Li, E-mail: luli7300@126.com; Song, Hui-Fang; Wei, Jiao-Long

2014-01-24

Highlights: • PSMB5 overexpression restores the differentiation potential of aged hBMSCs. • PSMB5 overexpression enhances the proteasomal activity of late-stage hBMSCs. • PSMB5 overexpression inhibits replicative senescence and improved cell viability. • PSMB5 overexpression promotes cell growth by upregulating the Cyclin D1/CDK4 complex. - Abstract: Multipotent human bone marrow stromal cells (hBMSCs) potentially serve as a source for cell-based therapy in regenerative medicine. However, in vitro expansion was inescapably accompanied with cell senescence, characterized by inhibited proliferation and compromised pluripotency. We have previously demonstrated that this aging process is closely associated with reduced 20S proteasomal activity, with down-regulation of rate-limitingmore » catalytic β-subunits particularly evident. In the present study, we confirmed that proteasomal activity directly contributes to senescence of hBMSCs, which could be reversed by overexpression of the β5-subunit (PSMB5). Knocking down PSMB5 led to decreased proteasomal activity concurrent with reduced cell proliferation in early-stage hBMSCs, which is similar to the senescent phenotype observed in late-stage cells. In contrast, overexpressing PSMB5 in late-stage cells efficiently restored the normal activity of 20S proteasomes and promoted cell growth, possibly via upregulating the Cyclin D1/CDK4 complex. Additionally, PSMB5 could enhance cell resistance to oxidative stress, as evidenced by the increased cell survival upon exposing senescent hBMSCs to hydrogen peroxide. Furthermore, PSMB5 overexpression retained the pluripotency of late-stage hBMSCs by facilitating their neural differentiation both in vitro and in vivo. Collectively, our work reveals a critical role of PSMB5 in 20S proteasome-mediated protection against replicative senescence, pointing to a possible strategy for maintaining the integrity of culture-expanded hBMSCs by manipulating the expression of PSMB5.« less

Paradoxical resistance of multiple myeloma to proteasome inhibitors by decreased levels of 19S proteasomal subunits

PubMed Central

Acosta-Alvear, Diego; Cho, Min Y; Wild, Thomas; Buchholz, Tonia J; Lerner, Alana G; Simakova, Olga; Hahn, Jamie; Korde, Neha; Landgren, Ola; Maric, Irina; Choudhary, Chunaram; Walter, Peter; Weissman, Jonathan S; Kampmann, Martin

2015-01-01

Hallmarks of cancer, including rapid growth and aneuploidy, can result in non-oncogene addiction to the proteostasis network that can be exploited clinically. The defining example is the exquisite sensitivity of multiple myeloma (MM) to 20S proteasome inhibitors, such as carfilzomib. However, MM patients invariably acquire resistance to these drugs. Using a next-generation shRNA platform, we found that proteostasis factors, including chaperones and stress-response regulators, controlled the response to carfilzomib. Paradoxically, 19S proteasome regulator knockdown induced resistance to carfilzomib in MM and non-MM cells. 19S subunit knockdown did not affect the activity of the 20S subunits targeted by carfilzomib nor their inhibition by the drug, suggesting an alternative mechanism, such as the selective accumulation of protective factors. In MM patients, lower 19S levels predicted a diminished response to carfilzomib-based therapies. Together, our findings suggest that an understanding of network rewiring can inform development of new combination therapies to overcome drug resistance. DOI: http://dx.doi.org/10.7554/eLife.08153.001 PMID:26327694

The proton pump inhibitor pantoprazole disrupts protein degradation systems and sensitizes cancer cells to death under various stresses.

PubMed

Cao, Yu; Chen, Min; Tang, Dehua; Yan, Hongli; Ding, Xiwei; Zhou, Fan; Zhang, Mingming; Xu, Guifang; Zhang, Weijie; Zhang, Shu; Zhuge, Yuzheng; Wang, Lei; Zou, Xiaoping

2018-05-22

Proton pump inhibitors (PPIs) play a role in antitumor activity, with studies showing specialized impacts of PPIs on cancer cell apoptosis, metastasis, and autophagy. In this study, we demonstrated that pantoprazole (PPI) increased autophagosomes formation and affected autophagic flux depending on the pH conditions. PPI specifically elevated SQSTM1 protein levels by increasing SQSTM1 transcription via NFE2L2 activation independent of the specific effect of PPI on autophagic flux. Via decreasing proteasome subunits expression, PPI significantly impaired the function of the proteasome, accompanied by the accumulation of undegraded poly-ubiquitinated proteins. Notably, PPI-induced autophagy functioned as a downstream response of proteasome inhibition by PPI, while suppressing protein synthesis abrogated autophagy. Blocking autophagic flux in neutral pH condition or further impairing proteasome function with proteasome inhibitors, significantly aggravated PPI cytotoxicity by worsening protein degradation ability. Interestingly, under conditions of mitochondrial stress, PPI showed significant synergism when combined with Bcl-2 inhibitors. Taken together, these findings provide a new understanding of the impact of PPIs on cancer cells' biological processes and highlight the potential to develop more efficient and effective combination therapies.

Human Antiviral Protein IFIX Suppresses Viral Gene Expression during Herpes Simplex Virus 1 (HSV-1) Infection and Is Counteracted by Virus-induced Proteasomal Degradation.

PubMed

Crow, Marni S; Cristea, Ileana M

2017-04-01

The interferon-inducible protein X (IFIX), a member of the PYHIN family, was recently recognized as an antiviral factor against infection with herpes simplex virus 1 (HSV-1). IFIX binds viral DNA upon infection and promotes expression of antiviral cytokines. How IFIX exerts its host defense functions and whether it is inhibited by the virus remain unknown. Here, we integrated live cell microscopy, proteomics, IFIX domain characterization, and molecular virology to investigate IFIX regulation and antiviral functions during HSV-1 infection. We find that IFIX has a dynamic localization during infection that changes from diffuse nuclear and nucleoli distribution in uninfected cells to discrete nuclear puncta early in infection. This is rapidly followed by a reduction in IFIX protein levels. Indeed, using immunoaffinity purification and mass spectrometry, we define IFIX interactions during HSV-1 infection, finding an association with a proteasome subunit and proteins involved in ubiquitin-proteasome processes. Using synchronized HSV-1 infection, microscopy, and proteasome-inhibition experiments, we demonstrate that IFIX co-localizes with nuclear proteasome puncta shortly after 3 h of infection and that its pyrin domain is rapidly degraded in a proteasome-dependent manner. We further demonstrate that, in contrast to several other host defense factors, IFIX degradation is not dependent on the E3 ubiquitin ligase activity of the viral protein ICP0. However, we show IFIX degradation requires immediate-early viral gene expression, suggesting a viral host suppression mechanism. The IFIX interactome also demonstrated its association with transcriptional regulatory proteins, including the 5FMC complex. We validate this interaction using microscopy and reciprocal isolations and determine it is mediated by the IFIX HIN domain. Finally, we show IFIX suppresses immediate-early and early viral gene expression during infection. Altogether, our study demonstrates that IFIX antiviral functions work in part via viral transcriptional suppression and that HSV-1 has acquired mechanisms to block its functions via proteasome-dependent degradation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Early Treatment in Shock

DTIC Science & Technology

2009-06-01

concentrations. e . In analyzing the protein subunits which comprise the proteasome, we found over-expression of LMP2 protease as compared with constitutively...and Enteral Nutrition, Jan 27-31, 2007 (Phoenix, Arizona). (Presentation and abstract) 10. Smit J, Kaltashov IA, Cotter RJ, Vinogradov E , Perry MB...of human TLR4 by Escherichia coli and Shigella flexneiri 2a lipopolysaccharide: Combined effects of lipid A acylation state and TRL4 polymorphisms on

Emodin potentiates the antiproliferative effect of interferon α/β by activation of JAK/STAT pathway signaling through inhibition of the 26S proteasome

PubMed Central

He, Yujiao; Huang, Junmei; Wang, Ping; Shen, Xiaofei; Li, Sheng; Yang, Lijuan; Liu, Wanli; Suksamrarn, Apichart; Zhang, Guolin; Wang, Fei

2016-01-01

The 26S proteasome is a negative regulator of type I interferon (IFN-α/β) signaling. Inhibition of the 26S proteasome by small molecules may be a new strategy to enhance the efficacy of type I IFNs and reduce their side effects. Using cell-based screening assay for new 26S proteasome inhibitors, we found that emodin, a natural anthraquinone, was a potent inhibitor of the human 26S proteasome. Emodin preferably inhibited the caspase-like and chymotrypsin-like activities of the human 26S proteasome and increased the ubiquitination of endogenous proteins in cells. Computational modeling showed that emodin exhibited an orientation/conformation favorable to nucleophilic attack in the active pocket of the β1, β2, and β5 subunits of the 26S proteasome. Emodin increased phosphorylation of STAT1, decreased phosphorylation of STAT3 and increased endogenous gene expression stimulated by IFN-α. Emodin inhibited IFN-α-stimulated ubiquitination and degradation of type I interferon receptor 1 (IFNAR1). Emodin also sensitized the antiproliferative effect of IFN-α in HeLa cervical carcinoma cells and reduced tumor growth in Huh7 hepatocellular carcinoma-bearing mice. These results suggest that emodin potentiates the antiproliferative effect of IFN-α by activation of JAK/STAT pathway signaling through inhibition of 26S proteasome-stimulated IFNAR1 degradation. Therefore, emodin warrants further investigation as a new means to enhance the efficacy of IFN-α/β. PMID:26683360

Emodin potentiates the antiproliferative effect of interferon α/β by activation of JAK/STAT pathway signaling through inhibition of the 26S proteasome.

PubMed

He, Yujiao; Huang, Junmei; Wang, Ping; Shen, Xiaofei; Li, Sheng; Yang, Lijuan; Liu, Wanli; Suksamrarn, Apichart; Zhang, Guolin; Wang, Fei

2016-01-26

The 26S proteasome is a negative regulator of type I interferon (IFN-α/β) signaling. Inhibition of the 26S proteasome by small molecules may be a new strategy to enhance the efficacy of type I IFNs and reduce their side effects. Using cell-based screening assay for new 26S proteasome inhibitors, we found that emodin, a natural anthraquinone, was a potent inhibitor of the human 26S proteasome. Emodin preferably inhibited the caspase-like and chymotrypsin-like activities of the human 26S proteasome and increased the ubiquitination of endogenous proteins in cells. Computational modeling showed that emodin exhibited an orientation/conformation favorable to nucleophilic attack in the active pocket of the β1, β2, and β5 subunits of the 26S proteasome. Emodin increased phosphorylation of STAT1, decreased phosphorylation of STAT3 and increased endogenous gene expression stimulated by IFN-α. Emodin inhibited IFN-α-stimulated ubiquitination and degradation of type I interferon receptor 1 (IFNAR1). Emodin also sensitized the antiproliferative effect of IFN-α in HeLa cervical carcinoma cells and reduced tumor growth in Huh7 hepatocellular carcinoma-bearing mice. These results suggest that emodin potentiates the antiproliferative effect of IFN-α by activation of JAK/STAT pathway signaling through inhibition of 26S proteasome-stimulated IFNAR1 degradation. Therefore, emodin warrants further investigation as a new means to enhance the efficacy of IFN-α/β.

Elastase-like Activity Is Dominant to Chymotrypsin-like Activity in 20S Proteasome's β5 Catalytic Subunit.

PubMed

Bensinger, Dennis; Neumann, Theresa; Scholz, Christoph; Voss, Constantin; Knorr, Sabine; Kuckelkorn, Ulrike; Hamacher, Kay; Kloetzel, Peter-Michael; Schmidt, Boris

2016-07-15

The ubiquitin/proteasome system is the major protein degradation pathway in eukaryotes with several key catalytic cores. Targeting the β5 subunit with small-molecule inhibitors is an established therapeutic strategy for hematologic cancers. Herein, we report a mouse-trap-like conformational change that influences molecular recognition depending on the substitution pattern of a bound ligand. Variation of the size of P1 residues from the highly β5-selective proteasome inhibitor BSc2118 allows for discrimination between inhibitory strength and substrate conversion. We found that increasing molecular size strengthens inhibition, whereas decreasing P1 size accelerates substrate conversion. Evaluation of substrate hydrolysis after silencing of β5 activity reveals significant residual activity for large residues exclusively. Thus, classification of the β5 subunit as chymotrypsin-like and the use of the standard tyrosine-containing substrate should be reconsidered.

Multiple assembly chaperones govern biogenesis of the proteasome regulatory particle base.

PubMed

Funakoshi, Minoru; Tomko, Robert J; Kobayashi, Hideki; Hochstrasser, Mark

2009-05-29

The central protease of eukaryotes, the 26S proteasome, has a 20S proteolytic core particle (CP) and an attached 19S regulatory particle (RP). The RP is further subdivided into lid and base subcomplexes. Little is known about RP assembly. Here, we show that four conserved assembly factors govern biogenesis of the yeast RP base. Nas2 forms a complex with the Rpt4 and Rpt5 ATPases and enhances 26S proteasome formation in vivo and in vitro. Other RP subcomplexes contain Hsm3, which is related to mammalian proteasome subunit S5b. Hsm3 also contributes to base assembly. Larger Hsm3-containing complexes include two additional proteins, Nas6 and Rpn14, which function as assembly chaperones as well. Specific deletion combinations affecting these four factors cause severe perturbations to RP assembly. Our results demonstrate that proteasomal RP biogenesis requires multiple, functionally overlapping chaperones and suggest a model in which subunits form specific subcomplexes that then assemble into the base.

Design, synthesis and docking studies of novel dipeptidyl boronic acid proteasome inhibitors constructed from αα- and αβ-amino acids.

PubMed

Shi, Jingmiao; Lei, Meng; Wu, Wenkui; Feng, Huayun; Wang, Jia; Chen, Shanshan; Zhu, Yongqiang; Hu, Shihe; Liu, Zhaogang; Jiang, Cheng

2016-04-15

A series of novel dipeptidyl boronic acid proteasome inhibitors constructed from αα- and αβ-amino acids were designed and synthesized. Their structures were elucidated by (1)H NMR, (13)C NMR, LC-MS and HRMS. These compounds were evaluated for their β5 subunit inhibitory activities of human proteasome. The results showed that dipeptidyl boronic acid inhibitors composed of αα-amino acids were as active as bortezomib. Interestingly, the activities of those derived from αβ-amino acids lost completely. Of all the inhibitors, compound 22 (IC50=4.82 nM) was the most potent for the inhibition of proteasome activity. Compound 22 was also the most active against three MM cell lines with IC50 values less than 5 nM in inhibiting cell growth assays. Molecular docking studies displayed that 22 fitted very well in the β5 subunit active pocket of proteasome. Copyright © 2016. Published by Elsevier Ltd.

Effect of ionizing radiation exposure on Trypanosoma cruzi ubiquitin-proteasome system.

PubMed

Cerqueira, Paula G; Passos-Silva, Danielle G; Vieira-da-Rocha, João P; Mendes, Isabela Cecilia; de Oliveira, Karla A; Oliveira, Camila F B; Vilela, Liza F F; Nagem, Ronaldo A P; Cardoso, Joseane; Nardelli, Sheila C; Krieger, Marco A; Franco, Glória R; Macedo, Andrea M; Pena, Sérgio D J; Schenkman, Sérgio; Gomes, Dawidson A; Guerra-Sá, Renata; Machado, Carlos R

2017-03-01

In recent years, proteasome involvement in the damage response induced by ionizing radiation (IR) became evident. However, whether proteasome plays a direct or indirect role in IR-induced damage response still unclear. Trypanosoma cruzi is a human parasite capable of remarkable high tolerance to IR, suggesting a highly efficient damage response system. Here, we investigate the role of T. cruzi proteasome in the damage response induced by IR. We exposed epimastigotes to high doses of gamma ray and we analyzed the expression and subcellular localization of several components of the ubiquitin-proteasome system. We show that proteasome inhibition increases IR-induced cell growth arrest and proteasome-mediated proteolysis is altered after parasite exposure. We observed nuclear accumulation of 19S and 20S proteasome subunits in response to IR treatments. Intriguingly, the dynamic of 19S particle nuclear accumulation was more similar to the dynamic observed for Rad51 nuclear translocation than the observed for 20S. In the other hand, 20S increase and nuclear translocation could be related with an increase of its regulator PA26 and high levels of proteasome-mediated proteolysis in vitro. The intersection between the opposed peaks of 19S and 20S protein levels was marked by nuclear accumulation of both 20S and 19S together with Ubiquitin, suggesting a role of ubiquitin-proteasome system in the nuclear protein turnover at the time. Our results revealed the importance of proteasome-mediated proteolysis in T. cruzi IR-induced damage response suggesting that proteasome is also involved in T. cruzi IR tolerance. Moreover, our data support the possible direct/signaling role of 19S in DNA damage repair. Based on these results, we speculate that spatial and temporal differences between the 19S particle and 20S proteasome controls proteasome multiple roles in IR damage response. Copyright © 2017 Elsevier B.V. All rights reserved.

Genetic myostatin decrease in the golden retriever muscular dystrophy model does not significantly affect the ubiquitin proteasome system despite enhancing the severity of disease.

PubMed

Cotten, Steven W; Kornegay, Joe N; Bogan, Daniel J; Wadosky, Kristine M; Patterson, Cam; Willis, Monte S

2013-01-01

Recent studies suggest that inhibiting the protein myostatin, a negative regulator of skeletal muscle mass, may improve outcomes in patients with Duchenne muscular dystrophy by enhancing muscle mass. When the dystrophin-deficient golden retriever muscular dystrophy (GRMD) dog was bred with whippets having a heterozygous mutation for the myostatin gene, affected GRMD dogs with decreased myostatin (GRippets) demonstrated an accelerated physical decline compared to related affected GRMD dogs with full myostatin. To examine the role of the ubiquitin proteasome and calpain systems in this accelerated decline, we determined the expression of the muscle ubiquitin ligases MuRF1, Atrogin-1, RNF25, RNF11, and CHIP: the proteasome subunits PSMA6, PSMB4, and PSME1: and calpain 1/2 by real time PCR in the cranial sartorius and vastus lateralis muscles in control, affected GRMD, and GRippet dogs. While individual affected GRMD and GRippet dogs contributed to an increased variability seen in ubiquitin ligase expression, neither group was significantly different from the control group. The affected GRMD dogs demonstrated significant increases in caspase-like and trypsin-like activity in the cranial sartorius; however, all three proteasome activities in the GRippet muscles did not differ from controls. Increased variability in calpain 1 and calpain 2 expression and activity in the affected GRMD and GRippet groups were identified, but no statistical differences from the control group were seen. These studies suggest a role of myostatin in the disease progression of GRMD, which does not significantly involve key components of the ubiquitin proteasome and calpain systems involved in the protein quality control of sarcomere and other structural skeletal muscle proteins.

A tolerance gene for prenylated flavonoid encodes a 26S proteasome regulatory subunit in Sophora flavescens.

PubMed

Shitan, Nobukazu; Kamimoto, Yoshihisa; Minami, Shota; Kubo, Mizuki; Ito, Kozue; Moriyasu, Masataka; Yazaki, Kazufumi

2011-01-01

Yeast functional screening with a Sophora flavescens cDNA library was performed to identify the genes involved in the tolerant mechanism to the self-producing prenylated flavonoid sophoraflavanone G (SFG). One cDNA, which conferred SFG tolerance, encoded a regulatory particle triple-A ATPase 2 (SfRPT2), a member of the 26S proteasome subunit. The yeast transformant of SfRPT2 showed reduced SFG accumulation in the cells.

Ubiquitin-like and ubiquitin-associated domain proteins: significance in proteasomal degradation

PubMed Central

Lau, Alan F.

2009-01-01

The ubiquitin–proteasome pathway of protein degradation is one of the major mechanisms that are involved in the maintenance of the proper levels of cellular proteins. The regulation of proteasomal degradation thus ensures proper cell functions. The family of proteins containing ubiquitin-like (UbL) and ubiquitin-associated (UBA) domains has been implicated in proteasomal degradation. UbL–UBA domain containing proteins associate with substrates destined for degradation as well as with subunits of the proteasome, thus regulating the proper turnover of proteins. PMID:19468686

Phosphorylation of Rpt6 regulates synaptic strength in hippocampal neurons

PubMed Central

Djakovic, Stevan N.; Marquez-Lona, Esther M.; Jakawich, Sonya K.; Wright, Rebecca; Chu, Carissa; Sutton, Michael A.; Patrick, Gentry N.

2012-01-01

It has become increasingly evident that protein degradation via the ubiquitin proteasome system plays a fundamental role in the development, maintenance and remodeling of synaptic connections in the central nervous system. We and others have recently described the activity-dependent regulation of proteasome activity (Djakovic et al., 2009) and recruitment of proteasomes into spine compartments (Bingol and Schuman, 2006) involving the phosphorylation of the 19S ATPase subunit, Rpt6, by the plasticity kinase Ca2+/calmodulin-dependent protein kinases II alpha CaMKIIα) (Bingol et al., 2010). Here, we investigated the role of Rpt6 phosphorylation on proteasome function and synaptic strength. Utilizing a phospho-specific antibody we verified that Rpt6 is phosphorylated at Serine 120 (S120) by CaMKIIα. In addition, we found that Rpt6 is phosphorylated by CaMKIIα in an activity-dependent manner. In addition, we showed that a serine 120 to aspartic acid phospho-mimetic mutant of Rpt6 (S120D) increases its resistance to detergent extraction in rat hippocampal dendrites, indicating phosphorylated Rpt6 may promote the tethering of proteasomes to scaffolds and cytoskeletal components. Interestingly, expression of Rpt6 S120D decreased miniature excitatory postsynaptic current (mEPSC) amplitude, while expression of a phospho-dead mutant (S120A) increased mEPSC amplitude. Surprisingly, homeostatic scaling of mEPSC amplitude produced by chronic application of bicuculline or tetrodotoxin is both mimicked and occluded by altered Rpt6 phosphorylation. Together these data suggest that CaMKII-dependent phosphorylation of Rpt6 at S120 may be an important regulatory mechanism for proteasome-dependent control of synaptic remodeling in slow homeostatic plasticity. PMID:22496558

DNA-repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a

PubMed Central

Walters, Kylie J.; Lech, Patrycja J.; Goh, Amanda M.; Wang, Qinghua; Howley, Peter M.

2003-01-01

The Rad23 family of proteins, including the human homologs hHR23a and hHR23b, stimulates nucleotide excision repair and has been shown to provide a novel link between proteasome-mediated protein degradation and DNA repair. In this work, we illustrate how the proteasomal subunit S5a regulates hHR23a protein structure. By using NMR spectroscopy, we have elucidated the structure and dynamic properties of the 40-kDa hHR23a protein and show it to contain four structured domains connected by flexible linker regions. In addition, we reveal that these domains interact in an intramolecular fashion, and by using residual dipolar coupling data in combination with chemical shift perturbation analysis, we present the hHR23a structure. By itself, hHR23a adopts a closed conformation defined by the interaction of an N-terminal ubiquitin-like domain with two ubiquitin-associated domains. Interestingly, binding of the proteasomal subunit S5a disrupts the hHR23a interdomain interactions and thereby causes it to adopt an opened conformation. PMID:14557549

Regulators of the Proteasome Pathway, Uch37 and Rpn13, Play Distinct Roles in Mouse Development

PubMed Central

Al-Shami, Amin; Jhaver, Kanchan G.; Vogel, Peter; Wilkins, Carrie; Humphries, Juliane; Davis, John J.; Xu, Nianhua; Potter, David G.; Gerhardt, Brenda; Mullinax, Robert; Shirley, Cynthia R.; Anderson, Stephen J.; Oravecz, Tamas

2010-01-01

Rpn13 is a novel mammalian proteasomal receptor that has recently been identified as an amplification target in ovarian cancer. It can interact with ubiquitin and activate the deubiquitinating enzyme Uch37 at the 26S proteasome. Since neither Rpn13 nor Uch37 is an integral proteasomal subunit, we explored whether either protein is essential for mammalian development and survival. Deletion of Uch37 resulted in prenatal lethality in mice associated with severe defect in embryonic brain development. In contrast, the majority of Rpn13-deficient mice survived to adulthood, although they were smaller at birth and fewer in number than wild-type littermates. Absence of Rpn13 produced tissue-specific effects on proteasomal function: increased proteasome activity in adrenal gland and lymphoid organs, and decreased activity in testes and brain. Adult Rpn13−/− mice reached normal body weight but had increased body fat content and were infertile due to defective gametogenesis. Additionally, Rpn13−/− mice showed increased T-cell numbers, resembling growth hormone-mediated effects. Indeed, serum growth hormone and follicular stimulating hormone levels were significantly increased in Rpn13−/− mice, while growth hormone receptor expression was reduced in the testes. In conclusion, this is the first report characterizing the physiological roles of Uch37 and Rpn13 in murine development and implicating a non-ATPase proteasomal protein, Rpn13, in the process of gametogenesis. PMID:21048919

Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues.

PubMed

Wyroba, E; Surmacz, L; Osinska, M; Wiejak, J

2007-01-01

Phagosome maturation is a complex process enabling degradation of internalised particles. Our data obtained at the gene, protein and cellular level indicate that the set of components involved in this process and known up to now in mammalian cells is functioning in unicellular eukaryote. Rab7-interacting partners: homologues of its effector RILP (Rab-interacting lysosomal protein) and LAMP-2 (lysosomal membrane protein 2) as well as alpha7 subunit of the 26S proteasome were revealed in Paramecium phagolysosomal compartment. We identified the gene/transcript fragments encoding RILP-related proteins (RILP1 and RILP2) in Paramecium by PCR/RT-PCR and sequencing. The deduced amino acid sequences of RILP1 and RILP2 show 60.5% and 58.3% similarity, respectively, to the region involved in regulating of lysosomal morphology and dynein-dynactin recruitment of human RILP. RILP colocalised with Rab7 in Paramecium lysosomes and at phagolysosomal membrane during phagocytosis of both the latex beads and bacteria. In the same compartment LAMP-2 was present and its expression during latex internalisation was 2.5-fold higher than in the control when P2 protein fractions (100,000 x g) of equal load were quantified by immunoblotting. LAMP-2 cross-reacting polypeptide of approximately106 kDa was glycosylated as shown by fluorescent and Western analysis of the same blot preceded by PNGase F treatment. The alpha7 subunit of 26S proteasome was detected close to the phagosomal membrane in the small vesicles, in some of which it colocalised with Rab7. Immunoblotting confirmed presence of RILP-related polypeptide and a7 subunit of 26S proteasome in Paramecium protein fractions. These results suggest that Rab7, RILP and LAMP-2 may be involved in phagosome maturation in Paramecium.

The XPB subunit of repair/transcription factor TFIIH directly interacts with SUG1, a subunit of the 26S proteasome and putative transcription factor.

PubMed

Weeda, G; Rossignol, M; Fraser, R A; Winkler, G S; Vermeulen, W; van 't Veer, L J; Ma, L; Hoeijmakers, J H; Egly, J M

1997-06-15

Mutations in the basal transcription initiation/DNA repair factor TFIIH are responsible for three human disorders: xeroderma pigmentosum (XP), cockayne syndrome (CS) and trichothiodystrophy (TTD). The non-repair features of CS and TTD are thought to be due to a partial inactivation of the transcription function of the complex. To search for proteins whose interaction with TFIIH subunits is disturbed by mutations in patients we used the yeast two-hybrid system and report the isolation of a novel XPB interacting protein, SUG1. The interaction was validated in vivo and in vitro in the following manner. (i) SUG1 interacts with XPB but not with the other core TFIIH subunits in the two-hybrid assay. (ii) Physical interaction is observed in a baculovirus co-expression system. (iii) In fibroblasts under non-overexpression conditions a portion of SUG1 is bound to the TFIIH holocomplex as deduced from co-purification, immunopurification and nickel-chelate affinity chromatography using functional tagged TFIIH. Furthermore, overexpression of SUG1 in normal fibroblasts induced arrest of transcription and a chromatin collapse in vivo. Interestingly, the interaction was diminished with a mutant form of XPB, thus providing a potential link with the clinical features of XP-B patients. Since SUG1 is an integral component of the 26S proteasome and may be part of the mediator, our findings disclose a SUG1-dependent link between TFIIH and the cellular machinery involved in protein modelling/degradation.

A novel interplay between the ubiquitin–proteasome system and serine proteases during Drosophila development.

PubMed

Lipinszki, Zoltán; Klement, Eva; Hunyadi-Gulyas, Eva; Medzihradszky, Katalin F; Márkus, Róbert; Pál, Margit; Deák, Péter; Udvardy, Andor

2013-09-15

The concentrations of the Drosophila proteasomal and extraproteasomal polyubiquitin receptors fluctuate in a developmentally regulated fashion. This fluctuation is generated by a previously unidentified proteolytic activity. In the present paper, we describe the purification, identification and characterization of this protease (endoproteinase I). Its expression increases sharply at the L1-L2 larval stages, remains high until the second half of the L3 stage, then declines dramatically. This sharp decrease coincides precisely with the increase of polyubiquitin receptor concentrations in late L3 larvae, which suggests a tight developmental co-regulation. RNAi-induced down-regulation of endoproteinase I results in pupal lethality. Interestingly, we found a cross-talk between the 26S proteasome and this larval protease: transgenic overexpression of the in vivo target of endoproteinase I, the C-terminal half of the proteasomal polyubiquitin receptor subunit p54/Rpn10 results in transcriptional down-regulation of endoproteinase I and consequently a lower level of proteolytic elimination of the polyubiquitin receptors. Another larval protease, Jonah65A-IV, which degrades only unfolded proteins and exhibits similar cross-talk with the proteasome has also been purified and characterized. It may prevent the accumulation of polyubiquitylated proteins in larvae contrary to the low polyubiquitin receptor concentration.

The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together

PubMed Central

Pathare, Ganesh Ramnath; Nagy, István; Bohn, Stefan; Unverdorben, Pia; Hubert, Agnes; Körner, Roman; Nickell, Stephan; Lasker, Keren; Sali, Andrej; Tamura, Tomohiro; Nishioka, Taiki; Förster, Friedrich; Baumeister, Wolfgang; Bracher, Andreas

2012-01-01

Proteasomes execute the degradation of most cellular proteins. Although the 20S core particle (CP) has been studied in great detail, the structure of the 19S regulatory particle (RP), which prepares ubiquitylated substrates for degradation, has remained elusive. Here, we report the crystal structure of one of the RP subunits, Rpn6, and we describe its integration into the cryo-EM density map of the 26S holocomplex at 9.1 Å resolution. Rpn6 consists of an α-solenoid-like fold and a proteasome COP9/signalosome eIF3 (PCI) module in a right-handed suprahelical configuration. Highly conserved surface areas of Rpn6 interact with the conserved surfaces of the Pre8 (alpha2) and Rpt6 subunits from the alpha and ATPase rings, respectively. The structure suggests that Rpn6 has a pivotal role in stabilizing the otherwise weak interaction between the CP and the RP. PMID:22187461

A yeast-based genetic screening to identify human proteins that increase homologous recombination.

PubMed

Collavoli, Anita; Comelli, Laura; Rainaldi, Giuseppe; Galli, Alvaro

2008-05-01

To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR.

The genetic basis for the biosynthesis of the pharmaceutically important class of epoxyketone proteasome inhibitors

PubMed Central

Schorn, Michelle; Zettler, Judith; Noel, Joseph P.; Dorrestein, Pieter C.; Moore, Bradley S.; Kaysser, Leonard

2013-01-01

The epoxyketone proteasome inhibitors are an established class of therapeutic agents for the treatment of cancer. Their unique α′,β′-epoxyketone pharmacophore allows binding to the catalytic β-subunits of the proteasome with extraordinary specificity. Here we report the characterization of the first gene clusters for the biosynthesis of natural peptidyl-epoxyketones. The clusters for epoxomicin, the lead compound for the anti-cancer drug Kyprolis™, and for eponemycin were identified in the actinobacterial producer strains ATCC 53904 and Streptomyces hygroscopicus ATCC 53709, respectively, using a modified protocol for Ion Torrent PGM genome sequencing. Both gene clusters code for a hybrid non-ribosomal peptide synthetase/polyketide synthase multifunctional enzyme complex and homologous redox enzymes. Epoxomicin and eponemycin were heterologously produced in Streptomyces albus J1046 via whole pathway expression. Moreover, we employed mass spectral molecular networking for a new comparative metabolomics approach in a heterologous system and discovered a number of putative epoxyketone derivatives. With this study we have definitively linked epoxyketone proteasome inhibitors and their biosynthesis genes for the first time in any organism, which will now allow for their detailed biochemical investigation. PMID:24168704

A Minimal Anaphase Promoting Complex/Cyclosome (APC/C) in Trypanosoma brucei

PubMed Central

Bessat, Mohamed; Knudsen, Giselle; Burlingame, Alma L.; Wang, Ching C.

2013-01-01

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C’s. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C. PMID:23533609

The devil is in the details: comparison between COP9 signalosome (CSN) and the LID of the 26S proteasome.

PubMed

Meister, Cindy; Gulko, Miriam Kolog; Köhler, Anna M; Braus, Gerhard H

2016-02-01

The COP9 signalosome (CSN) and the proteasomal LID are conserved macromolecular complexes composed of at least eight subunits with molecular weights of approximately 350 kDa. CSN and LID are part of the ubiquitin–proteasome pathway and cleave isopeptide linkages of lysine side chains on target proteins. CSN cleaves the isopeptide bond of ubiquitin-like protein Nedd8 from cullins, whereas the LID cleaves ubiquitin from target proteins sentenced for degradation. CSN and LID are structurally and functionally similar but the order of the assembly pathway seems to be different. The assembly differs in at least the last subunit joining the pre-assembled subcomplex. This review addresses the similarities and differences in structure, function and assembly of CSN and LID.

SAMe prevents the induction of the immunoproteasome and preserves the 26S proteasome in the DDC-induced MDB mouse model.

PubMed

Bardag-Gorce, Fawzia; Oliva, Joan; Li, Jun; French, Barbara A; French, Samuel W

2010-06-01

Mallory-Denk bodies (MDBs) form in the liver of alcoholic patients. This occurs because of the accumulation and aggregation of ubiquitinated cytokeratins, which hypothetically is due to the ubiquitin-proteasome pathway's (UPP) failure to degrade the cytokeratins. The experimental model of MDB formation was used in which MDBs were induced by refeeding DDC to drug-primed mice. The gene expression and protein levels of LMP2, LMP7 and MECL-1, the catalytic subunits in the immunoproteasome, as well as FAT10, were increased in the liver cells forming MDBs but not in the intervening normal hepatocytes. Chymotrypsin-like activity of the UPP was decreased by DDC refeeding, indicating that a switch from the UPP to the immunoproteasome had occurred at the expense of the 26S proteasome. The failure of the UPP to digest cytokeratins would explain MDB aggregate formation. SAMe prevented the decrease in UPP activity, the increase in LMP2, LMP7, and MECL-1 protein levels and MDB formation induced by DDC. DDC refeeding also induced the TNFalpha and IFNgamma receptors. SAMe prevented the increase in the TNFalpha and IFNgamma receptors, supporting the idea that TNFalpha and IFNgamma were responsible for the up regulation of LMP2, LPM7, and FAT10. These results support the conclusion that MDBs form in FAT10 over-expressing hepatocytes where the up regulation of the immunoproteasome occurs at the expense of the 26S proteasome. Copyright 2010 Elsevier Inc. All rights reserved.

The XPB subunit of repair/transcription factor TFIIH directly interacts with SUG1, a subunit of the 26S proteasome and putative transcription factor.

PubMed Central

Weeda, G; Rossignol, M; Fraser, R A; Winkler, G S; Vermeulen, W; van 't Veer, L J; Ma, L; Hoeijmakers, J H; Egly, J M

1997-01-01

Mutations in the basal transcription initiation/DNA repair factor TFIIH are responsible for three human disorders: xeroderma pigmentosum (XP), cockayne syndrome (CS) and trichothiodystrophy (TTD). The non-repair features of CS and TTD are thought to be due to a partial inactivation of the transcription function of the complex. To search for proteins whose interaction with TFIIH subunits is disturbed by mutations in patients we used the yeast two-hybrid system and report the isolation of a novel XPB interacting protein, SUG1. The interaction was validated in vivo and in vitro in the following manner. (i) SUG1 interacts with XPB but not with the other core TFIIH subunits in the two-hybrid assay. (ii) Physical interaction is observed in a baculovirus co-expression system. (iii) In fibroblasts under non-overexpression conditions a portion of SUG1 is bound to the TFIIH holocomplex as deduced from co-purification, immunopurification and nickel-chelate affinity chromatography using functional tagged TFIIH. Furthermore, overexpression of SUG1 in normal fibroblasts induced arrest of transcription and a chromatin collapse in vivo. Interestingly, the interaction was diminished with a mutant form of XPB, thus providing a potential link with the clinical features of XP-B patients. Since SUG1 is an integral component of the 26S proteasome and may be part of the mediator, our findings disclose a SUG1-dependent link between TFIIH and the cellular machinery involved in protein modelling/degradation. PMID:9173976

Plant WEE1 kinase is cell cycle regulated and removed at mitosis via the 26S proteasome machinery

PubMed Central

Cook, Gemma S.; Grønlund, Anne Lentz; Siciliano, Ilario; Spadafora, Natasha; Amini, Maryam; Herbert, Robert J.; Bitonti, M. Beatrice; Graumann, Katja; Francis, Dennis; Rogers, Hilary J.

2013-01-01

In yeasts and animals, premature entry into mitosis is prevented by the inhibitory phosphorylation of cyclin-dependent kinase (CDK) by WEE1 kinase, and, at mitosis, WEE1 protein is removed through the action of the 26S proteasome. Although in higher plants WEE1 function has been confirmed in the DNA replication checkpoint, Arabidopsis wee1 insertion mutants grow normally, and a role for the protein in the G2/M transition during an unperturbed plant cell cycle is yet to be confirmed. Here data are presented showing that the inhibitory effect of WEE1 on CDK activity in tobacco BY-2 cell cultures is cell cycle regulated independently of the DNA replication checkpoint: it is high during S-phase but drops as cells traverse G2 and enter mitosis. To investigate this mechanism further, a yeast two-hybrid screen was undertaken to identify proteins interacting with Arabidopsis WEE1. Three F-box proteins and a subunit of the proteasome complex were identified, and bimolecular fluorescence complementation confirmed an interaction between AtWEE1 and the F-box protein SKP1 INTERACTING PARTNER 1 (SKIP1). Furthermore, the AtWEE1–green fluorescent protein (GFP) signal in Arabidopsis primary roots treated with the proteasome inhibitor MG132 was significantly increased compared with mock-treated controls. Expression of AtWEE1–YFPC (C-terminal portion of yellow fluorescent protein) or AtWEE1 per se in tobacco BY-2 cells resulted in a premature increase in the mitotic index compared with controls, whereas co-expression of AtSKIP1–YFPN negated this effect. These data support a role for WEE1 in a normal plant cell cycle and its removal at mitosis via the 26S proteasome. PMID:23536609

Regulation of leaf organ size by the Arabidopsis RPT2a 19S proteasome subunit.

PubMed

Sonoda, Yutaka; Sako, Kaori; Maki, Yuko; Yamazaki, Naoko; Yamamoto, Hiroko; Ikeda, Akira; Yamaguchi, Junji

2009-10-01

The ubiquitin/26S proteasome pathway plays a central role in the degradation of short-lived regulatory proteins, to control many cellular events. To further understand this pathway, we focused on the RPT2 subunit of the 26S proteasome regulatory particle. The Arabidopsis genome contains two genes, AtRPT2a and AtRPT2b, which encode paralog molecules of the RPT2 subunit, with a difference of only three amino acids in the protein sequences. Both genes showed similar mRNA accumulation patterns. However, the rpt2a mutant showed a specific phenotype of enlarged leaves caused by increased cell size, in correlation with increased ploidy. Detailed analyses revealed that cell expansion is increased in the rpt2a mutant by extended endoreduplication early in leaf development. The transcription of genes encoding cell cycle-related components, for DNA replication licensing and the G2/M phase, was also promoted in the rpt2a mutant, suggesting that extended endoreduplication was caused by increased DNA replication, and disrupted regulation of the G2/M checkpoint, at the proliferation stage of leaf development.

Hsp27 and F-box protein β-TrCP promote degradation of mRNA decay factor AUF1.

PubMed

Li, Mei-Ling; Defren, Jennifer; Brewer, Gary

2013-06-01

Activation of the mitogen-activated protein (MAP) pathway kinases p38 and MK2 induces phosphorylation of the chaperone Hsp27 and stabilization of mRNAs containing AU-rich elements (AREs) (ARE-mRNAs). Likewise, expression of phosphomimetic mutant forms of Hsp27 also stabilizes ARE-mRNAs. It appears to perform this function by promoting degradation of the ARE-mRNA decay factor AUF1 by proteasomes. In this study, we examined the molecular mechanism linking Hsp27 phosphorylation to AUF1 degradation by proteasomes. AUF1 is a target of β-TrCP, the substrate recognition subunit of the E3 ubiquitin ligase Skp1-cullin-F-box protein complex, SCF(β-TrCP). Depletion of β-TrCP stabilized AUF1. In contrast, overexpression of β-TrCP enhanced ubiquitination and degradation of AUF1 and led to stabilization of reporter mRNAs containing cytokine AREs. Enhanced AUF1 degradation required expression of phosphomimetic mutant forms of both Hsp27 and AUF1. Our results suggest that a signaling axis composed of p38 MAP kinase-MK2-Hsp27-β-TrCP may promote AUF1 degradation by proteasomes and stabilization of cytokine ARE-mRNAs.

Functional study of hot pepper 26S proteasome subunit RPN7 induced by Tobacco mosaic virus from nuclear proteome analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Boo-Ja; Kwon, Sun Jae; Kim, Sung-Kyu

Two-dimensional gel electrophoresis (2-DE) was applied for the screening of Tobacco mosaic virus (TMV)-induced hot pepper (Capsicum annuum cv. Bugang) nuclear proteins. From differentially expressed protein spots, we acquired the matched peptide mass fingerprint (PMF) data, analyzed by MALDI-TOF MS, from the non-redundant hot pepper EST protein FASTA database using the VEMS 2.0 software. Among six identified nuclear proteins, the hot pepper 26S proteasome subunit RPN7 (CaRPN7) was subjected to further study. The level of CaRPN7 mRNA was specifically increased during incompatible TMV-P{sub 0} interaction, but not during compatible TMV-P{sub 1.2} interaction. When CaRPN7::GFP fusion protein was targeted in onionmore » cells, the nuclei had been broken into pieces. In the hot pepper leaves, cell death was exacerbated and genomic DNA laddering was induced by Agrobacterium-mediated transient overexpression of CaPRN7. Thus, this report presents that the TMV-induced CaRPN7 may be involved in programmed cell death (PCD) in the hot pepper plant.« less

The 11S Proteasome Subunit PSME3 Is a Positive Feedforward Regulator of NF-κB and Important for Host Defense against Bacterial Pathogens.

PubMed

Sun, Jinxia; Luan, Yi; Xiang, Dong; Tan, Xiao; Chen, Hui; Deng, Qi; Zhang, Jiaojiao; Chen, Minghui; Huang, Hongjun; Wang, Weichao; Niu, Tingting; Li, Wenjie; Peng, Hu; Li, Shuangxi; Li, Lei; Tang, Wenwen; Li, Xiaotao; Wu, Dianqing; Wang, Ping

2016-02-02

The NF-κB pathway plays important roles in immune responses. Although its regulation has been extensively studied, here, we report an unknown feedforward mechanism for the regulation of this pathway by Toll-like receptor (TLR) ligands in macrophages. During bacterial infections, TLR ligands upregulate the expression of the 11S proteasome subunit PSME3 via NF-κB-mediated transcription in macrophages. PSME3, in turn, enhances the transcriptional activity of NF-κB by directly binding to and destabilizing KLF2, a negative regulator of NF-κB transcriptional activity. Consistent with this positive role of PSME3 in NF-κB regulation and importance of the NF-κB pathway in host defense against bacterial infections, the lack of PSME3 in hematopoietic cells renders the hosts more susceptible to bacterial infections, accompanied by increased bacterial burdens in host tissues. Thus, this study identifies a substrate for PSME3 and elucidates a proteolysis-dependent, but ubiquitin-independent, mechanism for NF-κB regulation that is important for host defense and innate immunity. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Features of proteasome functioning in malignant tumors

NASA Astrophysics Data System (ADS)

Kondakova, I. V.; Spirina, L. V.; Shashova, E. E.; Kolegova, E. S.; Slonimskaya, E. M.; Kolomiets, L. A.; Afanas'ev, S. G.; Choinzonov, Y. L.

2017-09-01

Proteasome ubiquitin system is the important system of intracellular proteolysis. The activity of the proteasomes may undergo changes during cancer development. We studied the chymotrypsin-like activity of proteasomes, their subunit composition, and their association with tumor stage in breast cancer, head and neck squamous cell carcinoma, endometrial cancer, renal cancer, bladder cancer, stomach cancer, ovarian cancer, and colorectal cancer. The increase in chymotrypsin-like activity of proteasomes and decrease in total proteasome pool compared with adjacent tissues were shown in all malignant tumors excluding kidney cancer. The increase in chymotrypsin-like activity of proteasomes was found in primary tumors with all types of metastasis: lymphogenous of head and neck squamous cell carcinoma, intraperitoneal metastasis of ovarian cancer, hematogenous metastasis colorectal cancer. The exception was kidney cancer, in which there was a decrease in chymotrypsin-like activity with distant metastasis.

Inhibitor-binding mode of homobelactosin C to proteasomes: New insights into class I MHC ligand generation

PubMed Central

Groll, Michael; Larionov, Oleg V.; Huber, Robert; de Meijere, Armin

2006-01-01

Most class I MHC ligands are generated from the vast majority of cellular proteins by proteolysis within the ubiquitin–proteasome pathway and are presented on the cell surface by MHC class I molecules. Here, we present the crystallographic analysis of yeast 20S proteasome in complex with the inhibitor homobelactosin C. The structure reveals a unique inhibitor-binding mode and provides information about the composition of proteasomal primed substrate-binding sites. IFN-γ inducible substitution of proteasomal constitutive subunits by immunosubunits modulates characteristics of generated peptides, thus producing fragments with higher preference for binding to MHC class I molecules. The structural data for the proteasome:homobelactosin C complex provide an explanation for involvement of immunosubunits in antigen generation and open perspectives for rational design of ligands, inhibiting exclusively constitutive proteasomes or immunoproteasomes. PMID:16537370

Upregulation of PSMB4 is Associated with the Necroptosis after Spinal Cord Injury.

PubMed

Wu, Chunshuai; Chen, Jiajia; Liu, Yonghua; Zhang, Jinlong; Ding, Wensen; Wang, Song; Bao, Guofeng; Xu, Guanhua; Sun, Yuyu; Wang, Lingling; Chen, Limin; Gu, Haiyan; Cui, Baihong; Cui, Zhiming

2016-11-01

Spinal cord injury (SCI) is one of the most common and severe complications in spine injury. It is difficult to prevent cell necroptosis and promote the survival of residual neurons after SCI. Proteasome beta-4 subunit (PSMB4) is the first proteasomal subunit with oncogenic properties promoting cancer cell survival and tumor growth in vivo, and our previous study showed that PSMB4 is significantly associated with neuronal apoptosis in neuroinflammation. However, PSMB4 function in the necroptosis after SCI is unkown. RIP3, a key regulatory factor of necroptosis, correlates with the induction of necroptosis in various types of cells and signaling pathway. Upregulation of the RIP3 expression may play a role as a novel molecular mechanism in secondary neural tissue damage following SCI. In this study, we established an acute spinal cord contusion injury model in adult rats to investigate the potential role of PSMB4 during the pathological process of SCI. We found PSMB4 expression was significantly up-regulated 3 days after injury by western blot and immunohistochemical staining. Double immunofluorescent staining indicated obvious changes of PSMB4 expression occurred in neurons. Significant up-regulation of PSMB4 expression was observed in Rip3 positive neurons at 3 days after SCI, which indicated that PSMB4 might play a vital role in the regulation of Rip3. Overexpress and knockdown PSMB4 could intervene the RIP3 and Mixed lineage kinase domain-like protein (MLKL) pathway in Tumor necrosis factor-α (TNF-α) induced necroptosis cell model. Based on our experimental data, we boldly conclude that PSMB4 is associated with RIP3 involved necroptosis after SCI.

Denervation-Induced Activation of the Ubiquitin-Proteasome System Reduces Skeletal Muscle Quantity Not Quality.

PubMed

Baumann, Cory W; Liu, Haiming M; Thompson, LaDora V

2016-01-01

It is well known that the ubiquitin-proteasome system is activated in response to skeletal muscle wasting and functions to degrade contractile proteins. The loss of these proteins inevitably reduces skeletal muscle size (i.e., quantity). However, it is currently unknown whether activation of this pathway also affects function by impairing the muscle's intrinsic ability to produce force (i.e., quality). Therefore, the purpose of this study was twofold, (1) document how the ubiquitin-proteasome system responds to denervation and (2) identify the physiological consequences of these changes. To induce soleus muscle atrophy, C57BL6 mice underwent tibial nerve transection of the left hindlimb for 7 or 14 days (n = 6-8 per group). At these time points, content of several proteins within the ubiquitin-proteasome system were determined via Western blot, while ex vivo whole muscle contractility was specifically analyzed at day 14. Denervation temporarily increased several key proteins within the ubiquitin-proteasome system, including the E3 ligase MuRF1 and the proteasome subunits 19S, α7 and β5. These changes were accompanied by reductions in absolute peak force and power, which were offset when expressed relative to physiological cross-sectional area. Contrary to peak force, absolute and relative forces at submaximal stimulation frequencies were significantly greater following 14 days of denervation. Taken together, these data represent two keys findings. First, activation of the ubiquitin-proteasome system is associated with reductions in skeletal muscle quantity rather than quality. Second, shortly after denervation, it appears the muscle remodels to compensate for the loss of neural activity via changes in Ca2+ handling.

Crystal structure of human proteasome assembly chaperone PAC4 involved in proteasome formation.

PubMed

Kurimoto, Eiji; Satoh, Tadashi; Ito, Yuri; Ishihara, Eri; Okamoto, Kenta; Yagi-Utsumi, Maho; Tanaka, Keiji; Kato, Koichi

2017-05-01

The 26S proteasome is a large protein complex, responsible for degradation of ubiquinated proteins in eukaryotic cells. Eukaryotic proteasome formation is a highly ordered process that is assisted by several assembly chaperones. The assembly of its catalytic 20S core particle depends on at least five proteasome-specific chaperones, i.e., proteasome-assembling chaperons 1-4 (PAC1-4) and proteasome maturation protein (POMP). The orthologues of yeast assembly chaperones have been structurally characterized, whereas most mammalian assembly chaperones are not. In the present study, we determined a crystal structure of human PAC4 at 1.90-Å resolution. Our crystallographic data identify a hydrophobic surface that is surrounded by charged residues. The hydrophobic surface is complementary to that of its binding partner, PAC3. The surface also exhibits charge complementarity with the proteasomal α4-5 subunits. This will provide insights into human proteasome-assembling chaperones as potential anticancer drug targets. © 2017 The Protein Society.

Targeting proteasomes in infectious organisms to combat disease.

PubMed

Bibo-Verdugo, Betsaida; Jiang, Zhenze; Caffrey, Conor R; O'Donoghue, Anthony J

2017-05-01

Proteasomes are multisubunit, energy-dependent, proteolytic complexes that play an essential role in intracellular protein turnover. They are present in eukaryotes, archaea, and in some actinobacteria species. Inhibition of proteasome activity has emerged as a powerful strategy for anticancer therapy and three drugs have been approved for treatment of multiple myeloma. These compounds react covalently with a threonine residue located in the active site of a proteasome subunit to block protein degradation. Proteasomes in pathogenic organisms such as Mycobacterium tuberculosis and Plasmodium falciparum also have a nucleophilic threonine residue in the proteasome active site and are therefore sensitive to these anticancer drugs. This review summarizes efforts to validate the proteasome in pathogenic organisms as a therapeutic target. We describe several strategies that have been used to develop inhibitors with increased potency and selectivity for the pathogen proteasome relative to the human proteasome. In addition, we highlight a cell-based chemical screening approach that identified a potent, allosteric inhibitor of proteasomes found in Leishmania and Trypanosoma species. Finally, we discuss the development of proteasome inhibitors as anti-infective agents. © 2017 Federation of European Biochemical Societies.

Insulin alleviates degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system in septic rats.

PubMed

Chen, Qiyi; Li, Ning; Zhu, Weiming; Li, Weiqin; Tang, Shaoqiu; Yu, Wenkui; Gao, Tao; Zhang, Juanjuan; Li, Jieshou

2011-06-03

Hypercatabolism is common under septic conditions. Skeletal muscle is the main target organ for hypercatabolism, and this phenomenon is a vital factor in the deterioration of recovery in septic patients. In skeletal muscle, activation of the ubiquitin-proteasome system plays an important role in hypercatabolism under septic status. Insulin is a vital anticatabolic hormone and previous evidence suggests that insulin administration inhibits various steps in the ubiquitin-proteasome system. However, whether insulin can alleviate the degradation of skeletal muscle protein by inhibiting the ubiquitin-proteasome system under septic condition is unclear. This paper confirmed that mRNA and protein levels of the ubiquitin-proteasome system were upregulated and molecular markers of skeletal muscle proteolysis (tyrosine and 3-methylhistidine) simultaneously increased in the skeletal muscle of septic rats. Septic rats were infused with insulin at a constant rate of 2.4 mU.kg-1.min-1 for 8 hours. Concentrations of mRNA and proteins of the ubiquitin-proteasome system and molecular markers of skeletal muscle proteolysis were mildly affected. When the insulin infusion dose increased to 4.8 mU.kg-1.min-1, mRNA for ubiquitin, E2-14 KDa, and the C2 subunit were all sharply downregulated. At the same time, the levels of ubiquitinated proteins, E2-14KDa, and the C2 subunit protein were significantly reduced. Tyrosine and 3-methylhistidine decreased significantly. We concluded that the ubiquitin-proteasome system is important skeletal muscle hypercatabolism in septic rats. Infusion of insulin can reverse the detrimental metabolism of skeletal muscle by inhibiting the ubiquitin-proteasome system, and the effect is proportional to the insulin infusion dose.

Role of Rad23 and Dsk2 in Nucleotide Excision Repair and Spindle Pole Body Duplication

DTIC Science & Technology

2007-03-01

proteasome. Mol. Cell, 6, 409–419. 14. Saeki, Y., Saitoh, A., Toh-e, A. & Yokosawa , H. (2002). Ubiquitin-like proteins and Rpn10 play cooperative...Sone, T., Toh-e, A. & Yokosawa , H. (2002). Identification of ubiquitin-like protein-binding sub- units of the 26 S proteasome. Biochem. Biophys. Res...Proteasome subunit Rpn1 binds ubiquitin-like protein domains. Nat Cell Biol 2002, 4:725-30. 15. Saeki Y, Sone T, Toh-e A, Yokosawa H: Identification of

The Green Tea Polyphenol Epigallocatechin-3-Gallate (EGCg) Attenuates Skeletal Muscle Atrophy in a Rat Model of Sarcopenia.

PubMed

Meador, B M; Mirza, K A; Tian, M; Skelding, M B; Reaves, L A; Edens, N K; Tisdale, M J; Pereira, S L

2015-01-01

Sarcopenia-the loss of muscle mass and functionality occurring with age-is a pervasive problem with few effective treatments beyond exercise. We examined the ability of the green tea catechin, epigallocatechin-3-gallate (EGCg), to impact muscle mass and the molecular pathway involved in muscle atrophy in a rat model of sarcopenia. 20-month-old Sprague-Dawley rats were treated for 8 weeks with control diet or control plus 200mg/kg body weight of EGCg diet. EGCg-supplemented animals had significantly greater gastrocnemius muscle mass than the aged controls, and showed a trend for increased muscle fiber cross-sectional areas (CSA) (p=0.06). These changes were associated with significantly lower protein expressions of the intramuscular 19S and 20S proteasome subunits and the MuRF1 and MAFbx ubiquitin ligases in the EGCg-treated animals. Proteasome activity as determined by 'Chymotrypsin-like' enzyme activity was also significantly reduced by EGCg. Muscle mRNA expression of IL-15 and IGF-1 were significantly increased in the EGCg group vs. the aged controls. In comparison to younger adult animals (6 month), the protein expression of 19S, 20S, MuRF1, MAFbx, and myostatin were increased between approximately 4- and 12-fold in the aged controls, but only up to ~2-fold in the aged EGCg animals. EGCg supplementation was able to preserve muscle in sarcopenic rats, partly through attenuating protein degradation via the ubiquitin-proteasome pathway, together with increased expression of anabolic factors.

Integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity

PubMed Central

Wang, Evelyn W.; Kessler, Benedikt M.; Borodovsky, Anna; Cravatt, Benjamin F.; Bogyo, Matthew; Ploegh, Hidde L.; Glas, Rickard

2000-01-01

Cytosolic proteolysis is carried out predominantly by the proteasome. We show that a large oligopeptidase, tripeptidylpeptidase II (TPPII), can compensate for compromised proteasome activity. Overexpression of TPPII is sufficient to prevent accumulation of polyubiquitinated proteins and allows survival of EL-4 cells at otherwise lethal concentrations of the covalent proteasome inhibitor NLVS (NIP-leu-leu-leu-vinylsulfone). Elevated TPPII activity also partially restores peptide loading of MHC molecules. Purified proteasomes from adapted cells lack the chymotryptic-like activity, but still degrade longer peptide substrates via residual activity of their Z subunits. However, growth of adapted cells depends on induction of other proteolytic activities. Therefore, cytosolic oligopeptidases such as TPPII normalize rates of intracellular protein breakdown required for normal cellular function and viability. PMID:10954757

[A new mechanism of ubiquitin-dependent proteolytic pathway--polyubiquitin chain recognition and proteasomal targeting].

PubMed

Kawahara, Hiroyuki; Yokosawa, Hideyoshi

2008-01-01

The RPN10 subunit of 26S proteasome and several UBA domain proteins can bind to the polyubiquitin chain and play a role as ubiquitin receptors of the 26S proteasome. Although it was thought that substrate recognition is an essential step in the proteasome-mediated protein degradation, deletion of rpn10 genes in yeast does not influence the viability of cells but instead causes only a mild phenotype, suggesting that the above ubiquitin receptors are redundantly involved in substrate delivery to the proteasome. However, their functional difference is still enigmatic. In this review, we summarize recent advances in polyubiquitin chain recognition/delivery system and provide potential applications to modulate this system as a probable target for drug development.

Immunoproteasome subunit ß5i/LMP7-deficiency in atherosclerosis.

PubMed

Hewing, Bernd; Ludwig, Antje; Dan, Cristian; Pötzsch, Max; Hannemann, Carmen; Petry, Andreas; Lauer, Dilyara; Görlach, Agnes; Kaschina, Elena; Müller, Dominik N; Baumann, Gert; Stangl, Verena; Stangl, Karl; Wilck, Nicola

2017-10-17

Management of protein homeostasis by the ubiquitin-proteasome system is critical for atherosclerosis development. Recent studies showed controversial results on the role of immunoproteasome (IP) subunit β5i/LMP7 in maintenance of protein homeostasis under cytokine induced oxidative stress. The present study aimed to investigate the effect of β5i/LMP7-deficiency on the initiation and progression of atherosclerosis as a chronic inflammatory, immune cell driven disease. LDLR -/- LMP7 -/- and LDLR -/- mice were fed a Western-type diet for either 6 or 24 weeks to induce early and advanced stage atherosclerosis, respectively. Lesion burden was similar between genotypes in both stages. Macrophage content and abundance of polyubiquitin conjugates in aortic root plaques were unaltered by β5i/LMP7-deficiency. In vitro experiments using bone marrow-derived macrophages (BMDM) showed that β5i/LMP7-deficiency did not influence macrophage polarization or accumulation of polyubiquitinated proteins and cell survival upon hydrogen peroxide and interferon-γ treatment. Analyses of proteasome core particle composition by Western blot revealed incorporation of standard proteasome subunits in β5i/LMP7-deficient BMDM and spleen. Chymotrypsin-, trypsin- and caspase-like activities assessed by using short fluorogenic peptides in BMDM whole cell lysates were similar in both genotypes. Taken together, deficiency of IP subunit β5i/LMP7 does not disturb protein homeostasis and does not aggravate atherogenesis in LDLR -/- mice.

Changes in proteasome structure and function caused by HAMLET in tumor cells.

PubMed

Gustafsson, Lotta; Aits, Sonja; Onnerfjord, Patrik; Trulsson, Maria; Storm, Petter; Svanborg, Catharina

2009-01-01

Proteasomes control the level of endogenous unfolded proteins by degrading them in the proteolytic core. Insufficient degradation due to altered protein structure or proteasome inhibition may trigger cell death. This study examined the proteasome response to HAMLET, a partially unfolded protein-lipid complex, which is internalized by tumor cells and triggers cell death. HAMLET bound directly to isolated 20S proteasomes in vitro and in tumor cells significant co-localization of HAMLET and 20S proteasomes was detected by confocal microscopy. This interaction was confirmed by co-immunoprecipitation from extracts of HAMLET-treated tumor cells. HAMLET resisted in vitro degradation by proteasomal enzymes and degradation by intact 20S proteasomes was slow compared to fatty acid-free, partially unfolded alpha-lactalbumin. After a brief activation, HAMLET inhibited proteasome activity in vitro and in parallel a change in proteasome structure occurred, with modifications of catalytic (beta1 and beta5) and structural subunits (alpha2, alpha3, alpha6 and beta3). Proteasome inhibition was confirmed in extracts from HAMLET-treated cells and there were indications of proteasome fragmentation in HAMLET-treated cells. The results suggest that internalized HAMLET is targeted to 20S proteasomes, that the complex resists degradation, inhibits proteasome activity and perturbs proteasome structure. We speculate that perturbations of proteasome structure might contribute to the cytotoxic effects of unfolded protein complexes that invade host cells.

Early cysteine-dependent inactivation of 26S proteasomes does not involve particle disassembly.

PubMed

Hugo, Martín; Korovila, Ioanna; Köhler, Markus; García-García, Carlos; Cabrera-García, J Daniel; Marina, Anabel; Martínez-Ruiz, Antonio; Grune, Tilman

2018-06-01

Under oxidative stress 26S proteasomes suffer reversible disassembly into its 20S and 19S subunits, a process mediated by HSP70. This inhibits the degradation of polyubiquitinated proteins by the 26S proteasome and allows the degradation of oxidized proteins by a free 20S proteasome. Low fluxes of antimycin A-stimulated ROS production caused dimerization of mitochondrial peroxiredoxin 3 and cytosolic peroxiredoxin 2, but not peroxiredoxin overoxidation and overall oxidation of cellular protein thiols. This moderate redox imbalance was sufficient to inhibit the ATP stimulation of 26S proteasome activity. This process was dependent on reversible cysteine oxidation. Moreover, our results show that this early inhibition of ATP stimulation occurs previous to particle disassembly, indicating an intermediate step during the redox regulation of the 26S proteasome with special relevance under redox signaling rather than oxidative stress conditions. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Validation of the proteasome as a therapeutic target in Plasmodium using an epoxyketone inhibitor with parasite-specific toxicity

PubMed Central

Li, Hao; Ponder, Elizabeth L.; Verdoes, Martijn; Asbjornsdottir, Kristijana H.; Deu, Edgar; Edgington, Laura E.; Lee, Jeong Tae; Kirk, Christopher J.; Demo, Susan D.; Williamson, Kim C.; Bogyo, Matthew

2012-01-01

Summary The Plasmodium proteasome has been suggested to be a potential anti-malarial drug target, however toxicity of inhibitors has prevented validation of this enzyme in vivo. We report here a screen of a library of 670 analogs of the recently FDA approved inhibitor, carfilzomib, to identify compounds that selectively kill parasites. We identified one compound, PR3, that has significant parasite killing activity in vitro but dramatically reduced toxicity in host cells. We found that this parasite-specific toxicity is not due to selective targeting of the Plasmodium proteasome over the host proteasome, but instead is due to a lack of activity against one of the human proteasome subunits. Subsequently, we used PR3 to significantly reduce parasite load in P. berghei infected mice without host toxicity, thus validating the proteasome as a viable anti-malarial drug target. PMID:23142757

Proteasome subunit Rpn13 is a novel ubiquitin receptor

PubMed Central

Husnjak, Koraljka; Elsasser, Suzanne; Zhang, Naixia; Chen, Xiang; Randles, Leah; Shi, Yuan; Hofmann, Kay; Walters, Kylie; Finley, Daniel; Dikic, Ivan

2010-01-01

Proteasomal receptors that recognize ubiquitin chains attached to substrates are key mediators of selective protein degradation in eukaryotes. Here we report the identification of a new ubiquitin receptor, Rpn13/ARM1, a known component of the proteasome. Rpn13 binds ubiquitin via a conserved N-terminal region termed the Pru domain (Pleckstrin-like receptor for ubiquitin), which binds K48-linked diubiquitin with an affinity of ∼90 nM. Like proteasomal ubiquitin receptor Rpn10/S5a, Rpn13 also binds ubiquitin-like domains of the UBL/UBA family of ubiquitin receptors. A synthetic phenotype results in yeast when specific mutations of the ubiquitin binding sites of Rpn10 and Rpn13 are combined, indicating functional linkage between these ubiquitin receptors. Since Rpn13 is also the proteasomal receptor for Uch37, a deubiquitinating enzyme, our findings suggest a coupling of chain recognition and disassembly at the proteasome. PMID:18497817

Pathophysiological roles of aldo-keto reductases (AKR1C1 and AKR1C3) in development of cisplatin resistance in human colon cancers.

PubMed

Matsunaga, Toshiyuki; Hojo, Aki; Yamane, Yumi; Endo, Satoshi; El-Kabbani, Ossama; Hara, Akira

2013-02-25

Cisplatin (cis-diamminedichloroplatinum, CDDP) is widely used for treatment of patients with solid tumors formed in various organs including the lung, prostate and cervix, but is much less sensitive in colon and breast cancers. One major factor implicated in the ineffectiveness has been suggested to be acquisition of the CDDP resistance. Here, we established the CDDP-resistant phenotypes of human colon HCT15 cells by continuously exposing them to incremental concentrations of the drug, and monitored expressions of aldo-keto reductases (AKRs) 1A1, 1B1, 1B10, 1C1, 1C2 and 1C3. Among the six AKRs, AKR1C1 and AKR1C3 are highly induced with the CDDP resistance. The resistance lowered the sensitivity toward cellular damages evoked by oxidative stress-derived aldehydes, 4-hydroxy-2-nonenal and 4-oxo-2-nonenal that are detoxified by AKR1C1 and AKR1C3. Overexpression of AKR1C1 or AKR1C3 in the parental HCT15 cells mitigated the cytotoxicity of the aldehydes and CDDP. Knockdown of both AKR1C1 and AKR1C3 in the resistant cells or treatment of the cells with specific inhibitors of the AKRs increased the sensitivity to CDDP toxicity. Thus, the two AKRs participate in the mechanism underlying the CDDP resistance probably via detoxification of the aldehydes resulting from enhanced oxidative stress. The resistant cells also showed an enhancement in proteolytic activity of proteasome accompanied by overexpression of its catalytic subunits (PSMβ9 and PSMβ10). Pretreatment of the resistant cells with a potent proteasome inhibitor Z-Leu-Leu-Leu-al augmented the CDDP sensitization elicited by the AKR inhibitors. Additionally, the treatment of the cells with Z-Leu-Leu-Leu-al and the AKR inhibitors induced the expressions of the two AKRs and proteasome subunits. Collectively, these results suggest the involvement of up-regulated AKR1C1, AKR1C3 and proteasome in CDDP resistance of colon cancers and support a chemotherapeutic role for their inhibitors. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Regulation of the Proteasome by Neuronal Activity and Calcium/Calmodulin-dependent Protein Kinase II*

PubMed Central

Djakovic, Stevan N.; Schwarz, Lindsay A.; Barylko, Barbara; DeMartino, George N.; Patrick, Gentry N.

2009-01-01

Protein degradation via the ubiquitin proteasome system has been shown to regulate changes in synaptic strength that underlie multiple forms of synaptic plasticity. It is plausible, therefore, that the ubiquitin proteasome system is itself regulated by synaptic activity. By utilizing live-cell imaging strategies we report the rapid and dynamic regulation of the proteasome in hippocampal neurons by synaptic activity. We find that the blockade of action potentials (APs) with tetrodotoxin inhibited the activity of the proteasome, whereas the up-regulation of APs with bicuculline dramatically increased the activity of the proteasome. In addition, the regulation of the proteasome is dependent upon external calcium entry in part through N-methyl-d-aspartate receptors and L-type voltage-gated calcium channels and requires the activity of calcium/calmodulin-dependent protein kinase II (CaMKII). Using in vitro and in vivo assays we find that CaMKII stimulates proteasome activity and directly phosphorylates Rpt6, a subunit of the 19 S (PA700) subcomplex of the 26 S proteasome. Our data provide a novel mechanism whereby CaMKII may regulate the proteasome in neurons to facilitate remodeling of synaptic connections through protein degradation. PMID:19638347

Regulation of the proteasome by neuronal activity and calcium/calmodulin-dependent protein kinase II.

PubMed

Djakovic, Stevan N; Schwarz, Lindsay A; Barylko, Barbara; DeMartino, George N; Patrick, Gentry N

2009-09-25

Protein degradation via the ubiquitin proteasome system has been shown to regulate changes in synaptic strength that underlie multiple forms of synaptic plasticity. It is plausible, therefore, that the ubiquitin proteasome system is itself regulated by synaptic activity. By utilizing live-cell imaging strategies we report the rapid and dynamic regulation of the proteasome in hippocampal neurons by synaptic activity. We find that the blockade of action potentials (APs) with tetrodotoxin inhibited the activity of the proteasome, whereas the up-regulation of APs with bicuculline dramatically increased the activity of the proteasome. In addition, the regulation of the proteasome is dependent upon external calcium entry in part through N-methyl-D-aspartate receptors and L-type voltage-gated calcium channels and requires the activity of calcium/calmodulin-dependent protein kinase II (CaMKII). Using in vitro and in vivo assays we find that CaMKII stimulates proteasome activity and directly phosphorylates Rpt6, a subunit of the 19 S (PA700) subcomplex of the 26 S proteasome. Our data provide a novel mechanism whereby CaMKII may regulate the proteasome in neurons to facilitate remodeling of synaptic connections through protein degradation.

Conjugated linoleic acid synthesis-related protein proteasome subunit α 5 (PSMA5) is increased by vaccenic acid treatment in goat mammary tissue.

PubMed

Jin, Y C; Li, Z H; Hong, Z S; Xu, C X; Han, J A; Choi, S H; Yin, J L; Zhang, Q K; Lee, K B; Kang, S K; Song, M K; Kim, Y J; Kang, H S; Choi, Y J; Lee, H G

2012-08-01

This study was conducted to identify proteins associated with the endogenous synthesis of conjugated linoleic acid (CLA) from trans-vaccenic acid (TVA; trans-11 C18:1, a precursor for CLA endogenous synthesis) in mammary tissues. Six lactating goats were divided into 2 groups. One group was given an intravenous bolus injection of TVA (150mg) twice daily over 4 d; the other group received saline injections. Treatment with TVA increased the concentration of cis-9,trans-11 CLA and TVA in goat milk. Additionally, TVA treatment increased the expression of stearoyl-CoA desaturase (SCD) in mammary tissue. Using 2-dimensional gel electrophoresis and electrospray ionization quadrupole time-of-flight mass spectrometry, 3 proteins affected by infusions of TVA were identified. Proteasome (prosome, macropain) subunit α type 5 (PSMA5) was upregulated, whereas peroxiredoxin-1 and translationally controlled tumor protein 1 were downregulated in TVA-treated animals compared with the vehicle-injected controls. Only the effect of TVA on PSMA5 could be confirmed by Western blot analysis. To further explore the regulation of PSMA5 in mammary epithelial cells when TVA is converted into CLA, we used a differentiated bovine mammary epithelial cell line treated with TVA for 6h. Changes in cis-9,trans-11 CLA concentrations and mRNA expression patterns of both SCD and PSMA5 were monitored. The concentration of cis-9,trans-11 CLA increased after TVA treatment. The mRNA expression level of PSMA5 was significantly elevated to 6h, but SCD mRNA expression only increased in 2h after TVA treatment. These results indicate that PSMA5 is highly expressed in goat mammary tissue and bovine mammary epithelial cells when TVA is converted into CLA. Our data suggest that PSMA5 protein is associated with CLA biosynthesis in mammary tissue. Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Immunoproteasome Overexpression Underlies the Pathogenesis of Thyroid Oncocytes and Primary Hypothyroidism: Studies in Humans and Mice

PubMed Central

Kimura, Hiroaki J.; Chen, Cindy Y.; Tzou, Shey-Cherng; Rocchi, Roberto; Landek-Salgado, Melissa A.; Suzuki, Koichi; Kimura, Miho; Rose, Noel R.; Caturegli, Patrizio

2009-01-01

Background Oncocytes of the thyroid gland (Hürthle cells) are found in tumors and autoimmune diseases. They have a unique appearance characterized by abundant granular eosinophilic cytoplasm and hyperchromatic nucleus. Their pathogenesis has remained, thus far, unknown. Methodology/Principal Findings Using transgenic mice chronically expressing IFNγ in thyroid gland, we showed changes in the thyroid follicular epithelium reminiscent of the human oncocyte. Transcriptome analysis comparing transgenic to wild type thyrocytes revealed increased levels of immunoproteasome subunits like LMP2 in transgenics, suggesting an important role of the immunoproteasome in oncocyte pathogenesis. Pharmacologic blockade of the proteasome, in fact, ameliorated the oncocytic phenotype. Genetic deletion of LMP2 subunit prevented the development of the oncocytic phenotype and primary hypothyroidism. LMP2 was also found expressed in oncocytes from patients with Hashimoto thyroiditis and Hürthle cell tumors. Conclusions/Significance In summary, we report that oncocytes are the result of an increased immunoproteasome expression secondary to a chronic inflammatory milieu, and suggest LMP2 as a novel therapeutic target for the treatment of oncocytic lesions and autoimmune hypothyroidism. PMID:19924240

Activation of the Yeast UBI4 Polyubiquitin Gene by Zap1 Transcription Factor via an Intragenic Promoter Is Critical for Zinc-deficient Growth*

PubMed Central

MacDiarmid, Colin W.; Taggart, Janet; Jeong, Jeeyon; Kerdsomboon, Kittikhun; Eide, David J.

2016-01-01

Stability of many proteins requires zinc. Zinc deficiency disrupts their folding, and the ubiquitin-proteasome system may help manage this stress. In Saccharomyces cerevisiae, UBI4 encodes five tandem ubiquitin monomers and is essential for growth in zinc-deficient conditions. Although UBI4 is only one of four ubiquitin-encoding genes in the genome, a dramatic decrease in ubiquitin was observed in zinc-deficient ubi4Δ cells. The three other ubiquitin genes were strongly repressed under these conditions, contributing to the decline in ubiquitin. In a screen for ubi4Δ suppressors, a hypomorphic allele of the RPT2 proteasome regulatory subunit gene (rpt2E301K) suppressed the ubi4Δ growth defect. The rpt2E301K mutation also increased ubiquitin accumulation in zinc-deficient cells, and by using a ubiquitin-independent proteasome substrate we found that proteasome activity was reduced. These results suggested that increased ubiquitin supply in suppressed ubi4Δ cells was a consequence of more efficient ubiquitin release and recycling during proteasome degradation. Degradation of a ubiquitin-dependent substrate was restored by the rpt2E301K mutation, indicating that ubiquitination is rate-limiting in this process. The UBI4 gene was induced ∼5-fold in low zinc and is regulated by the zinc-responsive Zap1 transcription factor. Surprisingly, Zap1 controls UBI4 by inducing transcription from an intragenic promoter, and the resulting truncated mRNA encodes only two of the five ubiquitin repeats. Expression of a short transcript alone complemented the ubi4Δ mutation, indicating that it is efficiently translated. Loss of Zap1-dependent UBI4 expression caused a growth defect in zinc-deficient conditions. Thus, the intragenic UBI4 promoter is critical to preventing ubiquitin deficiency in zinc-deficient cells. PMID:27432887

Tau protein degradation is catalyzed by the ATP/ubiquitin-independent 20S proteasome under normal cell conditions

PubMed Central

Grune, Tilman; Botzen, Diana; Engels, Martina; Voss, Peter; Kaiser, Barbara; Jung, Tobias; Grimm, Stefanie; Ermak, Gennady; Davies, Kelvin J. A.

2010-01-01

Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degradation has also been postulated during stress. In the current studies, we utilized HT22 cells and tau-transfected E36 cells in order to test the relative importance or possible requirement of the ubiquitin-dependent 26S proteasomal system versus the ubiquitin-independent 20S proteasome, in tau degradation. By means of ATP-depletion, ubiquitinylation-deficient E36ts20 cells, a 19S proteasomal regulator subunit MSS1-siRNA approaches, and in vitro ubiquitinylation studies, we were able to demonstrate that ubiquitinylation is not required for normal tau degradation. PMID:20478262

Parkin binds the Rpn10 subunit of 26S proteasomes through its ubiquitin-like domain

PubMed Central

Sakata, Eri; Yamaguchi, Yoshiki; Kurimoto, Eiji; Kikuchi, Jun; Yokoyama, Shigeyuki; Yamada, Shingo; Kawahara, Hiroyuki; Yokosawa, Hideyoshi; Hattori, Nobutaka; Mizuno, Yoshikuni; Tanaka, Keiji; Kato, Koichi

2003-01-01

Parkin, a product of the causative gene of autosomal-recessive juvenile parkinsonism (AR-JP), is a RING-type E3 ubiquitin ligase and has an amino-terminal ubiquitin-like (Ubl) domain. Although a single mutation that causes an Arg to Pro substitution at position 42 of the Ubl domain (the Arg 42 mutation) has been identified in AR-JP patients, the function of this domain is not clear. In this study, we determined the three-dimensional structure of the Ubl domain of parkin by NMR, in particular by extensive use of backbone 15N-1H residual dipolar-coupling data. Inspection of chemical-shift-perturbation data showed that the parkin Ubl domain binds the Rpn10 subunit of 26S proteasomes via the region of parkin that includes position 42. Our findings suggest that the Arg 42 mutation induces a conformational change in the Rpn10-binding site of Ubl, resulting in impaired proteasomal binding of parkin, which could be the cause of AR-JP. PMID:12634850

Functional characterization of rpn3 uncovers a distinct 19S proteasomal subunit requirement for ubiquitin-dependent proteolysis of cell cycle regulatory proteins in budding yeast.

PubMed

Bailly, E; Reed, S I

1999-10-01

By selectively eliminating ubiquitin-conjugated proteins, the 26S proteasome plays a pivotal role in a large variety of cellular regulatory processes, particularly in the control of cell cycle transitions. Access of ubiquitinated substrates to the inner catalytic chamber within the 20S core particle is mediated by the 19S regulatory particle (RP), whose subunit composition in budding yeast has been recently elucidated. In this study, we have investigated the cell cycle defects resulting from conditional inactivation of one of these RP components, the essential non-ATPase Rpn3/Sun2 subunit. Using temperature-sensitive mutant alleles, we show that rpn3 mutations do not prevent the G(1)/S transition but cause a metaphase arrest, indicating that the essential Rpn3 function is limiting for mitosis. rpn3 mutants appear severely compromised in the ubiquitin-dependent proteolysis of several physiologically important proteasome substrates. Thus, RPN3 function is required for the degradation of the G(1)-phase cyclin Cln2 targeted by SCF; the S-phase cyclin Clb5, whose ubiquitination is likely to involve a combination of E3 (ubiquitin protein ligase) enzymes; and anaphase-promoting complex targets, such as the B-type cyclin Clb2 and the anaphase inhibitor Pds1. Our results indicate that the Pds1 degradation defect of the rpn3 mutants most likely accounts for the metaphase arrest phenotype observed. Surprisingly, but consistent with the lack of a G(1) arrest phenotype in thermosensitive rpn3 strains, the Cdk inhibitor Sic1 exhibits a short half-life regardless of the RPN3 genotype. In striking contrast, Sic1 turnover is severely impaired by a temperature-sensitive mutation in RPN12/NIN1, encoding another essential RP subunit. While other interpretations are possible, these data strongly argue for the requirement of distinct RP subunits for efficient proteolysis of specific cell cycle regulators. The potential implications of these data are discussed in the context of possible Rpn3 function in multiubiquitin-protein conjugate recognition by the 19S proteasomal regulatory particle.

Cables1 controls p21/Cip1 protein stability by antagonizing proteasome subunit alpha type 3.

PubMed

Shi, Z; Li, Z; Li, Z J; Cheng, K; Du, Y; Fu, H; Khuri, F R

2015-05-07

The cyclin-dependent kinase (CDK) inhibitor 1A, p21/Cip1, is a vital cell cycle regulator, dysregulation of which has been associated with a large number of human malignancies. One critical mechanism that controls p21 function is through its degradation, which allows the activation of its associated cell cycle-promoting kinases, CDK2 and CDK4. Thus delineating how p21 is stabilized and degraded will enhance our understanding of cell growth control and offer a basis for potential therapeutic interventions. Here we report a novel regulatory mechanism that controls the dynamic status of p21 through its interaction with Cdk5 and Abl enzyme substrate 1 (Cables1). Cables1 has a proposed role as a tumor suppressor. We found that upregulation of Cables1 protein was correlated with increased half-life of p21 protein, which was attributed to Cables1/p21 complex formation and supported by their co-localization in the nucleus. Mechanistically, Cables1 interferes with the proteasome (Prosome, Macropain) subunit alpha type 3 (PSMA3) binding to p21 and protects p21 from PSMA3-mediated proteasomal degradation. Moreover, silencing of p21 partially reverses the ability of Cables1 to induce cell death and inhibit cell proliferation. In further support of a potential pathophysiological role of Cables1, the expression level of Cables1 is tightly associated with p21 in both cancer cell lines and human lung cancer patient tumor samples. Together, these results suggest Cables1 as a novel p21 regulator through maintaining p21 stability and support the model that the tumor-suppressive function of Cables1 occurs at least in part through enhancing the tumor-suppressive activity of p21.

Rpn9 Is Required for Efficient Assembly of the Yeast 26S Proteasome

PubMed Central

Takeuchi, Junko; Fujimuro, Masahiro; Yokosawa, Hideyosi; Tanaka, Keiji; Toh-e, Akio

1999-01-01

We have isolated the RPN9 gene by two-hybrid screening with, as bait, RPN10 (formerly SUN1), which encodes a multiubiquitin chain receptor residing in the regulatory particle of the 26S proteasome. Rpn9 is a nonessential subunit of the regulatory particle of the 26S proteasome, but the deletion of this gene results in temperature-sensitive growth. At the restrictive temperature, the Δrpn9 strain accumulated multiubiquitinated proteins, indicating that the RPN9 function is needed for the 26S proteasome activity at a higher temperature. We analyzed the proteasome fractions separated by glycerol density gradient centrifugation by native polyacrylamide gel electrophoresis and found that a smaller amount of the 26S proteasome was produced in the Δrpn9 cells and that the 26S proteasome was shifted to lighter fractions than expected. The incomplete proteasome complexes were found to accumulate in the Δrpn9 cells. Furthermore, Rpn10 was not detected in the fractions containing proteasomes of the Δrpn9 cells. These results indicate that Rpn9 is needed for incorporating Rpn10 into the 26S proteasome and that Rpn9 participates in the assembly and/or stability of the 26S proteasome. PMID:10490597

Trefoil factor 2 (TFF2) deficiency in murine digestive tract influences the immune system.

PubMed

Baus-Loncar, Mirela; Schmid, Janinne; Lalani, El-Nasir; Rosewell, Ian; Goodlad, Robert A; Stamp, Gordon W H; Blin, Nikolaus; Kayademir, Tuncay

2005-01-01

The gastrointestinal trefoil factor family (TFF1, TFF2, TFF3) peptides are considered to play an important role in maintaining the integrity of the mucosa. The physiological role of TFF2 in the protection of the GI tract was investigated in TFF2 deficiency. TFF2-/- mice were generated and differential expression of various genes was assessed by using a mouse expression microarray, quantitative real time PCR, Northern blots or immunohistochemistry. On an mRNA level we found 128 differentially expressed genes. We observed modulation of a number of crucial genes involved in innate and adaptive immunity in the TFF2-/- mice. Expression of proteasomal subunits genes (LMP2, LMP7 and PSMB5) involved in the MHC class I presentation pathway were modulated indicating the formation of immunoproteasomes improving antigen presentation. Expression of one subunit of a transporter (TAP1) responsible for importing degraded antigens into ER was increased, similarly to the BAG2 gene that modulates chaperone activity in ER helping proper loading on MHC class I molecules. Several mouse defensin (cryptdin) genes coding important intestinal microbicidal proteins were up-regulated as a consequence of TFF2 deficiency. Normally moderate expression of TFF3 was highly increased in stomach. Copyright (c) 2005 S. Karger AG, Basel.

Biochemical and Physical Properties of the Methanococcus jannaschii 20S Proteasome and PAN, a Homolog of the ATPase (Rpt) Subunits of the Eucaryal 26S Proteasome†

PubMed Central

Wilson, Heather L.; Ou, Mark S.; Aldrich, Henry C.; Maupin-Furlow, Julie

2000-01-01

The 20S proteasome is a self-compartmentalized protease which degrades unfolded polypeptides and has been purified from eucaryotes, gram-positive actinomycetes, and archaea. Energy-dependent complexes, such as the 19S cap of the eucaryal 26S proteasome, are assumed to be responsible for the recognition and/or unfolding of substrate proteins which are then translocated into the central chamber of the 20S proteasome and hydrolyzed to polypeptide products of 3 to 30 residues. All archaeal genomes which have been sequenced are predicted to encode proteins with up to ∼50% identity to the six ATPase subunits of the 19S cap. In this study, one of these archaeal homologs which has been named PAN for proteasome-activating nucleotidase was characterized from the hyperthermophile Methanococcus jannaschii. In addition, the M. jannaschii 20S proteasome was purified as a 700-kDa complex by in vitro assembly of the α and β subunits and has an unusually high rate of peptide and unfolded-polypeptide hydrolysis at 100°C. The 550-kDa PAN complex was required for CTP- or ATP-dependent degradation of β-casein by archaeal 20S proteasomes. A 500-kDa complex of PAN(Δ1–73), which has a deletion of residues 1 to 73 of the deduced protein and disrupts the predicted N-terminal coiled-coil, also facilitated this energy-dependent proteolysis. However, this deletion increased the types of nucleotides hydrolyzed to include not only ATP and CTP but also ITP, GTP, TTP, and UTP. The temperature optimum for nucleotide (ATP) hydrolysis was reduced from 80°C for the full-length protein to 65°C for PAN(Δ1–73). Both PAN protein complexes were stable in the absence of ATP and were inhibited by N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid. Kinetic analysis reveals that the PAN protein has a relatively high Vmax for ATP and CTP hydrolysis of 3.5 and 5.8 μmol of Pi per min per mg of protein as well as a relatively low affinity for CTP and ATP with Km values of 307 and 497 μM compared to other proteins of the AAA family. Based on electron micrographs, PAN and PAN(Δ1–73) apparently associate with the ends of the 20S proteasome cylinder. These results suggest that the M. jannaschii as well as related archaeal 20S proteasomes require a nucleotidase complex such as PAN to mediate the energy-dependent hydrolysis of folded-substrate proteins and that the N-terminal 73 amino acid residues of PAN are not absolutely required for this reaction. PMID:10692374

Tetracapsuloides bryosalmonae infection affects the expression of genes involved in cellular signal transduction and iron metabolism in the kidney of the brown trout Salmo trutta.

PubMed

Kumar, Gokhlesh; Sarker, Subhodeep; Menanteau-Ledouble, Simon; El-Matbouli, Mansour

2015-06-01

Tetracapsuloides bryosalmonae is an enigmatic endoparasite which causes proliferative kidney disease in various species of salmonids in Europe and North America. The life cycle of the European strain of T. bryosalmonae generally completes in an invertebrate host freshwater bryozoan and vertebrate host brown trout (Salmo trutta) Linnaeus, 1758. Little is known about the gene expression in the kidney of brown trout during the developmental stages of T. bryosalmonae. In the present study, quantitative real-time PCR was applied to quantify the target genes of interest in the kidney of brown trout at different time points of T. bryosalmonae development. PCR primers specific for target genes were designed and optimized, and their gene expression levels were quantified in the cDNA kidney samples using SYBR Green Supermix. Expression of Rab GDP dissociation inhibitor beta, integral membrane protein 2B, NADH dehydrogenase 1 beta subcomplex subunit 6, and 26S protease regulatory subunit S10B were upregulated significantly in infected brown trout, while the expression of the ferritin M middle subunit was downregulated significantly. These results suggest that host genes involved in cellular signal transduction, proteasomal activities, including membrane transporters and cellular iron storage, are differentially upregulated or downregulated in the kidney of brown trout during parasite development. The gene expression pattern of infected renal tissue may support the development of intraluminal sporogonic stages of T. bryosalmonae in the renal tubular lumen of brown trout which may facilitate the release of viable parasite spores to transmit to the invertebrate host bryozoan.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vasuri, Francesco; Capizzi, Elisa; Bellavista, Elena

Despite the central role of proteasomes in relevant physiological pathways and pathological processes, this topic is unexpectedly largely unexplored in human liver. Here we present data on the presence of proteasome and immunoproteasome in human livers from normal adults, fetuses and patients affected by major hepatic diseases such as cirrhosis and chronic active hepatitis. Immunohistochemistry for constitutive ({alpha}4 and {beta}1) and inducible (LMP2 and LMP7) proteasome subunits, and for the PA28{alpha}{beta} regulator, was performed in liver samples from 38 normal subjects, 6 fetuses, 2 pediatric cases, and 19 pathological cases (10 chronic active hepatitis and 9 cirrhosis). The immunohistochemical datamore » have been validated and quantified by Western blotting analysis. The most striking result we found was the concomitant presence in hepatocyte cytoplasm of all healthy subjects, including the pediatric cases, of constitutive proteasome and immunoproteasome subunits, as well as PA28{alpha}{beta}. At variance, immunoproteasome was not present in hepatocytes from fetuses, while a strong cytoplasmic and nuclear positivity for LMP2 and LMP7 was found in pathological samples, directly correlated to the histopathological grade of inflammation. At variance from other organs such as the brain, immunoproteasome is present in livers from normal adult and pediatric cases, in apparent absence of pathological processes, suggesting the presence of a peculiar regulation of the proteasome/immunoproteasome system, likely related to the physiological stimuli derived from the gut microbiota after birth. Other inflammatory stimuli contribute in inducing high levels of immunoproteasome in pathological conditions, where its role deserve further attention.« less

On the Trails of the Proteasome Fold: Structural and Functional Analysis of the Ancestral β-Subunit Protein Anbu.

PubMed

Vielberg, Marie-Theres; Bauer, Verena C; Groll, Michael

2018-03-02

The 20S proteasome is a key player in eukaryotic and archaeal protein degradation, but its progenitor in eubacteria is unknown. Recently, the ancestral β-subunit protein (Anbu) was predicted to be the evolutionary precursor of the proteasome. We crystallized Anbu from Hyphomicrobium sp. strain MC1 in four different space groups and solved the structures by SAD-phasing and Patterson search calculation techniques. Our data reveal that Anbu adopts the classical fold of Ntn-hydrolases, but its oligomeric state differs from that of barrel-shaped proteases. In contrast to their typical architecture, the Anbu protomer is a tightly interacting dimer that can assemble into a helical superstructure. Although Anbu features a catalytic triad of Thr1O γ , Asp17O δ1 and Lys32N ε , it is unable to hydrolyze standard protease substrates. The lack of activity might be caused by the incapacity of Thr1NH 2 to function as a Brønsted acid during substrate cleavage due to its missing activation via hydrogen bonding. Altogether, we demonstrate that the topology of the proteasomal fold is conserved in Anbu, but whether it acts as a protease still needs to be clarified. Copyright © 2018 Elsevier Ltd. All rights reserved.

Alterations of the Intracellular Peptidome in Response to the Proteasome Inhibitor Bortezomib

PubMed Central

Berezniuk, Iryna; Dasgupta, Sayani; Castro, Leandro M.; Gozzo, Fabio C.; Ferro, Emer S.; Fricker, Lloyd D.

2013-01-01

Bortezomib is an antitumor drug that competitively inhibits proteasome beta-1 and beta-5 subunits. While the impact of bortezomib on protein stability is known, the effect of this drug on intracellular peptides has not been previously explored. A quantitative peptidomics technique was used to examine the effect of treating human embryonic kidney 293T (HEK293T) cells with 5–500 nM bortezomib for various lengths of time (30 minutes to 16 hours), and human neuroblastoma SH-SY5Y cells with 500 nM bortezomib for 1 hour. Although bortezomib treatment decreased the levels of some intracellular peptides, the majority of peptides were increased by 50–500 nM bortezomib. Peptides requiring cleavage at acidic and hydrophobic sites, which involve beta-1 and -5 proteasome subunits, were among those elevated by bortezomib. In contrast, the proteasome inhibitor epoxomicin caused a decrease in the levels of many of these peptides. Although bortezomib can induce autophagy under certain conditions, the rapid bortezomib-mediated increase in peptide levels did not correlate with the induction of autophagy. Taken together, the present data indicate that bortezomib alters the balance of intracellular peptides, which may contribute to the biological effects of this drug. PMID:23308178

Structure of the 26S proteasome with ATP-γS bound provides insights into the mechanism of nucleotide-dependent substrate translocation

PubMed Central

Śledź, Paweł; Unverdorben, Pia; Beck, Florian; Pfeifer, Günter; Schweitzer, Andreas; Förster, Friedrich; Baumeister, Wolfgang

2013-01-01

The 26S proteasome is a 2.5-MDa, ATP-dependent multisubunit proteolytic complex that processively destroys proteins carrying a degradation signal. The proteasomal ATPase heterohexamer is a key module of the 19S regulatory particle; it unfolds substrates and translocates them into the 20S core particle where degradation takes place. We used cryoelectron microscopy single-particle analysis to obtain insights into the structural changes of 26S proteasome upon the binding and hydrolysis of ATP. The ATPase ring adopts at least two distinct helical staircase conformations dependent on the nucleotide state. The transition from the conformation observed in the presence of ATP to the predominant conformation in the presence of ATP-γS induces a sliding motion of the ATPase ring over the 20S core particle ring leading to an alignment of the translocation channels of the ATPase and the core particle gate, a conformational state likely to facilitate substrate translocation. Two types of intersubunit modules formed by the large ATPase domain of one ATPase subunit and the small ATPase domain of its neighbor exist. They resemble the contacts observed in the crystal structures of ClpX and proteasome-activating nucleotidase, respectively. The ClpX-like contacts are positioned consecutively and give rise to helical shape in the hexamer, whereas the proteasome-activating nucleotidase-like contact is required to close the ring. Conformational switching between these forms allows adopting different helical conformations in different nucleotide states. We postulate that ATP hydrolysis by the regulatory particle ATPase (Rpt) 5 subunit initiates a cascade of conformational changes, leading to pulling of the substrate, which is primarily executed by Rpt1, Rpt2, and Rpt6. PMID:23589842

Proteasome inhibitors enhance endothelial thrombomodulin expression via induction of Krüppel-like transcription factors

PubMed Central

Hiroi, Toyoko; Deming, Clayton B.; Zhao, Haige; Hansen, Baranda S.; Arkenbout, Elisabeth K.; Myers, Thomas J.; McDevitt, Michael A.; Rade, Jeffrey J.

2009-01-01

Objective Impairment of the thrombomodulin-protein C anticoagulant pathway has been implicated in pathologic thrombosis associated with malignancy. Patients who receive proteasome inhibitors as part of their chemotherapeutic regimen appear to be at decreased risk for thromboembolic events. We investigated the effects of proteasome inhibitors on endothelial thrombomodulin expression and function. Methods and Results Proteasome inhibitors as a class markedly induced the expression thrombomodulin and enhanced the protein C activating capacity of endothelial cells. Thrombomodulin upregulation was independent of NF-κB signaling, a principal target of proteasome inhibitors, but was instead a direct consequence of increased expression of the Krüppel-like transcription factors, KLF2 and KLF4. These effects were confirmed in vivo, where systemic administration of a proteasome inhibitor enhanced thrombomodulin expression that was paralleled by changes in the expression of KLF2 and KLF4. Conclusions These findings identify a novel mechanism of action of proteasome inhibitors that may help to explain their clinically observed thromboprotective effects. PMID:19661484

Proteasome dynamics between proliferation and quiescence stages of Saccharomyces cerevisiae.

PubMed

Yedidi, Ravikiran S; Fatehi, Amatullah K; Enenkel, Cordula

The ubiquitin-proteasome system (UPS) plays a critical role in cellular protein homeostasis and is required for the turnover of short-lived and unwanted proteins, which are targeted by poly-ubiquitination for degradation. Proteasome is the key protease of UPS and consists of multiple subunits, which are organized into a catalytic core particle (CP) and a regulatory particle (RP). In Saccharomyces cerevisiae, proteasome holo-enzymes are engaged in degrading poly-ubiquitinated substrates and are mostly localized in the nucleus during cell proliferation. While in quiescence, the RP and CP are sequestered into motile and reversible storage granules in the cytoplasm, called proteasome storage granules (PSGs). The reversible nature of PSGs allows the proteasomes to be transported back into the nucleus upon exit from quiescence. Nuclear import of RP and CP through nuclear pores occurs via the canonical pathway that includes the importin-αβ heterodimer and takes advantage of the Ran-GTP gradient across the nuclear membrane. Dependent on the growth stage, either inactive precursor complexes or mature holo-enzymes are imported into the nucleus. The present review discusses the dynamics of proteasomes including their assembly, nucleo-cytoplasmic transport during proliferation and the sequestration of proteasomes into PSGs during quiescence. [Formula: see text].

Decreased expression of thymus-specific proteasome subunit β5t in Down syndrome patients.

PubMed

Tomaru, Utano; Tsuji, Takahiro; Kiuchi, Shizuka; Ishizu, Akihiro; Suzuki, Akira; Otsuka, Noriyuki; Ito, Tomoki; Ikeda, Hitoshi; Fukasawa, Yuichiro; Kasahara, Masanori

2015-08-01

The majority of patients with Down syndrome (DS), trisomy 21, have morphologically abnormal thymuses and present with intrinsic immunological abnormalities affecting mainly the cellular immune response. The aim of this study was to examine whether the expression of functionally important molecules is altered in thymic stromal cells in patients with DS. We analysed thymic tissues from patients with trisomy 13 (n = 4), trisomy 18 (n = 14) and trisomy 21 (n = 13) for histological alterations, and for the expression of functionally important molecules such as β5t, a thymoproteasome subunit, and cathepsins L and S. In patients with trisomy 13 and trisomy 18, the thymus was morphologically normal or showed only mild depletion of cortical thymocytes. In contrast, the thymus showed variable histological changes in patients with trisomy 21; six of 13 cases showed severe depletion of thymocytes accompanied by the disappearance of thymic lobular architecture. In such thymuses, spindle-shaped keratin-positive cells were densely distributed, and expression of β5t, but not of cathepsin L, was markedly decreased. The present study suggests that abnormal thymic architecture and decreased expression of functionally important molecules in thymic stromal cells may be involved in immunological abnormalities in DS patients. © 2015 John Wiley & Sons Ltd.

A Proteasome Cap Subunit Required for Spindle Pole Body Duplication in Yeast

PubMed Central

McDonald, Heather B.; Byers, Breck

1997-01-01

Proteasome-mediated protein degradation is a key regulatory mechanism in a diversity of complex processes, including the control of cell cycle progression. The selection of substrates for degradation clearly depends on the specificity of ubiquitination mechanisms, but further regulation may occur within the proteasomal 19S cap complexes, which attach to the ends of the 20S proteolytic core and are thought to control entry of substrates into the core. We have characterized a gene from Saccharomyces cerevisiae that displays extensive sequence similarity to members of a family of ATPases that are components of the 19S complex, including human subunit p42 and S. cerevisiae SUG1/ CIM3 and CIM5 products. This gene, termed PCS1 (for proteasomal cap subunit), is identical to the recently described SUG2 gene (Russell, S.J., U.G. Sathyanarayana, and S.A. Johnston. 1996. J. Biol. Chem. 271:32810– 32817). We have shown that PCS1 function is essential for viability. A temperature-sensitive pcs1 strain arrests principally in the second cycle after transfer to the restrictive temperature, blocking as large-budded cells with a G2 content of unsegregated DNA. EM reveals that each arrested pcs1 cell has failed to duplicate its spindle pole body (SPB), which becomes enlarged as in other monopolar mutants. Additionally, we have shown localization of a functional Pcs1–green fluorescent protein fusion to the nucleus throughout the cell cycle. We hypothesize that Pcs1p plays a role in the degradation of certain potentially nuclear component(s) in a manner that specifically is required for SPB duplication. PMID:9151663

Poly-Ub-Substrate-Degradative Activity of 26S Proteasome Is Not Impaired in the Aging Rat Brain

PubMed Central

Giannini, Carolin; Kloß, Alexander; Gohlke, Sabrina; Mishto, Michele; Nicholson, Thomas P.; Sheppard, Paul W.; Kloetzel, Peter-Michael; Dahlmann, Burkhardt

2013-01-01

Proteostasis is critical for the maintenance of life. In neuronal cells an imbalance between protein synthesis and degradation is thought to be involved in the pathogenesis of neurodegenerative diseases during aging. Partly, this seems to be due to a decrease in the activity of the ubiquitin-proteasome system, wherein the 20S/26S proteasome complexes catalyse the proteolytic step. We have characterised 20S and 26S proteasomes from cerebrum, cerebellum and hippocampus of 3 weeks old (young) and 24 month old (aged) rats. Our data reveal that the absolute amount of the proteasome is not dfferent between both age groups. Within the majority of standard proteasomes in brain the minute amounts of immuno-subunits are slightly increased in aged rat brain. While this goes along with a decrease in the activities of 20S and 26S proteasomes to hydrolyse synthetic fluorogenic tripeptide substrates from young to aged rats, the capacity of 26S proteasomes for degradation of poly-Ub-model substrates and its activation by poly-Ub-substrates is not impaired or even slightly increased in brain of aged rats. We conclude that these alterations in proteasome properties are important for maintaining proteostasis in the brain during an uncomplicated aging process. PMID:23667697

RA190, a Proteasome Subunit ADRM1 Inhibitor, Suppresses Intrahepatic Cholangiocarcinoma by Inducing NF-KB-Mediated Cell Apoptosis.

PubMed

Yu, Guang-Yang; Wang, Xuan; Zheng, Su-Su; Gao, Xiao-Mei; Jia, Qing-An; Zhu, Wen-Wei; Lu, Lu; Jia, Hu-Liang; Chen, Jin-Hong; Dong, Qiong-Zhu; Lu, Ming; Qin, Lun-Xiu

2018-06-15

Effective drug treatment for intrahepatic cholangiocarcinoma (ICC) is currently lacking. Therefore, there is an urgent need for new targets and new drugs that can prolong patient survival. Recently targeting the ubiquitin proteasome pathway has become an attractive anti-cancer strategy. In this study, we aimed to evaluate the therapeutic effect of and identify the potential mechanisms involved in targeting the proteasome subunit ADRM1 for ICC. The expression of ADRM1 and its prognostic value in ICC was analyzed using GEO and TCGA datasets, tumor tissues, and tumor tissue arrays. The effects of RA190 on the proliferation and survival of both established ICC cell lines and primary ICC cells were examined in vitro. Annexin V/propidium iodide staining, western blotting and immunohistochemical staining were performed. The in vivo anti-tumor effect of RA190 on ICC was validated in subcutaneous xenograft and patient-derived xenograft (PDX) models. ADRM1 levels were significantly higher in ICC tissues than in normal bile duct tissues. ICC patients with high ADRM1 levels had worse overall survival (hazard ratio [HR] = 2.383, 95% confidence interval [CI] =1.357 to 4.188) and recurrence-free survival (HR = 1.710, 95% CI =1.045 to 2.796). ADRM1 knockdown significantly inhibited ICC growth in vitro and in vivo. The specific inhibitor RA190 targeting ADRM1 suppressed proliferation and reduced cell vitality of ICC cell lines and primary ICC cells significantly in vitro. Furthermore, RA190 significantly inhibited the proteasome by inactivating ADRM1, and the consequent accumulation of ADRM1 substrates decreased the activating levels of NF-κB to aggravate cell apoptosis. The therapeutic benefits of RA190 treatment were further demonstrated in both subcutaneous implantation and PDX models. Our findings indicate that up-regulated ADRM1 was involved in ICC progression and suggest the potential clinical application of ADRM1 inhibitors (e.g., RA190 and KDT-11) for ICC treatment. © 2018 The Author(s). Published by S. Karger AG, Basel.

Carica papaya microRNAs are responsive to Papaya meleira virus infection.

PubMed

Abreu, Paolla M V; Gaspar, Clicia G; Buss, David S; Ventura, José A; Ferreira, Paulo C G; Fernandes, Patricia M B

2014-01-01

MicroRNAs are implicated in the response to biotic stresses. Papaya meleira virus (PMeV) is the causal agent of sticky disease, a commercially important pathology in papaya for which there are currently no resistant varieties. PMeV has a number of unusual features, such as residence in the laticifers of infected plants, and the response of the papaya to PMeV infection is not well understood. The protein levels of 20S proteasome subunits increase during PMeV infection, suggesting that proteolysis could be an important aspect of the plant defense response mechanism. To date, 10,598 plant microRNAs have been identified in the Plant miRNAs Database, but only two, miR162 and miR403, are from papaya. In this study, known plant microRNA sequences were used to search for potential microRNAs in the papaya genome. A total of 462 microRNAs, representing 72 microRNA families, were identified. The expression of 11 microRNAs, whose targets are involved in 20S and 26S proteasomal degradation and in other stress response pathways, was compared by real-time PCR in healthy and infected papaya leaf tissue. We found that the expression of miRNAs involved in proteasomal degradation increased in response to very low levels of PMeV titre and decreased as the viral titre increased. In contrast, miRNAs implicated in the plant response to biotic stress decreased their expression at very low level of PMeV and increased at high PMeV levels. Corroborating with this results, analysed target genes for this miRNAs had their expression modulated in a dependent manner. This study represents a comprehensive identification of conserved miRNAs inpapaya. The data presented here might help to complement the available molecular and genomic tools for the study of papaya. The differential expression of some miRNAs and identifying their target genes will be helpful for understanding the regulation and interaction of PMeV and papaya.

Carica papaya MicroRNAs Are Responsive to Papaya meleira virus Infection

PubMed Central

Abreu, Paolla M. V.; Gaspar, Clicia G.; Buss, David S.; Ventura, José A.; Ferreira, Paulo C. G.; Fernandes, Patricia M. B.

2014-01-01

MicroRNAs are implicated in the response to biotic stresses. Papaya meleira virus (PMeV) is the causal agent of sticky disease, a commercially important pathology in papaya for which there are currently no resistant varieties. PMeV has a number of unusual features, such as residence in the laticifers of infected plants, and the response of the papaya to PMeV infection is not well understood. The protein levels of 20S proteasome subunits increase during PMeV infection, suggesting that proteolysis could be an important aspect of the plant defense response mechanism. To date, 10,598 plant microRNAs have been identified in the Plant miRNAs Database, but only two, miR162 and miR403, are from papaya. In this study, known plant microRNA sequences were used to search for potential microRNAs in the papaya genome. A total of 462 microRNAs, representing 72 microRNA families, were identified. The expression of 11 microRNAs, whose targets are involved in 20S and 26S proteasomal degradation and in other stress response pathways, was compared by real-time PCR in healthy and infected papaya leaf tissue. We found that the expression of miRNAs involved in proteasomal degradation increased in response to very low levels of PMeV titre and decreased as the viral titre increased. In contrast, miRNAs implicated in the plant response to biotic stress decreased their expression at very low level of PMeV and increased at high PMeV levels. Corroborating with this results, analysed target genes for this miRNAs had their expression modulated in a dependent manner. This study represents a comprehensive identification of conserved miRNAs inpapaya. The data presented here might help to complement the available molecular and genomic tools for the study of papaya. The differential expression of some miRNAs and identifying their target genes will be helpful for understanding the regulation and interaction of PMeV and papaya. PMID:25072834

β-hydroxy-β-methylbutyrate (HMB) Prevents Sepsis-Induced Diaphragm Dysfunction in Mice

PubMed Central

Supinski, Gerald S.; Callahan, Leigh Ann

2014-01-01

Infections induce severe respiratory muscle weakness. Currently there are no treatments for this important clinical problem. We tested the hypothesis that β-hydroxy-β-methylbutyrate (HMB) would prevent sepsis-induced diaphragm weakness. Four groups of adult male mice were studied: controls (saline-injected), sepsis (intraperitoneal lipopolysaccharide), sepsis+HMB (injected intravenously), and HMB. Diaphragm force generation and indices of caspase 3, calpain, 20S proteasomal subunit, and double-stranded RNA-dependent protein kinase (PKR) activation were assessed after 24 hours. Sepsis elicited large reductions in diaphragm specific force generation at all stimulation frequencies. Endotoxin also activated caspase 3, calpain, the 20S proteasomal subunit and PKR in the diaphragm. HMB blocked sepsis-induced caspase 3, 20S proteasomal and PKR activation, but did not prevent calpain activation. Most importantly, HMB administration significantly attenuated sepsis-induced diaphragm weakness, preserving muscle force generation at all stimulation frequencies (p<0.01). We speculate that HMB may prove to be an important therapy in infected patients, with the potential to increase diaphragm strength, to reduce the duration of mechanical ventilation and to decrease mortality in this patient population PMID:24632527

Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit.

PubMed

Chojnacki, Michal; Mansour, Wissam; Hameed, Dharjath S; Singh, Rajesh K; El Oualid, Farid; Rosenzweig, Rina; Nakasone, Mark A; Yu, Zanlin; Glaser, Fabian; Kay, Lewis E; Fushman, David; Ovaa, Huib; Glickman, Michael H

2017-04-20

Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The best documented Ub signaling pathway is the one that delivers polyUb proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (Ub PT ), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb conjugates. Our work demonstrates the value of Ub PT , and we expect that its future uses will help define and investigate the ubiquitin interactome. Copyright © 2017 Elsevier Ltd. All rights reserved.

Three Immunoproteasome-Associated Subunits Cooperatively Generate a Cytotoxic T-Lymphocyte Epitope of Epstein-Barr Virus LMP2A by Overcoming Specific Structures Resistant to Epitope Liberation

PubMed Central

Ito, Yoshinori; Kondo, Eisei; Demachi-Okamura, Ayako; Akatsuka, Yoshiki; Tsujimura, Kunio; Tanimoto, Mitsune; Morishima, Yasuo; Takahashi, Toshitada; Kuzushima, Kiyotaka

2006-01-01

The precise roles of gamma interferon-inducible immunoproteasome-associated molecules in generation of cytotoxic T-lymphocyte (CTL) epitopes have yet to be fully elucidated. We describe here a unique epitope derived from the Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) presented by HLA-A*2402 molecules. Generation of the epitope, designated LMP2A222-230, from the full-length protein requires the immunoproteasome subunit low-molecular-weight protein 7 (ip-LMP7) and the proteasome activator 28-α subunit and is accelerated by ip-LMP2, as revealed by gene expression experiments using an LMP2A222-230-specific CTL clone as a responder in enzyme-linked immunospot assays. The unequivocal involvement of all three components was confirmed by RNA interference gene silencing. Interestingly, the LMP2A222-230 epitope could be efficiently generated from incomplete EBV-LMP2A fragments that were produced by puromycin treatment or gene-engineered shortened EBV-LMP2A lacking some of its hydrophobic domains. In addition, epitope generation was increased by a single amino acid substitution from leucine to alanine immediately flanking the C terminus, this being predicted by a web-accessible program to increase the cleavage strength. Taken together, the data indicate that the generation of LMP2A222-230 is influenced not only by extrinsic factors such as immunoproteasomes but also by intrinsic factors such as the length of the EBV-LMP2A protein and proteasomal cleavage strength at specific positions in the source antigen. PMID:16378990

Natural products inhibiting the ubiquitin-proteasome proteolytic pathway, a target for drug development.

PubMed

Tsukamoto, Sachiko; Yokosawa, Hideyoshi

2006-01-01

The ubiquitin-proteasome proteolytic pathway plays a major role in selective protein degradation and regulates various cellular events including cell cycle progression, transcription, DNA repair, signal transduction, and immune response. Ubiquitin, a highly conserved small protein in eukaryotes, attaches to a target protein prior to degradation. The polyubiquitin chain tagged to the target protein is recognized by the 26S proteasome, a high-molecular-mass protease subunit complex, and the protein portion is degraded by the 26S proteasome. The potential of specific proteasome inhibitors, which act as anti-cancer agents, is now under intensive investigation, and bortezomib (PS-341), a proteasome inhibitor, has been recently approved by FDA for multiple myeloma treatment. Since ubiquitination of proteins requires the sequential action of three enzymes, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3), and polyubiquitination is a prerequisite for proteasome-mediated protein degradation, inhibitors of E1, E2, and E3 are reasonably thought to be drug candidates for treatment of diseases related to ubiquitination. Recently, various compounds inhibiting the ubiquitin-proteasome pathway have been isolated from natural resources. We also succeeded in isolating inhibitors against the proteasome and E1 enzyme from marine natural resources. In this review, we summarize the structures and biological activities of natural products that inhibit the ubiquitin-proteasome proteolytic pathway.

Probing the cooperativity of Thermoplasma acidophilum proteasome core particle gating by NMR spectroscopy

PubMed Central

Huang, Rui; Pérez, Felipe; Kay, Lewis E.

2017-01-01

The 20S proteasome core particle (20S CP) plays an integral role in cellular homeostasis by degrading proteins no longer required for function. The process is, in part, controlled via gating residues localized to the ends of the heptameric barrel-like CP structure that occlude substrate entry pores, preventing unregulated degradation of substrates that might otherwise enter the proteasome. Previously, we showed that the N-terminal residues of the α-subunits of the CP from the archaeon Thermoplasma acidophilum are arranged such that, on average, two of the seven termini are localized inside the lumen of the proteasome, thereby plugging the entry pore and functioning as a gate. However, the mechanism of gating remains unclear. Using solution NMR and a labeling procedure in which a series of mixed proteasome rings are prepared such that the percentage of gate-containing subunits is varied, we address the energetics of gating and establish whether gating is a cooperative process involving the concerted action of residues from more than a single protomer. Our results establish that the intrinsic probability of a gate entering the lumen favors the in state by close to 20-fold, that entry of each gate is noncooperative, with the number of gates that can be accommodated inside the lumen a function of the substrate entry pore size and the bulkiness of the gating residues. Insight into the origin of the high affinity for the in state is obtained from spin-relaxation experiments. More generally, our approach provides an avenue for dissecting interactions of individual protomers in homo-oligomeric complexes. PMID:29087330

A HIF-LIMD1 negative feedback mechanism mitigates the pro-tumorigenic effects of hypoxia.

PubMed

Foxler, Daniel E; Bridge, Katherine S; Foster, John G; Grevitt, Paul; Curry, Sean; Shah, Kunal M; Davidson, Kathryn M; Nagano, Ai; Gadaleta, Emanuela; Rhys, Hefin I; Kennedy, Paul T; Hermida, Miguel A; Chang, Ting-Yu; Shaw, Peter E; Reynolds, Louise E; McKay, Tristan R; Wang, Hsei-Wei; Ribeiro, Paulo S; Plevin, Michael J; Lagos, Dimitris; Lemoine, Nicholas R; Rajan, Prabhakar; Graham, Trevor A; Chelala, Claude; Hodivala-Dilke, Kairbaan M; Spendlove, Ian; Sharp, Tyson V

2018-06-21

The adaptive cellular response to low oxygen tensions is mediated by the hypoxia-inducible factors (HIFs), a family of heterodimeric transcription factors composed of HIF-α and HIF-β subunits. Prolonged HIF expression is a key contributor to cellular transformation, tumorigenesis and metastasis. As such, HIF degradation under hypoxic conditions is an essential homeostatic and tumour-suppressive mechanism. LIMD1 complexes with PHD2 and VHL in physiological oxygen levels (normoxia) to facilitate proteasomal degradation of the HIF-α subunit. Here, we identify LIMD1 as a HIF-1 target gene, which mediates a previously uncharacterised, negative regulatory feedback mechanism for hypoxic HIF-α degradation by modulating PHD2-LIMD1-VHL complex formation. Hypoxic induction of LIMD1 expression results in increased HIF-α protein degradation, inhibiting HIF-1 target gene expression, tumour growth and vascularisation. Furthermore, we report that copy number variation at the LIMD1 locus occurs in 47.1% of lung adenocarcinoma patients, correlates with enhanced expression of a HIF target gene signature and is a negative prognostic indicator. Taken together, our data open a new field of research into the aetiology, diagnosis and prognosis of LIMD1 -negative lung cancers. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Enhanced Expression of Interferon-γ-Induced Antigen-Processing Machinery Components in a Spontaneously Occurring Cancer1

PubMed Central

Cerruti, Fulvia; Martano, Marina; Petterino, Claudio; Bollo, Enrico; Morello, Emanuela; Bruno, Renato; Buracco, Paolo; Cascio, Paolo

2007-01-01

In human tumors, changes in the surface expression and/or function of major histocompatibility complex (MHC) class I antigens are frequently found and may provide malignant cells with a mechanism to escape control of the immune system. This altered human lymphocyte antigen (HLA) class I phenotype can be caused by either structural alterations or dysregulation of genes encoding subunits of HLA class I antigens and/or components of the MHC class I antigen-processing machinery (APM). Herein we analyze the expression of several proteins involved in the generation of MHC class I epitopes in feline injection site sarcoma, a spontaneously occurring tumor in cats that is an informativemodel for the study of tumor biology in other species, including humans. Eighteen surgically removed primary fibrosarcoma lesions were analyzed, and an enhanced expression of two catalytic subunits of immunoproteasomes, PA28 and leucine aminopeptidase, was found in tumors compared to matched normal tissues. As a functional counterpart of these changes in protein levels, proteasomal activities were increased in tissue extracts from fibrosarcomas. Taken together, these results suggest that alterations in the APM system may account for reduced processing of selected tumor antigens and may potentially provide neoplastic fibroblasts with a mechanism for escape from T-cell recognition and destruction. PMID:18030364

Enhanced expression of interferon-gamma-induced antigen-processing machinery components in a spontaneously occurring cancer.

PubMed

Cerruti, Fulvia; Martano, Marina; Petterino, Claudio; Bollo, Enrico; Morello, Emanuela; Bruno, Renato; Buracco, Paolo; Cascio, Paolo

2007-11-01

In human tumors, changes in the surface expression and/or function of major histocompatibility complex (MHC) class I antigens are frequently found and may provide malignant cells with a mechanism to escape control of the immune system. This altered human lymphocyte antigen (HLA) class I phenotype can be caused by either structural alterations or dysregulation of genes encoding subunits of HLA class I antigens and/or components of the MHC class I antigen-processing machinery (APM). Herein we analyze the expression of several proteins involved in the generation of MHC class I epitopes in feline injection site sarcoma, a spontaneously occurring tumor in cats that is an informative model for the study of tumor biology in other species, including humans. Eighteen surgically removed primary fibrosarcoma lesions were analyzed, and an enhanced expression of two catalytic subunits of immunoproteasomes, PA28 and leucine aminopeptidase, was found in tumors compared to matched normal tissues. As a functional counterpart of these changes in protein levels, proteasomal activities were increased in tissue extracts from fibrosarcomas. Taken together, these results suggest that alterations in the APM system may account for reduced processing of selected tumor antigens and may potentially provide neoplastic fibroblasts with a mechanism for escape from T-cell recognition and destruction.

6-Shogaol induces caspase-independent paraptosis in cancer cells via proteasomal inhibition.

PubMed

Nedungadi, Divya; Binoy, Anupama; Pandurangan, Nanjan; Pal, Sanjay; Nair, Bipin G; Mishra, Nandita

2018-03-15

An α, β-unsaturated carbonyl compound of ginger, 6-Shogaol (6S), induced extensive cytoplasmic vacuolation and cell death in breast cancer cell (MDA-MB-231) and non-small lung cancer (A549) cells. In the presence of autophagic inhibitors the cells continued to exhibit cytoplasmic vacuolation and cell death clearly distinguishing it from the classic autophagic process. 6S induced death did not exhibit the characteristic apoptotic features like caspase cleavage, phosphatidyl serine exposure and DNA fragmentation. The immunofluorescence with the Endoplasmic Reticulum (ER) resident protein, calreticulin indicated that the vacuoles were of ER origin, typical of paraptosis. This was supported by the increase in level of microtubule associated protein light chain 3B (LC3 I and LC3 II) and polyubiquitin binding protein, p62. The level of ER stress markers like polyubiquitinated proteins, Bip and CHOP also consistently increased. We have found that 6S inhibits the 26S proteasome. The proteasomal inhibitory activity was elucidated by a) molecular docking of 6S onto the active site of β5 subunit and b) reduced fluorescence by the fluorogenic substrate of the chymotrypsin-like subunit. In conclusion these studies demonstrate for the first time that proteasomal inhibition by 6S induces cell death via paraptosis. So 6-shogaol may act as a template for anti-cancer lead discovery against the apoptosis resistant cancer cells. Copyright © 2018 Elsevier Inc. All rights reserved.

Reconfiguration of the proteasome during chaperone-mediated assembly

PubMed Central

Park, Soyeon; Li, Xueming; Kim, Ho Min; Singh, Chingakham Ranjit; Tian, Geng; Hoyt, Martin A.; Lovell, Scott; Battaile, Kevin P.; Zolkiewski, Michal; Coffino, Philip; Roelofs, Jeroen; Cheng, Yifan; Finley, Daniel

2013-01-01

The proteasomal ATPase ring, comprising Rpt1-Rpt6, associates with the heptameric α ring of the proteasome core particle (CP) in the mature proteasome, with the Rpt C-terminal tails inserting into pockets of the α ring1–4. Rpt ring assembly is mediated by four chaperones, each binding a distinct Rpt subunit5–10. We report that the base subassembly of the proteasome, which includes the Rpt ring, forms a high affinity complex with the CP. This complex is subject to active dissociation by the chaperones Hsm3, Nas6, and Rpn14. Chaperone-mediated dissociation was abrogated by a nonhydrolyzable ATP analog, indicating that chaperone action is coupled to nucleotide hydrolysis by the Rpt ring. Unexpectedly, synthetic Rpt tail peptides bound α pockets with poor specificity, except for Rpt6, which uniquely bound the α2/α3 pocket. Although the Rpt6 tail is not visualized within an α pocket in mature proteasomes2–4, it inserts into the α2/α3 pocket in the base-CP complex and is important for complex formation. Thus, the Rpt-CP interface is reconfigured when the lid complex joins the nascent proteasome to form the mature holoenzyme. PMID:23644457

Proteasome phosphorylation regulates cocaine-induced sensitization.

PubMed

Gonzales, Frankie R; Howell, Kristin K; Dozier, Lara E; Anagnostaras, Stephan G; Patrick, Gentry N

2018-04-01

Repeated exposure to cocaine produces structural and functional modifications at synapses from neurons in several brain regions including the nucleus accumbens. These changes are thought to underlie cocaine-induced sensitization. The ubiquitin proteasome system plays a crucial role in the remodeling of synapses and has recently been implicated in addiction-related behavior. The ATPase Rpt6 subunit of the 26S proteasome is phosphorylated by Ca 2+ /calmodulin-dependent protein kinases II alpha at ser120 which is thought to regulate proteasome activity and distribution in neurons. Here, we demonstrate that Rpt6 phosphorylation is involved in cocaine-induced locomotor sensitization. Cocaine concomitantly increases proteasome activity and Rpt6 S120 phosphorylation in cultured neurons and in various brain regions of wild type mice including the nucleus accumbens and prefrontal cortex. In contrast, cocaine does not increase proteasome activity in Rpt6 phospho-mimetic (ser120Asp) mice. Strikingly, we found a complete absence of cocaine-induced locomotor sensitization in the Rpt6 ser120Asp mice. Together, these findings suggest a critical role for Rpt6 phosphorylation and proteasome function in the regulation cocaine-induced behavioral plasticity. Copyright © 2017 Elsevier Inc. All rights reserved.

Feline immunodeficiency virus OrfA alters gene expression of splicing factors and proteasome-ubiquitination proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sundstrom, Magnus; Chatterji, Udayan; Schaffer, Lana

2008-02-20

Expression of the feline immunodeficiency virus (FIV) accessory protein OrfA (or Orf2) is critical for efficient viral replication in lymphocytes, both in vitro and in vivo. OrfA has been reported to exhibit functions in common with the human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) accessory proteins Vpr and Tat, although the function of OrfA has not been fully explained. Here, we use microarray analysis to characterize how OrfA modulates the gene expression profile of T-lymphocytes. The primary IL-2-dependent T-cell line 104-C1 was transduced to express OrfA. Functional expression of OrfA was demonstrated by trans complementation of the OrfA-defectivemore » clone, FIV-34TF10. OrfA-expressing cells had a slightly reduced cell proliferation rate but did not exhibit any significant alteration in cell cycle distribution. Reverse-transcribed RNA from cells expressing green fluorescent protein (GFP) or GFP + OrfA were hybridized to Affymetrix HU133 Plus 2.0 microarray chips representing more than 47,000 genome-wide transcripts. By using two statistical approaches, 461 (Rank Products) and 277 (ANOVA) genes were identified as modulated by OrfA expression. The functional relevance of the differentially expressed genes was explored by Ingenuity Pathway Analysis. The analyses revealed alterations in genes critical for RNA post-transcriptional modifications and protein ubiquitination as the two most significant functional outcomes of OrfA expression. In these two groups, several subunits of the spliceosome, cellular splicing factors and family members of the proteasome-ubiquitination system were identified. These findings provide novel information on the versatile function of OrfA during FIV infection and indicate a fine-tuning mechanism of the cellular environment by OrfA to facilitate efficient FIV replication.« less

Bladder cancer detection using a peptide substrate of the 20S proteasome.

PubMed

Gruba, Natalia; Wysocka, Magdalena; Brzezińska, Magdalena; Dębowski, Dawid; Sieńczyk, Marcin; Gorodkiewicz, Ewa; Guszcz, Tomasz; Czaplewski, Cezary; Rolka, Krzysztof; Lesner, Adam

2016-08-01

The 20S catalytic core of the human 26S proteasome can be secreted from cells, and high levels of extracellular 20S proteasome have been linked to many types of cancers and autoimmune diseases. Several diagnostic approaches have been developed that detect 20S proteasome activity in plasma, but these suffer from problems with efficiency and sensitivity. In this report, we describe the optimization and synthesis of an internally quenched fluorescent substrate of the 20S proteasome, and investigate its use as a potential diagnostic test in bladder cancer. This peptide, 2-aminobenzoic acid (ABZ)-Val-Val-Ser-Tyr-Ala-Met-Gly-Tyr(3-NO2 )-NH2 , is cleaved by the chymotrypsin 20S proteasome subunit and displays an excellent specificity constant value (9.7 × 10(5)  m(-1) ·s(-1) ) and a high kcat (8 s(-1) ). Using this peptide, we identified chymotrypsin-like proteasome activity in the majority of urine samples obtained from patients with bladder cancer, whereas the proteasome activity in urine samples from healthy volunteers was below the detection limit (0.5 pm). These findings were confirmed by an inhibitory study and immunochemistry methods. © 2016 Federation of European Biochemical Societies.

Formation and dissociation of proteasome storage granules are regulated by cytosolic pH.

PubMed

Peters, Lee Zeev; Hazan, Rotem; Breker, Michal; Schuldiner, Maya; Ben-Aroya, Shay

2013-05-27

The 26S proteasome is the major protein degradation machinery of the cell and is regulated at many levels. One mode of regulation involves accumulation of proteasomes in proteasome storage granules (PSGs) upon glucose depletion. Using a systematic robotic screening approach in yeast, we identify trans-acting proteins that regulate the accumulation of proteasomes in PSGs. Our dataset was enriched for subunits of the vacuolar adenosine triphosphatase (V-ATPase) complex, a proton pump required for vacuole acidification. We show that the impaired ability of V-ATPase mutants to properly govern intracellular pH affects the kinetics of PSG formation. We further show that formation of other protein aggregates upon carbon depletion also is triggered in mutants with impaired activity of the plasma membrane proton pump and the V-ATPase complex. We thus identify cytosolic pH as a specific cellular signal involved both in the glucose sensing that mediates PSG formation and in a more general mechanism for signaling carbon source exhaustion.

Arabidopsis cop8 and fus4 mutations define the same gene that encodes subunit 4 of the COP9 signalosome.

PubMed Central

Serino, G; Tsuge, T; Kwok, S; Matsui, M; Wei, N; Deng, X W

1999-01-01

The pleiotropic constitutive photomorphogenic/deetiolated/fusca (cop/det/fus) mutants of Arabidopsis exhibit features of light-grown seedlings when grown in the dark. Cloning and biochemical analysis of COP9 have revealed that it is a component of a multiprotein complex, the COP9 signalosome (previously known as the COP9 complex). Here, we compare the immunoaffinity and the biochemical purification of the COP9 signalosome from cauliflower and confirm its eight-subunit composition. Molecular cloning of subunit 4 of the complex revealed that it is a proteasome-COP9 complex-eIF3 domain protein encoded by a gene that maps to chromosome 5, near the chromosomal location of the cop8 and fus4 mutations. Genetic complementation tests showed that the cop8 and fus4 mutations define the same locus, now designated as COP8. Molecular analysis of the subunit 4-encoding gene in both cop8 and fus4 mutants identified specific molecular lesions, and overexpression of the subunit 4 cDNA in a cop8 mutant background resulted in complete rescue of the mutant phenotype. Thus, we conclude that COP8 encodes subunit 4 of the COP9 signalosome. Examination of possible molecular interactions by using the yeast two-hybrid assay indicated that COP8 is capable of strong self-association as well as interaction with COP9, FUS6/COP11, FUS5, and Arabidopsis JAB1 homolog 1, the latter four proteins being previously defined subunits of the Arabidopsis COP9 signalosome. A comparative sequence analysis indicated that COP8 is highly conserved among multicellular eukaryotes and is also similar to a subunit of the 19S regulatory particle of the 26S proteasome. PMID:10521526

The Xanthomonas campestris type III effector XopJ proteolytically degrades proteasome subunit RPT6.

PubMed

Üstün, Suayib; Börnke, Frederik

2015-05-01

Many animal and plant pathogenic bacteria inject type III effector (T3E) proteins into their eukaryotic host cells to suppress immunity. The Yersinia outer protein J (YopJ) family of T3Es is a widely distributed family of effector proteins found in both animal and plant pathogens, and its members are highly diversified in virulence functions. Some members have been shown to possess acetyltransferase activity; however, whether this is a general feature of YopJ family T3Es is currently unknown. The T3E Xanthomonas outer protein J (XopJ), a YopJ family effector from the plant pathogen Xanthomonas campestris pv vesicatoria, interacts with the proteasomal subunit Regulatory Particle AAA-ATPase6 (RPT6) in planta to suppress proteasome activity, resulting in the inhibition of salicylic acid-related immune responses. Here, we show that XopJ has protease activity to specifically degrade RPT6, leading to reduced proteasome activity in the cytoplasm as well as in the nucleus. Proteolytic degradation of RPT6 was dependent on the localization of XopJ to the plasma membrane as well as on its catalytic triad. Mutation of the Walker B motif of RPT6 prevented XopJ-mediated degradation of the protein but not XopJ interaction. This indicates that the interaction of RPT6 with XopJ is dependent on the ATP-binding activity of RPT6, but proteolytic cleavage additionally requires its ATPase activity. Inhibition of the proteasome impairs the proteasomal turnover of Nonexpressor of Pathogenesis-Related1 (NPR1), the master regulator of salicylic acid responses, leading to the accumulation of ubiquitinated NPR1, which likely interferes with the full induction of NPR1 target genes. Our results show that YopJ family T3Es are not only highly diversified in virulence function but also appear to possess different biochemical activities. © 2015 American Society of Plant Biologists. All Rights Reserved.

β-hydroxy-β-methylbutyrate (HMB) prevents sepsis-induced diaphragm dysfunction in mice.

PubMed

Supinski, Gerald S; Callahan, Leigh A

2014-06-01

Infections induce severe respiratory muscle weakness. Currently there are no treatments for this important clinical problem. We tested the hypothesis that β-hydroxy-β-methylbutyrate (HMB) would prevent sepsis-induced diaphragm weakness. Four groups of adult male mice were studied: controls (saline-injected), sepsis (intraperitoneal lipopolysaccharide), sepsis+HMB (injected intravenously), and HMB. Diaphragm force generation and indices of caspase 3, calpain, 20S proteasomal subunit, and double-stranded RNA-dependent protein kinase (PKR) activation were assessed after 24h. Sepsis elicited large reductions in diaphragm specific force generation at all stimulation frequencies. Endotoxin also activated caspase 3, calpain, the 20S proteasomal subunit and PKR in the diaphragm. HMB blocked sepsis-induced caspase 3, 20S proteasomal and PKR activation, but did not prevent calpain activation. Most importantly, HMB administration significantly attenuated sepsis-induced diaphragm weakness, preserving muscle force generation at all stimulation frequencies (p<0.01). We speculate that HMB may prove to be an important therapy in infected patients, with the potential to increase diaphragm strength, to reduce the duration of mechanical ventilation and to decrease mortality in this patient population. Copyright © 2014 Elsevier B.V. All rights reserved.

Immunofluorescent localization of ubiquitin and proteasomes in nucleolar vacuoles of soybean root meristematic cells

PubMed Central

Stępiński, D.

2012-01-01

In this study, using the immunofluorescent method, the immunopositive signals to ubiquitin and proteasomes in nucleoli of root meristematic cells of soybean seedlings have been observed. In fact, those signals were present exclusively in nucleolar vacuoles. No signals were observed in the nucleolar territory out of the nucleolar vacuoles or in the nucleoli without vacuoles. The ubiquitin-proteasome system (UPS) may act within the nucleoli of plants with high metabolic activities and may provide an additional level of regulation of intracellular proteolysis via compartment-specific activities of their components. It is suggested that the presence of the UPS solely in vacuolated nucleoli serves as a mechanism that enhances the speed of ribosome subunit production in very actively transcribing nucleoli. On the other hand, nucleolar vacuoles in a cell/nucleus could play additional roles associated with temporary sequestration or storage of some cellular factors, including components of the ubiquitin-proteasome system. PMID:22688294

Genome-wide screen identifies novel machineries required for both ciliogenesis and cell cycle arrest upon serum starvation

PubMed Central

Kim, Ji Hyun; Ki, Soo Mi; Joung, Je-Gun; Scott, Eric; Heynen-Genel, Susanne; Aza-Blanc, Pedro; Kwon, Chang Hyuk; Kim, Joon; Gleeson, Joseph G.; Lee, Ji Eun

2016-01-01

Biogenesis of the primary cilium, a cellular organelle mediating various signaling pathways, is generally coordinated with cell cycle exit/re-entry. Although the dynamic cell cycle-associated profile of the primary cilium has been largely accepted, the mechanism governing the link between ciliogenesis and cell cycle progression has been poorly understood. Using a human genome-wide RNAi screen, we identify genes encoding subunits of the spliceosome and proteasome as novel regulators of ciliogenesis. We demonstrate that 1) the mRNA processing-related hits are essential for RNA expression of molecules acting in cilia disassembly, such as AURKA and PLK1, and 2) the ubiquitin-proteasome systems (UPS)-involved hits are necessary for proteolysis of molecules acting in cilia assembly, such as IFT88 and CPAP. In particular, we show that these screen hit-associated mechanisms are crucial for both cilia assembly and cell cycle arrest in response to serum withdrawal. Finally, our data suggest that the mRNA processing mechanism may modulate the UPS-dependent decay of cilia assembly regulators to control ciliary resorption-coupled cell cycle re-entry. PMID:27033521

Sperm proteasome and fertilization.

PubMed

Sutovsky, Peter

2011-07-01

The omnipresent ubiquitin-proteasome system (UPS) is an ATP-dependent enzymatic machinery that targets substrate proteins for degradation by the 26S proteasome by tagging them with an isopeptide chain composed of covalently linked molecules of ubiquitin, a small chaperone protein. The current knowledge of UPS involvement in the process of sperm penetration through vitelline coat (VC) during human and animal fertilization is reviewed in this study, with attention also being given to sperm capacitation and acrosome reaction/exocytosis. In ascidians, spermatozoa release ubiquitin-activating and conjugating enzymes, proteasomes, and unconjugated ubiquitin to first ubiquitinate and then degrade the sperm receptor on the VC; in echinoderms and mammals, the VC (zona pellucida/ZP in mammals) is ubiquitinated during oogenesis and the sperm receptor degraded during fertilization. Various proteasomal subunits and associated enzymes have been detected in spermatozoa and localized to sperm acrosome and other sperm structures. By using specific fluorometric substrates, proteasome-specific proteolytic and deubiquitinating activities can be measured in live, intact spermatozoa and in sperm protein extracts. The requirement of proteasomal proteolysis during fertilization has been documented by the application of various proteasome-specific inhibitors and antibodies. A similar effect was achieved by depletion of sperm-surface ATP. Degradation of VC/ZP-associated sperm receptor proteins by sperm-borne proteasomes has been demonstrated in ascidians and sea urchins. On the applied side, polyspermy has been ameliorated by modulating sperm-associated deubiquitinating enzymes. Diagnostic and therapeutic applications could emerge in human reproductive medicine. Altogether, the studies on sperm proteasome indicate that animal fertilization is controlled in part by a unique, gamete associated, extracellular UPS.

Shikonin Exerts Antitumor Activity via Proteasome Inhibition and Cell Death Induction in vitro and in vivo

PubMed Central

Yang, Huanjie; Zhou, Ping; Huang, Hongbiao; Chen, Di; Ma, Ningfang; Cui, Cindy Qiuzhi; Shen, Shouxing; Dong, Weihua; Zhang, Xiaoyan; Lian, Wen; Wang, Xuejun; Dou, Q. Ping; Liu, Jinbao

2009-01-01

Dysregulation of the ubiquitin-proteasome pathway plays an essential role in tumor growth and development. Shikonin, a natural naphthoquinone isolated from the traditional Chinese medicine Zi Cao (gromwell), has been reported to possess tumor cell-killing activity, and results from a clinical study using a shikonin-containing mixture demonstrated its safety and efficacy for the treatment of late-stage lung cancer. In the present study, we reported that shikonin is an inhibitor of tumor proteasome activity in vitro and in vivo. Our computational modeling predicts that the carbonyl carbons C1 and C4 of shikonin potentially interact with the catalytic site of β5 chymotryptic subunit of the proteasome. Indeed, shikonin potently inhibits the chymotrypsin-like activity of purified 20S proteasome (IC50 12.5 μmol/L) and tumor cellular 26S proteasome (IC50 between 2-16 μmol/L). Inhibition of the proteasome by shikonin in murine hepatoma H22, leukemia P388 and human prostate cancer PC-3 cultures resulted in accumulation of ubiquitinated proteins and several proteasome target proapoptotic proteins (IκB-α, Bax and p27), followed by induction of cell death. Shikonin treatment resulted in tumor growth inhibition in both H22 allografts and PC-3 xenografts, associated with suppression of the proteasomal activity and induction of cell death in vivo. Finally, shikonin treatment significantly prolonged the survival period of mice bearing P388 leukemia. Our results indicate that the tumor proteasome is one of the cellular targets of shikonin, and inhibition of the proteasome activity by shikonin contributes to its anti-tumor property. PMID:19165859

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue.

PubMed

Trugilho, Monique Ramos de Oliveira; Hottz, Eugenio Damaceno; Brunoro, Giselle Villa Flor; Teixeira-Ferreira, André; Carvalho, Paulo Costa; Salazar, Gustavo Adolfo; Zimmerman, Guy A; Bozza, Fernando A; Bozza, Patrícia T; Perales, Jonas

2017-05-01

Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses.

Mismatch in epitope specificities between IFNγ inflamed and uninflamed conditions leads to escape from T lymphocyte killing in melanoma.

PubMed

Woods, Katherine; Knights, Ashley J; Anaka, Matthew; Schittenhelm, Ralf B; Purcell, Anthony W; Behren, Andreas; Cebon, Jonathan

2016-01-01

A current focus in cancer treatment is to broaden responses to immunotherapy. One reason these therapies may prove inadequate is that T lymphocytes fail to recognize the tumor due to differences in immunogenic epitopes presented by the cancer cells under inflammatory or non-inflammatory conditions. The antigen processing machinery of the cell, the proteasome, cleaves proteins into peptide epitopes for presentation on MHC complexes. Immunoproteasomes in inflammatory melanomas, and in antigen presenting cells of the immune system, are enzymatically different to standard proteasomes expressed by tumors with no inflammation. This corresponds to alterations in protein cleavage between proteasome subtypes, and a disparate repertoire of MHC-presented epitopes. We assessed steady state and IFNγ-induced immunoproteasome expression in melanoma cells. Using epitope specific T-lymphocyte clones, we studied processing and presentation of three NY-ESO-1 HLA-Cw3 restricted epitopes by melanoma cell lines. Our experimental model allowed comparison of the processing of three distinct epitopes from a single antigen presented on the same HLA complex. We further investigated processing of these epitopes by direct inhibition, or siRNA mediated knockdown, of the immunoproteasome catalytic subunit LMP7. Our data demonstrated a profound difference in the way in which immunogenic T-lymphocyte epitopes are presented by melanoma cells under IFNγ inflammatory versus non-inflammatory conditions. These alterations led to significant changes in the ability of T-lymphocytes to recognize and target melanoma cells. Our results illustrate a little-studied mechanism of immune escape by tumor cells which, with appropriate understanding and treatment, may be reversible. These data have implications for the design of cancer vaccines and adoptive T cell therapies.

Platelet proteome reveals novel pathways of platelet activation and platelet-mediated immunoregulation in dengue

PubMed Central

Teixeira-Ferreira, André; Carvalho, Paulo Costa; Salazar, Gustavo Adolfo; Zimmerman, Guy A.; Perales, Jonas

2017-01-01

Dengue is the most prevalent human arbovirus disease worldwide. Dengue virus (DENV) infection causes syndromes varying from self-limiting febrile illness to severe dengue. Although dengue pathophysiology is not completely understood, it is widely accepted that increased inflammation plays important roles in dengue pathogenesis. Platelets are blood cells classically known as effectors of hemostasis which have been increasingly recognized to have major immune and inflammatory activities. Nevertheless, the phenotype and effector functions of platelets in dengue pathogenesis are not completely understood. Here we used quantitative proteomics to investigate the protein content of platelets in clinical samples from patients with dengue compared to platelets from healthy donors. Our assays revealed a set of 252 differentially abundant proteins. In silico analyses associated these proteins with key molecular events including platelet activation and inflammatory responses, and with events not previously attributed to platelets during dengue infection including antigen processing and presentation, proteasome activity, and expression of histones. From these results, we conducted functional assays using samples from a larger cohort of patients and demonstrated evidence for platelet activation indicated by P-selectin (CD62P) translocation and secretion of granule-stored chemokines by platelets. In addition, we found evidence that DENV infection triggers HLA class I synthesis and surface expression by a mechanism depending on functional proteasome activity. Furthermore, we demonstrate that cell-free histone H2A released during dengue infection binds to platelets, increasing platelet activation. These findings are consistent with functional importance of HLA class I, proteasome subunits, and histones that we found exclusively in proteome analysis of platelets in samples from dengue patients. Our study provides the first in-depth characterization of the platelet proteome in dengue, and sheds light on new mechanisms of platelet activation and platelet-mediated immune and inflammatory responses. PMID:28542641

Esophageal cancer alters the expression of nuclear pore complex binding protein Hsc70 and eIF5A-1.

PubMed

Moghanibashi, Mehdi; Rastgar Jazii, Ferdous; Soheili, Zahra-Soheila; Zare, Maryam; Karkhane, Aliasghar; Parivar, Kazem; Mohamadynejad, Parisa

2013-06-01

Nuclear pore complex (NPC) is the only corridor for macromolecules exchange between nucleus and cytoplasm. NPC and its components, nucleoporins, play important role in the diverse physiological processes including macromolecule exchange, chromosome segregation, apoptosis and gene expression. Recent reports also suggest involvement of nucleoporins in carcinogenesis. Applying proteomics, we analyzed expression pattern of the NPC components in a newly established esophageal cancer cell line from Persia (Iran), the high-risk region for esophageal cancer. Our results indicate overexpression of Hsc70 and downregulation of subunit alpha type-3 of proteasome, calpain small subunit 1, and eIF5A-1. Among these proteins, Hsc70 and eIF5A-1 are in direct interaction with NPC and involved in the nucleocytoplasmic exchange. Hsc70 plays a critical role as a chaperone in the formation of a cargo-receptor complex in nucleocytoplasmic transport. On the other hand, it is an NPC-associated protein that binds to nucleoporins and contributes in recycling of the nucleocytoplasmic transport receptors in mammals and affects transport of proteins between nucleus and cytoplasm. The other nuclear pore interacting protein: eIF5A-1 binds to the several nucleoporins and participates in nucleocytoplasmic transport. Altered expression of Hsc70 and eIF5A-1 may cause defects in nucleocytoplasmic transport and play a role in esophageal carcinogenesis.

FOXOs modulate proteasome activity in human-induced pluripotent stem cells of Huntington's disease and their derived neural cells.

PubMed

Liu, Yanying; Qiao, Fangfang; Leiferman, Patricia C; Ross, Alan; Schlenker, Evelyn H; Wang, Hongmin

2017-11-15

Although it has been speculated that proteasome dysfunction may contribute to the pathogenesis of Huntington's disease (HD), a devastating neurodegenerative disorder, how proteasome activity is regulated in HD affected stem cells and somatic cells remains largely unclear. To better understand the pathogenesis of HD, we analyzed proteasome activity and the expression of FOXO transcription factors in three wild-type (WT) and three HD induced-pluripotent stem cell (iPSC) lines. HD iPSCs exhibited elevated proteasome activity and higher levels of FOXO1 and FOXO4 proteins. Knockdown of FOXO4 but not FOXO1 expression decreased proteasome activity. Following neural differentiation, the HD-iPSC-derived neural progenitor cells (NPCs) demonstrated lower levels of proteasome activity and FOXO expressions than their WT counterparts. More importantly, overexpression of FOXO4 but not FOXO1 in HD NPCs dramatically enhanced proteasome activity. When HD NPCs were further differentiated into DARPP32-positive neurons, these HD neurons were more susceptible to death than WT neurons and formed Htt aggregates under the condition of oxidative stress. Similar to HD NPCs, HD-iPSC-derived neurons showed reduced proteasome activity and diminished FOXO4 expression compared to WT-iPSC-derived neurons. Furthermore, HD iPSCs had lower AKT activities than WT iPSCs, whereas the neurons derived from HD iPSC had higher AKT activities than their WT counterparts. Inhibiting AKT activity increased both FOXO4 level and proteasome activity, indicating a potential role of AKT in regulating FOXO levels. These data suggest that FOXOs modulate proteasome activity, and thus represents a potentially valuable therapeutic target for HD. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Differentially expressed proteins in nitric oxide-stimulated NIH/3T3 fibroblasts: implications for inhibiting cancer development.

PubMed

Shim, Dong Hwi; Lim, Joo Weon; Kim, Hyeyoung

2015-03-01

Recent evidence shows that nitric oxide (NO) may exhibit both pro-cancer and anti-cancer activities. The present study aimed to determine the differentially expressed proteins in NO-treated NIH/3T3 fibroblasts in order to investigate whether NO induces proteins with pro-cancer or anti-cancer effects. The cells were treated with 300 μM of an NO donor 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18) for 12 h. The changed protein patterns, which were separated by two-dimensional electrophoresis using pH gradients of 4-7, were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. Seventeen differentially expressed proteins were identified in NOC-18-treated cells. Nine proteins [vinculin protein, keratin 19, ubiquitous tropomodulin, F-actin capping protein (α1 subunit), tropomyosin 3, 26S proteasome-associated pad1 homolog, T-complex protein 1 (ε subunit) N(G)-dimethylarginine dimethylaminohydrolase, and heat shock protein 90] were increased and eight proteins (heat shock protein 70, glucosidase II, lamin B1, calreticulin, nucleophosmin 1, microtubule-associated protein retinitis pigmentosa/end binding family member 1, 150 kD oxygen-regulated protein precursor, and heat shock 70-related protein albino or pale green 2) were decreased by NOC-18 in the cells. Thirteen proteins are related to the suppression of cancer cell proliferation, invasion, and metastasis while two proteins (heat shock protein 90 and N(G)-dimethylarginine dimethylaminohydrolase) are related to carcinogenesis. The functions of 150 kD oxygen-regulated protein precursor and T-complex protein 1 (ε subunit) are unknown in relation to carcinogenesis. Most proteins differentially expressed by NOC-18 are involved in inhibiting cancer development.

The conserved RNA recognition motif and C3H1 domain of the Not4 ubiquitin ligase regulate in vivo ligase function.

PubMed

Chen, Hongfeng; Sirupangi, Tirupataiah; Wu, Zhao-Hui; Johnson, Daniel L; Laribee, R Nicholas

2018-05-25

The Ccr4-Not complex controls RNA polymerase II (Pol II) dependent gene expression and proteasome function. The Not4 ubiquitin ligase is a Ccr4-Not subunit that has both a RING domain and a conserved RNA recognition motif and C3H1 domain (referred to as the RRM-C domain) with unknown function. We demonstrate that while individual Not4 RING or RRM-C mutants fail to replicate the proteasomal defects found in Not4 deficient cells, mutation of both exhibits a Not4 loss of function phenotype. Transcriptome analysis revealed that the Not4 RRM-C affects a specific subset of Pol II-regulated genes, including those involved in transcription elongation, cyclin-dependent kinase regulated nutrient responses, and ribosomal biogenesis. The Not4 RING, RRM-C, or RING/RRM-C mutations cause a generalized increase in Pol II binding at a subset of these genes, yet their impact on gene expression does not always correlate with Pol II recruitment which suggests Not4 regulates their expression through additional mechanisms. Intriguingly, we find that while the Not4 RRM-C is dispensable for Ccr4-Not association with RNA Pol II, the Not4 RING domain is required for these interactions. Collectively, these data elucidate previously unknown roles for the conserved Not4 RRM-C and RING domains in regulating Ccr4-Not dependent functions in vivo.

Bortezomib: a novel therapy approved for multiple myeloma.

PubMed

Richardson, Paul G; Anderson, Kenneth C

2003-10-01

Cellular homeostasis requires routine degradation of key regulatory proteins, including tumor suppressor gene products, transcription factors, cell-cycle proteins and their inhibitors, as well as damaged and misfolded proteins. A critical part of this process is mediated by the 26S proteasome, a multi-subunit enzyme found in the nucleus and cytoplasm of all eukaryotic cells. Because of its essential role in many cellular processes controlling growth and survival, the proteasome has been identified as a potential target for cancer therapy. Drugs known to inhibit proteasome activity have been shown to induce cell-cycle arrest and programmed cell death (apoptosis). The impact of this finding is heightened by research showing that cancer cells are more sensitive to the proapoptotic effects of proteasome inhibition than normal cells. Preclinical evidence using bortezomib, the only proteasome inhibitor to enter clinical trials, suggests that proteasome inhibition may be effective in the treatment of hematologic and solid malignancies by promoting apoptosis, retarding angiogenesis, and inhibiting tumor cell adhesion and production of growth factors by acting on molecules such as nuclear factor-kappaB. Further preclinical evidence suggests that the antitumor effects of cytotoxic chemotherapy or radiotherapy may be enhanced by the addition of a proteasome inhibitor. Bortezomib was recently approved for the treatment of multiple myeloma. It is currently being investigated, both as a single agent and in combination, in phase I and II trials in a variety of tumor types.

Activation of Cell Surface Bound 20S Proteasome Inhibits Vascular Cell Growth and Arteriogenesis

PubMed Central

Ito, Wulf D.; Lund, Natalie; Zhang, Ziyang; Buck, Friedrich; Lellek, Heinrich; Horst, Andrea; Machens, Hans-Günther; Schunkert, Heribert; Schaper, Wolfgang; Meinertz, Thomas

2015-01-01

Arteriogenesis is an inflammatory process associated with rapid cellular changes involving vascular resident endothelial progenitor cells (VR-EPCs). Extracellular cell surface bound 20S proteasome has been implicated to play an important role in inflammatory processes. In our search for antigens initially regulated during collateral growth mAb CTA 157-2 was generated against membrane fractions of growing collateral vessels. CTA 157-2 stained endothelium of growing collateral vessels and the cell surface of VR-EPCs. CTA 157-2 bound a protein complex (760 kDa) that was identified as 26 kDa α7 and 21 kDa β3 subunit of 20S proteasome in mass spectrometry. Furthermore we demonstrated specific staining of 20S proteasome after immunoprecipitation of VR-EPC membrane extract with CTA 157-2 sepharose beads. Functionally, CTA 157-2 enhanced concentration dependently AMC (7-amino-4-methylcoumarin) cleavage from LLVY (N-Succinyl-Leu-Leu-Val-Tyr) by recombinant 20S proteasome as well as proteasomal activity in VR-EPC extracts. Proliferation of VR-EPCs (BrdU incorporation) was reduced by CTA 157-2. Infusion of the antibody into the collateral circulation reduced number of collateral arteries, collateral proliferation, and collateral conductance in vivo. In conclusion our results indicate that extracellular cell surface bound 20S proteasome influences VR-EPC function in vitro and collateral growth in vivo. PMID:26146628

PUB22 and PUB23 U-BOX E3 ligases directly ubiquitinate RPN6, a 26S proteasome lid subunit, for subsequent degradation in Arabidopsis thaliana.

PubMed

Cho, Seok Keun; Bae, Hansol; Ryu, Moon Young; Wook Yang, Seong; Kim, Woo TaeK

2015-09-04

Drought stress strongly affects plant growth and development, directly connected with crop yields, accordingly. However, related to the function of U-BOX E3 ligases, the underlying molecular mechanisms of desiccation stress response in plants are still largely unknown. Here we report that PUB22 and PUB23, two U-box E3 ligase homologs, tether ubiquitins to 19S proteasome regulatory particle (RP) subunit RPN6, leading to its degradation. RPN6 was identified as an interacting substrate of PUB22 by yeast two-hybrid screening, and in vitro pull-down assay confirmed that RPN6 interacts not only with PUB22, but also with PUB23. Both PUB22 and PUB23 were able to conjugate ubiquitins on RPN6 in vitro. Furthermore, RPN6 showed a shorter protein half-life in PUB22 overexpressing plants than in wild-type, besides RPN6 was significantly stabilized in pub22pub23 double knockout plants. Taken together, these results solidify a notion that PUB22 and PUB23 can alter the activity of 26S proteasome in response to drought stress. Copyright © 2015 Elsevier Inc. All rights reserved.

Curcumin inhibits the proteasome activity in human colon cancer cells in vitro and in vivo.

PubMed

Milacic, Vesna; Banerjee, Sanjeev; Landis-Piwowar, Kristin R; Sarkar, Fazlul H; Majumdar, Adhip P N; Dou, Q Ping

2008-09-15

Curcumin (diferuloylmethane) is the major active ingredient of turmeric (Curcuma longa) used in South Asian cuisine for centuries. Curcumin has been shown to inhibit the growth of transformed cells and to have a number of potential molecular targets. However, the essential molecular targets of curcumin under physiologic conditions have not been completely defined. Herein, we report that the tumor cellular proteasome is most likely an important target of curcumin. Nucleophilic susceptibility and in silico docking studies show that both carbonyl carbons of the curcumin molecule are highly susceptible to a nucleophilic attack by the hydroxyl group of the NH(2)-terminal threonine of the proteasomal chymotrypsin-like (CT-like) subunit. Consistently, curcumin potently inhibits the CT-like activity of a purified rabbit 20S proteasome (IC(50) = 1.85 micromol/L) and cellular 26S proteasome. Furthermore, inhibition of proteasome activity by curcumin in human colon cancer HCT-116 and SW480 cell lines leads to accumulation of ubiquitinated proteins and several proteasome target proteins, and subsequent induction of apoptosis. Furthermore, treatment of HCT-116 colon tumor-bearing ICR SCID mice with curcumin resulted in decreased tumor growth, associated with proteasome inhibition, proliferation suppression, and apoptosis induction in tumor tissues. Our study shows that proteasome inhibition could be one of the mechanisms for the chemopreventive and/or therapeutic roles of curcumin in human colon cancer. Based on its ability to inhibit the proteasome and induce apoptosis in both HCT-116 and metastatic SW480 colon cancer cell lines, our study suggests that curcumin could potentially be used for treatment of both early-stage and late-stage/refractory colon cancer.

Curcumin inhibits the proteasome activity in human colon cancer cells in vitro and in vivo

PubMed Central

Milacic, Vesna; Banerjee, Sanjeev; Landis-Piwowar, Kristin R.; Sarkar, Fazlul H.; Majumdar, Adhip P.N.; Dou, Q. Ping

2008-01-01

Curcumin (diferuloylmethane) is the major active ingredient of turmeric (curcuma longa) used in South Asian cuisine for centuries. Curcumin has been shown to inhibit the growth of transformed cells and to have a number of potential molecular targets. However, the essential molecular targets of curcumin under physiological conditions have not been completely defined. Herein, we report that the tumor cellular proteasome is most likely an important target of curcumin. Nucleophilic susceptibility and in silico docking studies show that both carbonyl carbons of the curcumin molecule are highly susceptible to a nucleophilic attack by the hydroxyl group of the N-terminal threonine of the proteasomal chymotrypsin-like subunit. Consistently, curcumin potently inhibits the chymotrypsin-like activity of a purified rabbit 20S proteasome (IC50=1.85 µM) and cellular 26S proteasome. Furthermore, inhibition of proteasome activity by curcumin in human colon cancer HCT-116 and SW480 cell lines leads to accumulation of ubiquitinated proteins and several proteasome target proteins, and subsequent induction of apoptosis. Furthermore, treatment of HCT-116 colon tumor–bearing ICR SCID mice with curcumin resulted in decreased tumor growth, associated with proteasome inhibition, proliferation suppression and apoptosis induction in tumor tissues. Our study demonstrates that proteasome inhibition could be one of the mechanisms for the chemopreventive and/or therapaeutic roles of curcumin in human colon cancer. Based on its ability to inhibit the proteasome and induce apoptosis in both HCT-116 and metastatic SW480 colon cancer cell lines, our study suggests that curcumin could potentially be used for treatment of both early stage and late stage/refractory colon cancer. PMID:18794115

Discovery of an Inhibitor of the Proteasome Subunit Rpn11.

PubMed

Perez, Christian; Li, Jing; Parlati, Francesco; Rouffet, Matthieu; Ma, Yuyong; Mackinnon, Andrew L; Chou, Tsui-Fen; Deshaies, Raymond J; Cohen, Seth M

2017-02-23

The proteasome plays a crucial role in degradation of normal proteins that happen to be constitutively or inducibly unstable, and in this capacity it plays a regulatory role. Additionally, it degrades abnormal/damaged/mutant/misfolded proteins, which serves a quality-control function. Inhibitors of the proteasome have been validated in the treatment of multiple myeloma, with several FDA-approved therapeutics. Rpn11 is a Zn 2+ -dependent metalloisopeptidase that hydrolyzes ubiquitin from tagged proteins that are trafficked to the proteasome for degradation. A fragment-based drug discovery (FBDD) approach was utilized to identify fragments with activity against Rpn11. Screening of a library of metal-binding pharmacophores (MBPs) revealed that 8-thioquinoline (8TQ, IC 50 value ∼2.5 μM) displayed strong inhibition of Rpn11. Further synthetic elaboration of 8TQ yielded a small molecule compound (35, IC 50 value ∼400 nM) that is a potent and selective inhibitor of Rpn11 that blocks proliferation of tumor cells in culture.

Structure and function based design of Plasmodium-selective proteasome inhibitors

PubMed Central

Li, Hao; O'Donoghue, Anthony J.; van der Linden, Wouter A.; Xie, Stanley C.; Yoo, Euna; Foe, Ian T.; Tilley, Leann; Craik, Charles S.; da Fonseca, Paula C. A.; Bogyo, Matthew

2016-01-01

The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation1. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle2-5. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome resulting in toxicity that precludes their use as therapeutic agents2,6. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, we used a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We designed inhibitors based on amino acid preferences specific to the parasite proteasome, and found that they preferentially inhibit the β 2 subunit. We determined the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy (cryo-EM) and single particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information regarding active site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin (ART) family anti-malarials7,8, we observed growth inhibition synergism with low doses of this β 2 selective inhibitor in ART sensitive and resistant parasites. Finally, we demonstrated that a parasite selective inhibitor could be used to attenuate parasite growth in vivo without significant toxicity to the host. Thus, the Plasmodium proteasome is a chemically tractable target that could be exploited by next generation anti-malarial agents. PMID:26863983

Structure- and function-based design of Plasmodium-selective proteasome inhibitors.

PubMed

Li, Hao; O'Donoghue, Anthony J; van der Linden, Wouter A; Xie, Stanley C; Yoo, Euna; Foe, Ian T; Tilley, Leann; Craik, Charles S; da Fonseca, Paula C A; Bogyo, Matthew

2016-02-11

The proteasome is a multi-component protease complex responsible for regulating key processes such as the cell cycle and antigen presentation. Compounds that target the proteasome are potentially valuable tools for the treatment of pathogens that depend on proteasome function for survival and replication. In particular, proteasome inhibitors have been shown to be toxic for the malaria parasite Plasmodium falciparum at all stages of its life cycle. Most compounds that have been tested against the parasite also inhibit the mammalian proteasome, resulting in toxicity that precludes their use as therapeutic agents. Therefore, better definition of the substrate specificity and structural properties of the Plasmodium proteasome could enable the development of compounds with sufficient selectivity to allow their use as anti-malarial agents. To accomplish this goal, here we use a substrate profiling method to uncover differences in the specificities of the human and P. falciparum proteasome. We design inhibitors based on amino-acid preferences specific to the parasite proteasome, and find that they preferentially inhibit the β2-subunit. We determine the structure of the P. falciparum 20S proteasome bound to the inhibitor using cryo-electron microscopy and single-particle analysis, to a resolution of 3.6 Å. These data reveal the unusually open P. falciparum β2 active site and provide valuable information about active-site architecture that can be used to further refine inhibitor design. Furthermore, consistent with the recent finding that the proteasome is important for stress pathways associated with resistance of artemisinin family anti-malarials, we observe growth inhibition synergism with low doses of this β2-selective inhibitor in artemisinin-sensitive and -resistant parasites. Finally, we demonstrate that a parasite-selective inhibitor could be used to attenuate parasite growth in vivo without appreciable toxicity to the host. Thus, the Plasmodium proteasome is a chemically tractable target that could be exploited by next-generation anti-malarial agents.

PSMB5 plays a dual role in cancer development and immunosuppression

PubMed Central

Wang, Chih-Yang; Li, Chung-Yen; Hsu, Hui-Ping; Cho, Chien-Yu; Yen, Meng-Chi; Weng, Tzu-Yang; Chen, Wei-Ching; Hung, Yu-Hsuan; Lee, Kuo-Ting; Hung, Jui-Hsiang; Chen, Yi-Ling; Lai, Ming-Derg

2017-01-01

Tumor progression and metastasis are dependent on the intrinsic properties of tumor cells and the influence of microenvironment including the immune system. It would be important to identify target drug that can inhibit cancer cell and activate immune cells. Proteasome β subunits (PSMB) family, one component of the ubiquitin-proteasome system, has been demonstrated to play an important role in tumor cells and immune cells. Therefore, we used a bioinformatics approach to examine the potential role of PSMB family. Analysis of breast TCGA and METABRIC database revealed that high expression of PSMB5 was observed in breast cancer tissue and that high expression of PSMB5 predicted worse survival. In addition, high expression of PSMB5 was observed in M2 macrophages. Based on our bioinformatics analysis, we hypothesized that PSMB5 contained immunosuppressive and oncogenic characteristics. To study the effects of PSMB5 on the cancer cell and macrophage in vitro, we silenced PSMB5 expression with shRNA in THP-1 monocytes and MDA-MB-231 cells respectively. Knockdown of PSMB5 promoted human THP-1 monocyte differentiation into M1 macrophage. On the other hand, knockdown PSMB5 gene expression inhibited MDA-MB-231 cell growth and migration by colony formation assay and boyden chamber. Collectively, our data demonstrated that delivery of PSMB5 shRNA suppressed cell growth and activated defensive M1 macrophages in vitro. Furthermore, lentiviral delivery of PSMB5 shRNA significantly decreased tumor growth in a subcutaneous mouse model. In conclusion, our bioinformatics study and functional experiments revealed that PSMB5 served as novel cancer therapeutic targets. These results also demonstrated a novel translational approach to improve cancer immunotherapy. PMID:29218236

Protease signaling through protease activated receptor 1 mediate nerve activation by mucosal supernatants from irritable bowel syndrome but not from ulcerative colitis patients

PubMed Central

Buhner, Sabine; Hahne, Hannes; Hartwig, Kerstin; Li, Qin; Vignali, Sheila; Ostertag, Daniela; Meng, Chen; Hörmannsperger, Gabriele; Braak, Breg; Pehl, Christian; Frieling, Thomas; Barbara, Giovanni; De Giorgio, Roberto; Demir, Ihsan Ekin; Ceyhan, Güralp Onur; Zeller, Florian; Boeckxstaens, Guy; Haller, Dirk; Kuster, Bernhard

2018-01-01

Background & aims The causes of gastrointestinal complaints in irritable bowel syndrome (IBS) remain poorly understood. Altered nerve function has emerged as an important pathogenic factor as IBS mucosal biopsy supernatants consistently activate enteric and sensory neurons. We investigated the neurally active molecular components of such supernatants from patients with IBS and quiescent ulcerative colitis (UC). Method Effects of supernatants from 7 healthy controls (HC), 20 IBS and 12 UC patients on human and guinea pig submucous neurons were studied with neuroimaging techniques. We identify differentially expressed proteins with proteome analysis. Results Nerve activation by IBS supernatants was prevented by the protease activated receptor 1 (PAR1) antagonist SCHE79797. UC supernatants also activated enteric neurons through protease dependent mechanisms but without PAR1 involvement. Proteome analysis of the supernatants identified 204 proteins, among them 17 proteases as differentially expressed between IBS, UC and HC. Of those the four proteases elastase 3a, chymotrypsin C, proteasome subunit type beta-2 and an unspecified isoform of complement C3 were significantly more abundant in IBS compared to HC and UC supernatants. Of eight proteases, which were upregulated in IBS, the combination of elastase 3a, cathepsin L and proteasome alpha subunit-4 showed the highest prediction accuracy of 98% to discriminate between IBS and HC groups. Elastase synergistically potentiated the effects of histamine and serotonin–the two other main neuroactive substances in the IBS supernatants. A serine protease inhibitor isolated from the probiotic Bifidobacterium longum NCC2705 (SERPINBL), known to inhibit elastase-like proteases, prevented nerve activation by IBS supernatants. Conclusion Proteases in IBS and UC supernatants were responsible for nerve activation. Our data demonstrate that proteases, particularly those signalling through neuronal PAR1, are biomarker candidates for IBS, and protease profiling may be used to characterise IBS. PMID:29529042

Aggresomes formed by alpha-synuclein and synphilin-1 are cytoprotective.

PubMed

Tanaka, Mikiei; Kim, Yong Man; Lee, Gwang; Junn, Eunsung; Iwatsubo, Takeshi; Mouradian, M Maral

2004-02-06

Lewy bodies (LBs), which are the hallmark pathologic features of Parkinson's disease and of dementia with LBs, have several morphologic and molecular similarities to aggresomes. Whether such cytoplasmic inclusions contribute to neuronal death or protect cells from the toxic effects of misfolded proteins remains controversial. In this report, the role of aggresomes in cell viability was addressed in the context of over-expressing alpha-synuclein and its interacting partner synphilin-1 using engineered 293T cells. Inhibition of proteasome activity elicited the formation of juxtanuclear aggregates with characteristics of aggresomes including immunoreactivity for vimentin, gamma-tubulin, ubiquitin, proteasome subunit, and hsp70. As expected from the properties of aggresomes, the microtubule disrupting agents, vinblastin and nocodazole, markedly prevented the formation of these inclusions. Similar to LBs, the phosphorylated form of alpha-synuclein co-localized in these synphilin-1-containing aggresomes. Although the caspase inhibitor z-VAD-fmk significantly reduced the number of apoptotic cells, it had no impact on the percentage of aggresome-positive cells. Finally, quantitative analysis revealed aggresomes in 60% of nonapoptotic cells but only in 10% of apoptotic cells. Additionally, alpha-synuclein-induced apoptosis was not coupled with increased prevalence of aggresome-bearing cells. Taken together, these observations indicate a disconnection between aggresome formation and apoptosis, and support a protective role for these inclusions from the toxicity associated with the combined over-expression of alpha-synuclein and synphilin-1.

Chronic stress targets posttranscriptional mechanisms to rapidly upregulate α1C-subunit of Cav1.2b calcium channels in colonic smooth muscle cells.

PubMed

Li, Qingjie; Sarna, Sushil K

2011-01-01

Chronic stress elevates plasma norepinephrine, which enhances expression of the α(1C)-subunit of Ca(v)1.2b channels in colonic smooth muscle cells within 1 h. Transcriptional upregulation usually does not explain such rapid protein synthesis. We investigated whether chronic stress-induced release of norepinephrine utilizes posttranscriptional mechanisms to enhance the α(1C)-subunit. We performed experiments on colonic circular smooth muscle strips and in conscious rats, using a 9-day chronic intermittent stress protocol. Incubation of rat colonic muscularis externa with norepinephrine enhanced α(1C)-protein expression within 45 min, without a concomitant increase in α(1C) mRNA, indicating posttranscriptional regulation of α(1C)-protein by norepinephrine. We found that norepinephrine activates the PI3K/Akt/GSK-3β pathway to concurrently enhance α(1C)-protein translation and block its polyubiquitination and proteasomal degradation. Incubation of colonic muscularis externa with norepinephrine or LiCl, which inhibits GSK-3β, enhanced p-GSK-3β and α(1C)-protein time dependently. Using enrichment of phosphoproteins and ubiquitinated proteins, we found that both norepinephrine and LiCl decrease α(1C) phosphorylation and polyubiquitination. Concurrently, they suppress eIF2α (Ser51) phosphorylation and 4E-BP1 expression, which stimulates gene-specific translation. The antagonism of two upstream kinases, PI3K and Akt, inhibits the induction of α(1C)-protein by norepinephrine. Cyanopindolol (β(3)-AR-antagonist) almost completely suppresses and propranolol (β(1/2)-AR antagonist) partially suppresses norepinephrine-induced α(1C)-protein expression, whereas phentolamine and prazosin (α-AR and α(1)-AR antagonist, respectively) have no significant effect. Experiments in conscious animals showed that chronic stress activates the PI3K/Akt/GSK-3β signaling. We conclude that norepinephrine released by chronic stress rapidly enhances the protein expression of α(1C)-subunit of Ca(v)1.2b channels by concurrently suppressing its degradation and enhancing translation of existing transcripts to maintain homeostasis.

Association of growth factors, HIF-1 and NF-κB expression with proteasomes in endometrial cancer.

PubMed

Spirina, Ludmila V; Yunusova, Nataliya V; Kondakova, Irina V; Kolomiets, Larisa A; Koval, Valeriya D; Chernyshova, Alena L; Shpileva, Olga V

2012-09-01

Insulin-like growth factors (IGFs), vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1 (HIF-1), and nuclear factor kappa-B (NF-κB) are known to play an important role in endometrial cancer pathogenesis. However, the proteolytic regulation of these factors is still poorly understood. We studied the correlation between chymotrypsin-like activity of proteasomes and IGF-I, IGF-II, VEGF, HIF-1, and NF-κB levels in endometrial cancer tissues. It was shown that the total activity of proteasomes and the activity of the 20S and 26S proteasomes in malignant tumors were significantly higher than those observed in the normal endometrium. Negative relationships between the proteasome activity and IGF-I, HIF-1, and NF-κB p50 expressions were found. High 20S proteasome activity was associated with increase of HIF-1 level. Positive relationships between IGF-I expression and two classic forms of NF-κB p50 and p65 in endometrial cancer were revealed. The data obtained indicate the possible proteasomal regulation of growth and transcription factors. The major pool of IGF-I is located in the extracellular space, and it is likely that extracellular proteasomes also take part in the regulation of the IGF-I content. The present data show the evidence of proteasome regulation of growth and nuclear factors that can play an important role in cancer pathogenesis.

Betulinic acid inhibits colon cancer cell and tumor growth and induces proteasome-dependent and -independent downregulation of specificity proteins (Sp) transcription factors

PubMed Central

2011-01-01

Background Betulinic acid (BA) inhibits growth of several cancer cell lines and tumors and the effects of BA have been attributed to its mitochondriotoxicity and inhibition of multiple pro-oncogenic factors. Previous studies show that BA induces proteasome-dependent degradation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 in prostate cancer cells and this study focused on the mechanism of action of BA in colon cancer cells. Methods The effects of BA on colon cancer cell proliferation and apoptosis and tumor growth in vivo were determined using standardized assays. The effects of BA on Sp proteins and Sp-regulated gene products were analyzed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a) and ZBTB10 mRNA expression. Results BA inhibited growth and induced apoptosis in RKO and SW480 colon cancer cells and inhibited tumor growth in athymic nude mice bearing RKO cells as xenograft. BA also decreased expression of Sp1, Sp3 and Sp4 transcription factors which are overexpressed in colon cancer cells and decreased levels of several Sp-regulated genes including survivin, vascular endothelial growth factor, p65 sub-unit of NFκB, epidermal growth factor receptor, cyclin D1, and pituitary tumor transforming gene-1. The mechanism of action of BA was dependent on cell context, since BA induced proteasome-dependent and proteasome-independent downregulation of Sp1, Sp3 and Sp4 in SW480 and RKO cells, respectively. In RKO cells, the mechanism of BA-induced repression of Sp1, Sp3 and Sp4 was due to induction of reactive oxygen species (ROS), ROS-mediated repression of microRNA-27a, and induction of the Sp repressor gene ZBTB10. Conclusions These results suggest that the anticancer activity of BA in colon cancer cells is due, in part, to downregulation of Sp1, Sp3 and Sp4 transcription factors; however, the mechanism of this response is cell context-dependent. PMID:21864401

Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes

PubMed Central

Ebstein, F.; Textoris-Taube, K.; Keller, C.; Golnik, R.; Vigneron, N.; Van den Eynde, B. J.; Schuler-Thurner, B.; Schadendorf, D.; Lorenz, F. K. M.; Uckert, W.; Urban, S.; Lehmann, A.; Albrecht-Koepke, N.; Janek, K.; Henklein, P.; Niewienda, A.; Kloetzel, P. M.; Mishto, M.

2016-01-01

Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8+ T cell response. Importantly, we demonstrate that different gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes. PMID:27049119

High Levels of Serum Ubiquitin and Proteasome in a Case of HLA-B27 Uveitis.

PubMed

Rossi, Settimio; Gesualdo, Carlo; Maisto, Rosa; Trotta, Maria Consiglia; Di Carluccio, Nadia; Brigida, Annalisa; Di Iorio, Valentina; Testa, Francesco; Simonelli, Francesca; D'Amico, Michele; Di Filippo, Clara

2017-02-26

In this paper, the authors describe a case of high serum levels of ubiquitin and proteasome in a woman under an acute attack of autoimmune uveitis. The woman was 52 years old, diagnosed as positive for the Human leukocyte antigen-B27 gene, and came to our observation in January 2013 claiming a severe uveitis attack that involved the right eye. During the acute attack of uveitis, this woman had normal serum biochemical parameters but higher levels of serum ubiquitin and proteasome 20S subunit, with respect to a healthy volunteer matched for age and sex. These levels correlated well with the clinical score attributed to uveitis. After the patient was admitted to therapy, she received oral prednisone in a de-escalation protocol (doses from 50 to 5 mg/day) for four weeks. Following this therapy, she had an expected reduction of clinical signs and score for uveitis, but concomitantly she had a reduction of the serum levels of ubiquitin, poliubiquitinated proteins (MAb-FK1) and proteasome 20S activity. Therefore, a role for ubiquitin and proteasome in the development of human autoimmune uveitis has been hypothesized.

The genomic landscape of human cellular circadian variation points to a novel role for the signalosome

PubMed Central

Gaspar, Ludmila; Howald, Cedric; Popadin, Konstantin; Maier, Bert; Mauvoisin, Daniel; Moriggi, Ermanno; Gutierrez-Arcelus, Maria; Falconnet, Emilie; Borel, Christelle; Kunz, Dieter; Kramer, Achim; Gachon, Frederic; Dermitzakis, Emmanouil T; Antonarakis, Stylianos E

2017-01-01

The importance of natural gene expression variation for human behavior is undisputed, but its impact on circadian physiology remains mostly unexplored. Using umbilical cord fibroblasts, we have determined by genome-wide association how common genetic variation impacts upon cellular circadian function. Gene set enrichment points to differences in protein catabolism as one major source of clock variation in humans. The two most significant alleles regulated expression of COPS7B, a subunit of the COP9 signalosome. We further show that the signalosome complex is imported into the nucleus in timed fashion to stabilize the essential circadian protein BMAL1, a novel mechanism to oppose its proteasome-mediated degradation. Thus, circadian clock properties depend in part upon a genetically-encoded competition between stabilizing and destabilizing forces, and genetic alterations in these mechanisms provide one explanation for human chronotype. PMID:28869038

Teaching resources. Model of the TIR1 pathway for auxin-mediated gene expression.

PubMed

Laskowski, Marta

2006-02-14

Auxin mediates numerous plant responses, some of which have been shown to require transcriptional regulation. One auxin response pathway, which depends on the relief of transcriptional repression, is mediated by TIR1 (transport inhibitor response protein 1). TIR1 is an auxin receptor and also a subunit of an SCF-type ubiquitin ligase. In the presence of a low concentration of auxin in the nucleus, members of the Aux/IAA family of transcriptional repressors bind to ARF proteins and inhibit the transcription of specific auxin response genes. Increased nuclear concentrations of auxin promote auxin binding to TIR1, causing the Aux/IAA proteins to associate with TIR1 and leading to their degradation by a proteasome-mediated pathway. This decreases the concentration of Aux/IAA proteins in the nucleus and thereby enables the expression of certain auxin response genes.

Proteasome Activation is Mediated via a Functional Switch of the Rpt6 C-terminal Tail Following Chaperone-dependent Assembly

PubMed Central

Sokolova, Vladyslava; Li, Frances; Polovin, George; Park, Soyeon

2015-01-01

In the proteasome, the proteolytic 20S core particle (CP) associates with the 19S regulatory particle (RP) to degrade polyubiquitinated proteins. Six ATPases (Rpt1-Rpt6) of the RP form a hexameric Rpt ring and interact with the heptameric α ring (α1–α7) of the CP via the Rpt C-terminal tails individually binding to the α subunits. Importantly, the Rpt6 tail has been suggested to be crucial for RP assembly. Here, we show that the interaction of the CP and Rpt6 tail promotes a CP-Rpt3 tail interaction, and that they jointly mediate proteasome activation via opening the CP gate for substrate entry. The Rpt6 tail forms a novel relationship with the Nas6 chaperone, which binds to Rpt3 and regulates the CP-Rpt3 tail interaction, critically influencing cell growth and turnover of polyubiquitinated proteins. CP-Rpt6 tail binding promotes the release of Nas6 from the proteasome. Based on disulfide crosslinking that detects cognate α3-Rpt6 tail and α2-Rpt3 tail interactions in the proteasome, decreased α3-Rpt6 tail interaction facilitates robust α2-Rpt3 tail interaction that is also strongly ATP-dependent. Together, our data support the reported role of Rpt6 during proteasome assembly, and suggest that its function switches from anchoring for RP assembly into promoting Rpt3-dependent activation of the mature proteasome. PMID:26449534

Patterns of gene expression in atrophying skeletal muscles: response to food deprivation

NASA Technical Reports Server (NTRS)

Jagoe, R. Thomas; Lecker, Stewart H.; Gomes, Marcelo; Goldberg, Alfred L.

2002-01-01

During fasting and many systemic diseases, muscle undergoes rapid loss of protein and functional capacity. To define the transcriptional changes triggering muscle atrophy and energy conservation in fasting, we used cDNA microarrays to compare mRNAs from muscles of control and food-deprived mice. Expression of >94% of genes did not change, but interesting patterns emerged among genes that were differentially expressed: 1) mRNAs encoding polyubiquitin, ubiquitin extension proteins, and many (but not all) proteasome subunits increased, which presumably contributes to accelerated protein breakdown; 2) a dramatic increase in mRNA for the ubiquitin ligase, atrogin-1, but not most E3s; 3) a significant suppression of mRNA for myosin binding protein H (but not other myofibrillar proteins) and IGF binding protein 5, which may favor cell protein loss; 4) decreases in mRNAs for several glycolytic enzymes and phosphorylase kinase subunits, and dramatic increases in mRNAs for pyruvate dehydrogenase kinase 4 and glutamine synthase, which should promote glucose sparing and gluconeogenesis. During fasting, metallothionein mRNA increased dramatically, mRNAs for extracellular matrix components fell, and mRNAs that may favor cap-independent mRNA translation rose. Significant changes occurred in mRNAs for many growth-related proteins and transcriptional regulators. These transcriptional changes indicate a complex adaptive program that should favor protein degradation and suppress glucose oxidation in muscle. Similar analysis of muscles atrophying for other causes is allowing us to identify a set of atrophy-specific changes in gene expression.

Expression of regulatory proteins in choroid plexus changes in early stages of Alzheimer disease.

PubMed

Krzyzanowska, Agnieszka; García-Consuegra, Inés; Pascual, Consuelo; Antequera, Desiree; Ferrer, Isidro; Carro, Eva

2015-04-01

Recent studies indicate that the choroid plexus has important physiologic and pathologic roles in Alzheimer disease (AD). To obtain additional insight on choroid plexus function, we performed a proteomic analysis of choroid plexus samples from patients with AD stages I to II (n = 16), III to IV (n = 16), and V to VI (n = 11) and 7 age-matched control subjects. We used 2-dimensional differential gel electrophoresis coupled with mass spectrometry to generate a complete picture of changes in choroid plexus protein expression occurring in AD patients. We identified 6 proteins: 14-3-3 β/α, 14-3-3 ε, moesin, proteasome activator complex subunit 1, annexin V, and aldehyde dehydrogenase, which were significantly regulated in AD patient samples (p < 0.05, >1.5-fold variation in expression vs control samples). These proteins are implicated in major physiologic functions including mitochondrial dysfunction and apoptosis regulation. These findings contribute additional significance to the emerging importance of molecular and functional changes of choroid plexus function in the pathophysiology of AD.

Upregulation of Immunoproteasome Subunits in Myositis Indicates Active Inflammation with Involvement of Antigen Presenting Cells, CD8 T-Cells and IFNγ

PubMed Central

Ghannam, Khetam; Martinez-Gamboa, Lorena; Spengler, Lydia; Krause, Sabine; Smiljanovic, Biljana; Bonin, Marc; Bhattarai, Salyan; Grützkau, Andreas; Burmester, Gerd-R.

2014-01-01

Objective In idiopathic inflammatory myopathies (IIM) infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers. Methods Expression of constitutive (PSMB5, -6, -7) and corresponding immunoproteasomal subunits (PSMB8, -9, -10) was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM) and healthy donors (HD). Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses. Results Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10) in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood. Conclusions Immunoproteasomes seem to indicate IIM activity and suggest that dominant involvement of antigen processing and presentation may qualify these diseases exemplarily for the evolving therapeutic concepts of immunoproteasome specific inhibition. PMID:25098831

Upregulation of immunoproteasome subunits in myositis indicates active inflammation with involvement of antigen presenting cells, CD8 T-cells and IFNΓ.

PubMed

Ghannam, Khetam; Martinez-Gamboa, Lorena; Spengler, Lydia; Krause, Sabine; Smiljanovic, Biljana; Bonin, Marc; Bhattarai, Salyan; Grützkau, Andreas; Burmester, Gerd-R; Häupl, Thomas; Feist, Eugen

2014-01-01

In idiopathic inflammatory myopathies (IIM) infiltration of immune cells into muscle and upregulation of MHC-I expression implies increased antigen presentation and involvement of the proteasome system. To decipher the role of immunoproteasomes in myositis, we investigated individual cell types and muscle tissues and focused on possible immune triggers. Expression of constitutive (PSMB5, -6, -7) and corresponding immunoproteasomal subunits (PSMB8, -9, -10) was analyzed by real-time RT-PCR in muscle biopsies and sorted peripheral blood cells of patients with IIM, non-inflammatory myopathies (NIM) and healthy donors (HD). Protein analysis in muscle biopsies was performed by western blot. Affymetrix HG-U133 platform derived transcriptome data from biopsies of different muscle diseases and from immune cell types as well as monocyte stimulation experiments were used for validation, coregulation and coexpression analyses. Real-time RT-PCR revealed significantly increased expression of immunoproteasomal subunits (PSMB8/-9/-10) in DC, monocytes and CD8+ T-cells in IIM. In muscle biopsies, the immunosubunits were elevated in IIM compared to NIM and exceeded levels of matched blood samples. Proteins of PSMB8 and -9 were found only in IIM but not NIM muscle biopsies. Reanalysis of 78 myositis and 20 healthy muscle transcriptomes confirmed these results and revealed involvement of the antigen processing and presentation pathway. Comparison with reference profiles of sorted immune cells and healthy muscle confirmed upregulation of PSMB8 and -9 in myositis biopsies beyond infiltration related changes. This upregulation correlated highest with STAT1, IRF1 and IFNγ expression. Elevation of T-cell specific transcripts in active IIM muscles was accompanied by increased expression of DC and monocyte marker genes and thus reflects the cell type specific involvement observed in peripheral blood. Immunoproteasomes seem to indicate IIM activity and suggest that dominant involvement of antigen processing and presentation may qualify these diseases exemplarily for the evolving therapeutic concepts of immunoproteasome specific inhibition.

Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome

PubMed Central

Shi, Yuan; Chen, Xiang; Elsasser, Suzanne; Stocks, Bradley B.; Tian, Geng; Lee, Byung-Hoon; Shi, Yanhong; Zhang, Naixia; de Poot, Stefanie A. H.; Tuebing, Fabian; Sun, Shuangwu; Vannoy, Jacob; Tarasov, Sergey G.; Engen, John R.; Finley, Daniel; Walters, Kylie J.

2016-01-01

Structured Abstract INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly-associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced, multipoint binding of ubiquitin chains. The structures of the T1 site in its free state and complexed with monoubiquitin or K48-linked diubiquitin were solved, revealing that three neighboring outer helices from the T1 toroid engage two ubiquitins. This binding mode leads to a preference for certain ubiquitin chain types, especially K6- and K48-linked chains, in a distinct configuration that can position substrates close to the entry port of the proteasome. The fate of proteasome-docked ubiquitin conjugates is determined by a competition between deubiquitination and substrate degradation. We find that proximal to the T1 site within the Rpn1 toroid is a second UBL-binding site, T2, that does not assist in ubiquitin chain recognition, but rather in chain disassembly, by binding to the UBL domain of deubiquitinating enzyme Ubp6. Importantly, the UBL interactors at T1 and T2 are distinct, assigning substrate localization to T1 and substrate deubiquitination to T2. CONCLUSION A ligand-binding hotspot was identified in the Rpn1 toroid, consisting of two adjacent receptor sites, T1 and T2. The Rpn1 toroid represents a novel class of binding domains for ubiquitin and UBL proteins. This study thus defines a novel two-site recognition domain intrinsic to the proteasome that uses homologous ubiquitin/UBL-class ligands to assemble substrates, substrate shuttling factors, and a deubiquitinating enzyme in close proximity. A ligand-binding hotspot in the proteasome for assembling substrates and cofactors Schematic (top) and model structure (bottom, left) mapping the UBL-binding Rpn1 T1 (indigo) and T2 (orange) sites. (Bottom, right) Enlarged region of the proteasome to illustrate the Rpn1 T1 and T2 sites bound to a ubiquitin chain (yellow) and deubiquitinating enzyme Ubp6 (green), respectively. PDB 4CR2 and 2B9R were used for this figure. Hundreds of pathways for degradation converge at ubiquitin recognition by proteasome. Here we found that the five known proteasomal ubiquitin receptors are collectively nonessential for ubiquitin recognition, and identified a sixth receptor, Rpn1. A site (T1) in the Rpn1 toroid recognized ubiquitin and ubiquitin-like (UBL) domains of substrate shuttling factors. T1 structures with monoubiquitin or K48 diubiquitin show three neighboring outer helices engaging two ubiquitins. T1 contributes a distinct substrate-binding pathway with preference for K48-linked chains. Proximal to T1 within the Rpn1 toroid is a second UBL-binding site (T2) that assists in ubiquitin chain disassembly, by binding the UBL of deubiquitinating enzyme Ubp6. Thus a two-site recognition domain intrinsic to the proteasome uses homologous ubiquitin/UBL-class ligands to assemble substrates, shuttling factors, and a deubiquitinating enzyme. PMID:26912900

The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

PubMed Central

Halter, David; Collart, Martine A.; Panasenko, Olesya O.

2014-01-01

Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome. PMID:24465968

Activation of the ATP-ubiquitin-proteasome pathway in skeletal muscle of cachectic rats bearing a hepatoma

NASA Technical Reports Server (NTRS)

Baracos, V. E.; DeVivo, C.; Hoyle, D. H.; Goldberg, A. L.

1995-01-01

Rats implanted with Yoshida ascites hepatoma (YAH) show a rapid and selective loss of muscle protein due mainly to a marked increase (63-95%) in the rate of protein degradation (compared with rates in muscles of pair-fed controls). To define which proteolytic pathways contribute to this increase, epitrochlearis muscles from YAH-bearing and control rats were incubated under conditions that modify different proteolytic systems. Overall proteolysis in either group of rats was not affected by removal of Ca2+ or by blocking the Ca(2+)-dependent proteolytic system. Inhibition of lysosomal function with methylamine reduced proteolysis (-12%) in muscles from YAH-bearing rats, but not in muscles of pair-fed rats. When ATP production was also inhibited, the remaining accelerated proteolysis in muscles of tumor-bearing rats fell to control levels. Muscles of YAH-bearing rats showed increased levels of ubiquitin-conjugated proteins and a 27-kDa proteasome subunit in Western blot analysis. Levels of mRNA encoding components of proteolytic systems were quantitated using Northern hybridization analysis. Although their total RNA content decreased 20-38%, pale muscles of YAH-bearing rats showed increased levels of ubiquitin mRNA (590-880%) and mRNA for multiple subunits of the proteasome (100-215%). Liver, kidney, heart, and brain showed no weight loss and no change in these mRNA species. Muscles of YAH-bearing rats also showed small increases (30-40%) in mRNA for cathepsins B and D, but not for calpain I or heat shock protein 70. Our findings suggest that accelerated muscle proteolysis and muscle wasting in tumor-bearing rats result primarily from activation of the ATP-dependent pathway involving ubiquitin and the proteasome.

Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence

DOE Office of Scientific and Technical Information (OSTI.GOV)

Byrne, Ann; McLaren, Rajashree P.; Mason, Paul

2010-01-15

The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21{sup /Cip} and p27{sup /Kip1}. Mostmore » notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.« less

The yeast genome may harbor hypoxia response elements (HRE).

PubMed

Ferreira, Túlio César; Hertzberg, Libi; Gassmann, Max; Campos, Elida Geralda

2007-01-01

The hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor activated when cells are submitted to hypoxia. The heterodimer is composed of two subunits, HIF-1alpha and the constitutively expressed HIF-1beta. During normoxia, HIF-1alpha is degraded by the 26S proteasome, but hypoxia causes HIF-1alpha to be stabilized, enter the nucleus and bind to HIF-1beta, thus forming the active complex. The complex then binds to the regulatory sequences of various genes involved in physiological and pathological processes. The specific regulatory sequence recognized by HIF-1 is the hypoxia response element (HRE) that has the consensus sequence 5'BRCGTGVBBB3'. Although the basic transcriptional regulation machinery is conserved between yeast and mammals, Saccharomyces cerevisiae does not express HIF-1 subunits. However, we hypothesized that baker's yeast has a protein analogous to HIF-1 which participates in the response to changes in oxygen levels by binding to HRE sequences. In this study we screened the yeast genome for HREs using probabilistic motif search tools. We described 24 yeast genes containing motifs with high probability of being HREs (p-value<0.1) and classified them according to biological function. Our results show that S. cerevisiae may harbor HREs and indicate that a transcription factor analogous to HIF-1 may exist in this organism.

Differential global structural changes in the core particle of yeast and mouse proteasome induced by ligand binding

PubMed Central

Arciniega, Marcelino; Beck, Philipp; Lange, Oliver F.; Groll, Michael; Huber, Robert

2014-01-01

Two clusters of configurations of the main proteolytic subunit β5 were identified by principal component analysis of crystal structures of the yeast proteasome core particle (yCP). The apo-cluster encompasses unliganded species and complexes with nonpeptidic ligands, and the pep-cluster comprises complexes with peptidic ligands. The murine constitutive CP structures conform to the yeast system, with the apo-form settled in the apo-cluster and the PR-957 (a peptidic ligand) complex in the pep-cluster. In striking contrast, the murine immune CP classifies into the pep-cluster in both the apo and the PR-957–liganded species. The two clusters differ essentially by multiple small structural changes and a domain motion enabling enclosure of the peptidic ligand and formation of specific hydrogen bonds in the pep-cluster. The immune CP species is in optimal peptide binding configuration also in its apo form. This favors productive ligand binding and may help to explain the generally increased functional activity of the immunoproteasome. Molecular dynamics simulations of the representative murine species are consistent with the experimentally observed configurations. A comparison of all 28 subunits of the unliganded species with the peptidic liganded forms demonstrates a greatly enhanced plasticity of β5 and suggests specific signaling pathways to other subunits. PMID:24979800

Tripeptidyl peptidase II is dispensable for the generation of both proteasome-dependent and proteasome-independent ligands of HLA-B27 and other class I molecules.

PubMed

Marcilla, Miguel; Villasevil, Eugenia M; de Castro, José Antonio López

2008-03-01

A significant fraction of the HLA-B27-bound peptide repertoire is resistant to proteasome inhibitors. The possible implication of tripeptidyl peptidase II (TPPII) in generating this subset was analyzed by quantifying the surface re-expression of HLA-B*2705 after acid stripping in the presence of two TPPII inhibitors, butabindide and Ala-Ala-Phe-chloromethylketone. Neither decreased HLA-B27 re-expression under conditions in which TPPII activity was largely inhibited. This was in contrast to a significant effect of the proteasome inhibitor epoxomicin. The failure of TPPII inhibition to decrease surface re-expression was not limited to HLA-B27, since it was also observed in several HLA-B27-negative cell lines, including Mel JuSo. Actually, HLA class I re-expression in Mel JuSo cells increased as a function of butabindide concentration, which is consistent with an involvement of TPPII in destroying HLA class I ligands. Inhibition of TPPII with small interfering RNA also failed to decrease the surface expression of HLA class I molecules on 143B cells. Our results indicate that TPPII is dispensable for the generation of proteasome-dependent HLA class I ligands and, without excluding its role in producing some individual epitopes, this enzyme is not involved to any quantitatively significant extent, in generating the proteasome-independent HLA-B27-bound peptide repertoire.

Silencing Hsp25/Hsp27 gene expression augments proteasome activity and increases CD8+ T-cell-mediated tumor killing and memory responses.

PubMed

Nagaraja, Ganachari M; Kaur, Punit; Neumann, William; Asea, Edwina E; Bausero, María A; Multhoff, Gabriele; Asea, Alexzander

2012-01-01

Relatively high expression of Hsp27 in breast and prostate cancer is a predictor of poor clinical outcome. This study elucidates a hitherto unknown mechanism by which Hsp27 regulates proteasome function and modulates tumor-specific T-cell responses. Here, we showed that short-term silencing of Hsp25 or Hsp27 using siRNA or permanent silencing of Hsp25 using lentivirus RNA interference technology enhanced PA28α mRNA expression, PA28α protein expression, and proteasome activity; abrogated metastatic potential; induced the regression of established breast tumors by tumor-specific CD8(+) T cells; and stimulated long-lasting memory responses. The adoptive transfer of reactive CD8(+) T cells from mice bearing Hsp25-silenced tumors efficiently induced the regression of established tumors in nontreated mice which normally succumb to tumor burden. The overexpression of Hsp25 and Hsp27 resulted in the repression of normal proteasome function, induced poor antigen presentation, and resulted in increased tumor burden. Taken together, this study establishes a paradigm shift in our understanding of the role of Hsp27 in the regulation of proteasome function and tumor-specific T-cell responses and paves the way for the development of molecular targets to enhance proteasome function and concomitantly inhibit Hsp27 expression in tumors for therapeutic gain. ©2011 AACR.

Silencing hsp25/hsp27 gene expression augments proteasome activity and increases CD8+ T cell-mediated tumor killing and memory responses

PubMed Central

Nagaraja, Ganachari M.; Kaur, Punit; Neumann, William; Asea, Edwina E.; Bausero, María A.; Multhoff, Gabriele; Asea, Alexzander

2011-01-01

Relatively high expression of Hsp27 in breast and prostate cancer is a predictor of poor clinical outcome. This study elucidates a hitherto unknown mechanism by which Hsp27 regulates proteasome function and modulates tumor-specific T cell responses. Here we demonstrated that short term silencing of Hsp25 or Hsp27 using siRNA or permanent silencing of Hsp25 using lentivirus-RNAi technology enhanced PA28α mRNA expression, PA28α protein expression, proteasome activity, abrogated metastatic potential, induced the regression of established breast tumors by tumor-specific CD8+ T cells and stimulated long-lasting memory responses. The adoptive transfer of reactive CD8+ T cells from mice bearing Hsp25-silenced tumors efficiently induced the regression of established tumors in non-treated mice which normally succumb to tumor burden. The overexpression of Hsp25 and Hsp27 resulted in the repression of normal proteasome function, induced poor antigen presentation and resulted in increased tumor burden. Taken together, this study establishes a paradigm shift in our understanding of the role of Hsp27 in the regulation of proteasome function and tumor-specific T cell responses and paves the way for the development of molecular targets to enhance proteasome function and concomitantly inhibit Hsp27 expression in tumors for therapeutic gain. PMID:22185976

Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants

PubMed Central

Greenwood, Edward JD; Matheson, Nicholas J; Wals, Kim; van den Boomen, Dick JH; Antrobus, Robin; Williamson, James C; Lehner, Paul J

2016-01-01

Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function. DOI: http://dx.doi.org/10.7554/eLife.18296.001 PMID:27690223

PROTEASOME INHIBITOR TREATMENT REDUCED FATTY ACID, TRIACYLGLYCEROL AND CHOLESTEROL SYNTHESIS

PubMed Central

Oliva, Joan; French, Samuel W.; Li, Jun; Bardag-Gorce, Fawzia

2014-01-01

In the present study, the beneficial effects of proteasome inhibitor treatment in reducing ethanol-induced steatosis were investigated. A microarray analysis was performed on the liver of rats injected with PS-341 (Bortezomib, Velcade®), and the results showed that proteasome inhibitor treatment significantly reduced the mRNA expression of SREBP-1c, and the downstream lipogenic enzymes, such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), which catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid synthesis. ELOVL6, which is responsible for fatty acids long chain elongation, was also significantly down regulated by proteasome inhibitor treatment. Moreover, PS-341 administration significantly reduced the expression of acyl-glycerol-3-phosphate acyltransferase (AGPAT), and diacylglycerol acyltransferase (DGAT), enzyme involved in triacylglycerol (TAG) synthesis. Finally, PS-341 was found to down regulate the enzymes 3-hydroxy-3-methylglutaryl-CoenzymeA synthase (HMG-CoA synthase) that is responsible for cholesterol synthesis. Proteasome inhibitor was also found to play a role in intestinal lipid adsorption because apolipoproteins A (apoA-I, apoAII, apoA-IV and ApoCIII) were down regulated by proteasome inhibitor treatment, especially ApoA-II that is known to be a marker of alcohol consumption. Proteasome inhibitor treatment also decreased apobec-1 complementation factor (ACF) leading to lower level of editing and production of ApoB protein. Moreover apolipoprotein C-III, a major component of chylomicrons was significantly down regulated. However, lipoprotein lipase (Lpl) and High density lipoprotein binding protein (Hdlbp) mRNA levels were increased by proteasome inhibitor treatment. These results suggested that proteasome inhibitor treatment could be used to reduce the alcohol-enhanced lipogenesis and alcohol-induced liver steatosis. A morphologic analysis, performed on the liver of rats fed ethanol for one month and treated with PS-341, showed that proteasome inhibitor treatment significantly decreased ethanol-induced liver steatosis. SREBP-1c, FAS and ACC were increased by ethanol feeding alone, but were significantly decreased when proteasome inhibitor was administered to rats fed ethanol. Our results also show that both mRNA and protein levels of these lipogenic enzymes, up regulated by ethanol, were then down regulated when proteasome inhibitor was administered to rats fed ethanol. It was also confirmed that alcohol feeding caused an increase in AGPAT and DGAT, which was prevented by proteasome inhibitor treatment of the animal fed ethanol. Chronic alcohol feeding did not affect the gene expression of HMG-CoA synthase. However, PS341 administration significantly reduced the HMG-CoA synthase mRNA levels, confirming the results obtained with the microarray analysis. C/EBP transcription factors alpha (CCAAT/enhancer-binding protein alpha) has been shown to positively regulate SREBP-1c mRNA expression, thus regulating lipogenesis. Proteasome inhibition caused a decrease in C/EBP alpha mRNA expression, indicating that C/EBP down regulation may be the mechanism by which proteasome inhibitor treatment reduced lipogenesis. In conclusion, our results indicate that proteasome activity is not only involved in down regulating fatty acid synthesis and triacylglycerol synthesis, but also cholesterol synthesis and intestinal lipid adsorption. Proteasome inhibitor, administrated at a non-toxic low dose, played a beneficial role in reducing lipogenesis caused by chronic ethanol feeding and these beneficial effects are obtained because of the specificity and reversibility of the proteasome inhibitor used. PMID:22445925

Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism

DOE PAGES

VanderLinden, Ryan T.; Hemmis, Casey W.; Yao, Tingting; ...

2017-04-25

This work presents that the 26S proteasome is a large cellular assembly that mediates the selective degradation of proteins in the nucleus and cytosol and is an established target for anticancer therapeutics. Protein substrates are typically targeted to the proteasome through modification with a polyubiquitin chain, which can be recognized by several proteasome-associated ubiquitin receptors. One of these receptors, RPN13/ADRM1, is recruited to the proteasome through direct interaction with the large scaffolding protein RPN2 within the 19S regulatory particle. To better understand the interactions between RPN13, RPN2, and ubiquitin, we used human proteins to map the RPN13-binding epitope to themore » C-terminal 14 residues of RPN2, which, like ubiquitin, binds the N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain of RPN13. We also report the crystal structures of the RPN13 PRU domain in complex with peptides corresponding to the RPN2 C terminus and ubiquitin. Through mutational analysis, we validated the RPN2-binding interface revealed by our structures and quantified binding interactions with surface plasmon resonance and fluorescence polarization. In contrast to a previous report, we find that RPN13 binds ubiquitin with an affinity similar to that of other proteasome-associated ubiquitin receptors and that RPN2, ubiquitin, and the deubiquitylase UCH37 bind to RPN13 with independent energetics. In conclusion, these findings provide a detailed characterization of interactions that are important for proteasome function, indicate ubiquitin affinities that are consistent with the role of RPN13 as a proteasomal ubiquitin receptor, and have major implications for the development of novel anticancer therapeutics.« less

Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism.

PubMed

VanderLinden, Ryan T; Hemmis, Casey W; Yao, Tingting; Robinson, Howard; Hill, Christopher P

2017-06-09

The 26S proteasome is a large cellular assembly that mediates the selective degradation of proteins in the nucleus and cytosol and is an established target for anticancer therapeutics. Protein substrates are typically targeted to the proteasome through modification with a polyubiquitin chain, which can be recognized by several proteasome-associated ubiquitin receptors. One of these receptors, RPN13/ADRM1, is recruited to the proteasome through direct interaction with the large scaffolding protein RPN2 within the 19S regulatory particle. To better understand the interactions between RPN13, RPN2, and ubiquitin, we used human proteins to map the RPN13-binding epitope to the C-terminal 14 residues of RPN2, which, like ubiquitin, binds the N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain of RPN13. We also report the crystal structures of the RPN13 PRU domain in complex with peptides corresponding to the RPN2 C terminus and ubiquitin. Through mutational analysis, we validated the RPN2-binding interface revealed by our structures and quantified binding interactions with surface plasmon resonance and fluorescence polarization. In contrast to a previous report, we find that RPN13 binds ubiquitin with an affinity similar to that of other proteasome-associated ubiquitin receptors and that RPN2, ubiquitin, and the deubiquitylase UCH37 bind to RPN13 with independent energetics. These findings provide a detailed characterization of interactions that are important for proteasome function, indicate ubiquitin affinities that are consistent with the role of RPN13 as a proteasomal ubiquitin receptor, and have major implications for the development of novel anticancer therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Impairment of the ubiquitin-proteasome pathway in RPE alters the expression of inflammation related genes

USDA-ARS?s Scientific Manuscript database

The ubiquitin-proteasome pathway (UPP) plays an important role in regulating gene expression. Retinal pigment epithelial cells (RPE) are a major source of ocular inflammatory cytokines. In this work we determined the relationship between impairment of the UPP and expression of inflammation-related f...

Localization of proteasomes and proteasomal proteolysis in the mammalian interphase cell nucleus by systematic application of immunocytochemistry.

PubMed

Scharf, Andrea; Rockel, Thomas Dino; von Mikecz, Anna

2007-06-01

Proteasomes are ATP-driven, multisubunit proteolytic machines that degrade endogenous proteins into peptides and play a crucial role in cellular events such as the cell cycle, signal transduction, maintenance of proper protein folding and gene expression. Recent evidence indicates that the ubiquitin-proteasome system is an active component of the cell nucleus. A characteristic feature of the nucleus is its organization into distinct domains that have a unique composition of macromolecules and dynamically form as a response to the requirements of nuclear function. Here, we show by systematic application of different immunocytochemical procedures and comparison with signature proteins of nuclear domains that during interphase endogenous proteasomes are localized diffusely throughout the nucleoplasm, in speckles, in nuclear bodies, and in nucleoplasmic foci. Proteasomes do not occur in the nuclear envelope region or the nucleolus, unless nucleoplasmic invaginations expand into this nuclear body. Confirmedly, proteasomal proteolysis is detected in nucleoplasmic foci, but is absent from the nuclear envelope or nucleolus. The results underpin the idea that the ubiquitin-proteasome system is not only located, but also proteolytically active in distinct nuclear domains and thus may be directly involved in gene expression, and nuclear quality control.

Analysis of the hypoxia-sensing pathway in Drosophila melanogaster

PubMed Central

Arquier, Nathalie; Vigne, Paul; Duplan, Eric; Hsu, Tien; Therond, Pascal P.; Frelin, Christian; D'Angelo, Gisela

2005-01-01

The mechanism by which hypoxia induces gene transcription involves the inhibition of HIF-1α (hypoxia-inducible factor-1 α subunit) PHD (prolyl hydroxylase) activity, which prevents the VHL (von Hippel-Lindau)-dependent targeting of HIF-1α to the ubiquitin/proteasome pathway. HIF-1α thus accumulates and promotes gene transcription. In the present study, first we provide direct biochemical evidence for the presence of a conserved hypoxic signalling pathway in Drosophila melanogaster. An assay for 2-oxoglutarate-dependent dioxygenases was developed using Drosophila embryonic and larval homogenates as a source of enzyme. Drosophila PHD has a low substrate specificity and hydroxylates key proline residues in the ODD (oxygen-dependent degradation) domains of human HIF-1α and Similar, the Drosophila homologue of HIF-1α. The enzyme promotes human and Drosophila [35S]VHL binding to GST (glutathione S-transferase)–ODD-domain fusion protein. Hydroxylation is enhanced by proteasomal inhibitors and was ascertained using an anti-hydroxyproline antibody. Secondly, by using transgenic flies expressing a fusion protein that combined an ODD domain and the green fluorescent protein (ODD–GFP), we analysed the hypoxic cascade in different embryonic and larval tissues. Hypoxic accumulation of the reporter protein was observed in the whole tracheal tree, but not in the ectoderm. Hypoxic stabilization of ODD–GFP in the ectoderm was restored by inducing VHL expression in these cells. These results show that Drosophila tissues exhibit different sensitivities to hypoxia. PMID:16176182

Proteomic analysis of testis biopsies in men treated with injectable testosterone undecanoate alone or in combination with oral levonorgestrel as potential male contraceptive.

PubMed

Cui, Yugui; Zhu, Hui; Zhu, Yefei; Guo, Xuejiang; Huo, Ran; Wang, Xinghai; Tong, Jiansun; Qian, Lixin; Zhou, Zuomin; Jia, Yue; Lue, Yan-He; Hikim, Amiya Sinha; Wang, Christina; Swerdloff, Ronald S; Sha, Jiahao

2008-09-01

Treatment with injectable testosterone undecanoate (TU) alone or in combination with oral levonorgestrel (LNG) resulted in marked decreases in sperm concentrations. In this study, we used proteomic analyses to examine the cellular/molecular events occurring in the human testis after TU or TU + LNG treatment. We conducted a global proteomic analysis of the human testicular biopsies before and at 2 weeks after TU alone or TU + LNG treatment. Proteins showing significant changes in expression were identified and analyzed. As a result, 17 and 46 protein spots were found with significant differential expression after the treatment with TU alone and TU + LNG, respectively. TU treatment changed the expression of heterogeneous nuclear ribonucleoprotein K (hnRNP K), proteasome inhibitor PI31 subunit (PSMF1), and superoxide dismutase [Mn] mitochondrial precursor (SOD2). These proteins inhibit "assembly", induce cell death, and promote compensatory "cell survival" in the testis. After TU + LNG treatment, "proliferation/cell survival" and "apoptosis/death" were the predominant responses in the testis. TU + LNG treatment inhibited the expression of Prolyl 4-hydroxylase beta subunit (P4HB) and Annexin A2 (Annexin II). These proteins are involved in apoptosis and cell proliferation, respectively. TU + LNG treatment also enhanced the expression of SOD2 and Parvalbumin alpha (Pvalb). These two proteins may protect testicular cells against apoptosis/death and promote cell survival. In conclusion, TU and TU + LNG treatments suppress spermatogenesis through different pathways by changing the expression of different proteins. hnRNP K, PSMF1, SOD2, P4HB, Annexin II, and Pvalb, are key proteins that may be early molecular targets responsible for spermatogenesis suppression induced by hormone treatment.

Paraquat, but Not Maneb, Induces Synucleinopathy and Tauopathy in Striata of Mice through Inhibition of Proteasomal and Autophagic Pathways

PubMed Central

Wills, Jonathan; Credle, Joel; Oaks, Adam W.; Duka, Valeriy; Lee, Jae-Hoon; Jones, Jessica; Sidhu, Anita

2012-01-01

SNCA and MAPT genes and environmental factors are important risk factors of Parkinson's disease [PD], the second-most common neurodegenerative disease. The agrichemicals maneb and paraquat selectively target dopaminergic neurons, leading to parkinsonism, through ill-defined mechanisms. In the current studies we have analyzed the ability of maneb and paraquat, separately and together, to induce synucleinopathy and tauopathy in wild type mice. Maneb was ineffective in increasing α-synuclein [α-Syn] or p-Tau levels. By contrast, paraquat treatment of mice resulted in robust accumulation of α-Syn and hyperphosphorylation of Tau in striata, through activation of p-GSK-3β, a major Tau kinase. Co-treatment with maneb did not enhance the effects of paraquat. Increased hyperacetylation of α-tubulin was observed in paraquat-treated mice, suggesting cytoskeleton remodeling. Paraquat, but not maneb, inhibited soluble proteasomal activity on a peptide substrate but this was not associated with a decreased expression of 26S proteasome subunits. Both paraquat and maneb treatments increased levels of the autophagy inhibitor, mammalian target of rapamycin, mTOR, suggesting impaired axonal autophagy, despite increases in certain autophagic proteins, such as beclin 1 and Agt12. Autophagic flux was also impaired, as ratios of LC3 II to LC3 I were reduced in treated animals. Increased mTOR was also observed in postmortem human PD striata, where there was a reduction in the LC3 II to LC3 I ratio. Heat shock proteins were either increased or unchanged upon paraquat-treatment suggesting that chaperone-mediated autophagy is not hampered by the agrichemicals. These studies provide novel insight into the mechanisms of action of these agrichemicals, which indicate that paraquat is much more toxic than maneb, via its inhibitory effects on proteasomes and autophagy, which lead to accumulation of α-Syn and p-Tau. PMID:22292029

Both the autophagy and proteasomal pathways facilitate the Ubp3p-dependent depletion of a subset of translation and RNA turnover factors during nitrogen starvation in Saccharomyces cerevisiae

PubMed Central

Kelly, Shane P.; Bedwell, David M.

2015-01-01

Protein turnover is an important regulatory mechanism that facilitates cellular adaptation to changing environmental conditions. Previous studies have shown that ribosome abundance is reduced during nitrogen starvation by a selective autophagy mechanism termed ribophagy, which is dependent upon the deubiquitinase Ubp3p. In this study, we asked whether the abundance of various translation and RNA turnover factors are reduced following the onset of nitrogen starvation in Saccharomyces cerevisiae. We found distinct differences in the abundance of the proteins tested following nitrogen starvation: (1) The level of some did not change; (2) others were reduced with kinetics similar to ribophagy, and (3) a few proteins were rapidly depleted. Furthermore, different pathways differentially degraded the various proteins upon nitrogen starvation. The translation factors eRF3 and eIF4GI, and the decapping enhancer Pat1p, required an intact autophagy pathway for their depletion. In contrast, the deadenylase subunit Pop2p and the decapping enzyme Dcp2p were rapidly depleted by a proteasome-dependent mechanism. The proteasome-dependent depletion of Dcp2p and Pop2p was also induced by rapamycin, suggesting that the TOR1 pathway influences this pathway. Like ribophagy, depletion of eIF4GI, eRF3, Dcp2p, and Pop2p was dependent upon Ubp3p to varying extents. Together, our results suggest that the autophagy and proteasomal pathways degrade distinct translation and RNA turnover factors in a Ubp3p-dependent manner during nitrogen starvation. While ribophagy is thought to mediate the reutilization of scarce resources during nutrient limitation, our results suggest that the selective degradation of specific proteins could also facilitate a broader reprogramming of the post-transcriptional control of gene expression. PMID:25795416

Effect of cellular ubiquitin levels on the regulation of oxidative stress response and proteasome function via Nrf1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Donghee; Ryu, Kwon-Yul

The polyubiquitin genes Ubb and Ubc are upregulated under oxidative stress induced by arsenite [As(III)]. However, the role of ubiquitin (Ub) under As(III) exposure is not known in detail. In a previous study, we showed that the reduced viability observed in Ubc{sup −/−} mouse embryonic fibroblasts under As(III) exposure was not due to dysregulation of the Nrf2–Keap1 pathway, which prompted us to investigate another NFE2 family protein, nuclear factor erythroid 2-related factor 1 (Nrf1). In this study, we found that Ub deficiency due to Ubc knockdown in N2a cells reduced cell viability and proteasome activity under As(III) exposure. Furthermore, mRNAmore » levels of the proteasome subunit Psma1 were also reduced. In addition, Ub deficiency led to the nuclear accumulation of the p65 isoform of Nrf1 under As(III) exposure. Interestingly, the overexpression of p65-Nrf1 recapitulated the phenotypes of Ub-deficient N2a cells under As(III) exposure. On the other hand, Nrf1 knockdown suppressed the death of Ub-deficient N2a cells upon exposure to As(III). Therefore, the levels of p65-Nrf1 may play an important role in the maintenance of cell viability under oxidative stress induced by As(III). - Highlights: • N2a cells exhibit reduced viability upon exposure to As(III) via Ubc knockdown. • As(III)-induced proteasomal regulation is impaired in Ub-deficient N2a cells. • Ub deficiency leads to the nuclear accumulation of p65-Nrf1 under As(III) exposure. • p65 expression recapitulates As(III)-induced phenotypes of Ub-deficient N2a cells. • Nrf1 knockdown suppressed As(III)-induced death of Ub-deficient N2a cells.« less

RMND5 from Xenopus laevis is an E3 ubiquitin-ligase and functions in early embryonic forebrain development.

PubMed

Pfirrmann, Thorsten; Villavicencio-Lorini, Pablo; Subudhi, Abinash K; Menssen, Ruth; Wolf, Dieter H; Hollemann, Thomas

2015-01-01

In Saccharomyces cerevisiae the Gid-complex functions as an ubiquitin-ligase complex that regulates the metabolic switch between glycolysis and gluconeogenesis. In higher organisms six conserved Gid proteins form the CTLH protein-complex with unknown function. Here we show that Rmnd5, the Gid2 orthologue from Xenopus laevis, is an ubiquitin-ligase embedded in a high molecular weight complex. Expression of rmnd5 is strongest in neuronal ectoderm, prospective brain, eyes and ciliated cells of the skin and its suppression results in malformations of the fore- and midbrain. We therefore suggest that Xenopus laevis Rmnd5, as a subunit of the CTLH complex, is a ubiquitin-ligase targeting an unknown factor for polyubiquitination and subsequent proteasomal degradation for proper fore- and midbrain development.

Phosphorylation and Methylation of Proteasomal Proteins of the Haloarcheon Haloferax volcanii

DOE PAGES

Humbard, Matthew A.; Reuter, Christopher J.; Zuobi-Hasona, Kheir; ...

2010-01-01

Promore » teasomes are composed of 20S core particles (CPs) of α - and β -type subunits that associate with regulatory particle AAA ATPases such as the proteasome-activating nucleotidase (PAN) complexes of archaea. In this study, the roles and additional sites of post-translational modification of proteasomes were investigated using the archaeonHaloferax volcaniias a model. Indicative of phosphorylation, phosphatase-sensitive isoforms of α 1 and α 2 were detected by 2-DE immunoblot. To map these and other potential sites of post-translational modification, proteasomes were purified and analyzed by tandem mass spectrometry (MS/MS). Using this approach, several phosphosites were mapped including α 1 Thr147, α 2 Thr13/Ser14 and PAN-A Ser340. Multiple methylation sites were also mapped to α 1 , thus, revealing a new type of proteasomal modification. bing the biological role of α 1 and PAN-A phosphorylation by site-directed mutagenesis revealed dominant negative phenotypes for cell viability and/or pigmentation for α 1 variants including Thr147Ala, Thr158Ala and Ser58Ala. AnH. volcaniiRio1p Ser/Thr kinase homolog was purified and shown to catalyze autophosphorylation and phosphotransfer to α 1 . The α 1 variants in Thr and Ser residues that displayed dominant negative phenotypes were significantly reduced in their ability to accept phosphoryl groups from Rio1p, thus, providing an important link between cell physiology and proteasomal phosphorylation.« less

CLONING AND MOLECULAR CHARACTERIZATION OF A GENE ENCODING A CRYPTOSPORIDIUM PARVUM PUTATIVE 20S PROTEASOME B1-TYPE SUBUNIT. (R825148)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Focal dysfunction of the proteasome: a pathogenic factor in a mouse model of amyotrophic lateral sclerosis.

PubMed

Kabashi, Edor; Agar, Jeffrey N; Taylor, David M; Minotti, Sandra; Durham, Heather D

2004-06-01

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis (fALS). The present study demonstrated impaired proteasomal function in the lumbar spinal cord of transgenic mice expressing human SOD-1 with the ALS-causing mutation G93A (SOD-1(G93A)) compared to non-transgenic littermates (LM) and SOD-1(WT) transgenic mice. Chymotrypsin-like activity was decreased as early as 45 days of age. By 75 days, chymotrypsin-, trypsin-, and caspase-like activities of the proteasome were impaired, at about 50% of control activity in lumbar spinal cord, but unchanged in thoracic spinal cord and liver. Both total and specific activities of the proteasome were reduced to a similar extent, indicating that a change in proteasome function, rather than a decrease in proteasome levels, had occurred. Similar decreases of total and specific activities of the proteasome were observed in NIH 3T3 cell lines expressing fALS mutants SOD-1(G93A) and SOD-1(G41S), but not in SOD-1(WT) controls. Although overall levels of proteasome were maintained in spinal cord of SOD-1(G93A) transgenic mice, the level of 20S proteasome was substantially reduced in lumbar spinal motor neurons relative to the surrounding neuropil. It is concluded that impairment of the proteasome is an early event and contributes to ALS pathogenesis.

Small molecule therapeutics targeting F-box proteins in cancer.

PubMed

Liu, Yuan; Mallampalli, Rama K

2016-02-01

The ubiquitin proteasome system (UPS) plays vital roles in maintaining protein equilibrium mainly through proteolytic degradation of targeted substrates. The archetypical SCF ubiquitin E3 ligase complex contains a substrate recognition subunit F-box protein that recruits substrates to the catalytic ligase core for its polyubiquitylation and subsequent proteasomal degradation. Several well-characterized F-box proteins have been demonstrated that are tightly linked to neoplasia. There is mounting information characterizing F-box protein-substrate interactions with the rationale to develop unique therapeutics for cancer treatment. Here we review that how F-box proteins function in cancer and summarize potential small molecule inhibitors for cancer therapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Inhibition of the 26S proteasome blocks progesterone receptor-dependent transcription through failed recruitment of RNA polymerase II.

PubMed

Dennis, Andrew P; Lonard, David M; Nawaz, Zafar; O'Malley, Bert W

2005-03-01

In the present study, we investigated the involvement of protein degradation via the 26S proteasome during progesterone receptor (PR)-mediated transcription in T-47D cells containing a stably integrated MMTV-CAT reporter construct (CAT0 cells). Progesterone induced CAT and HSD11beta2 transcription while co-treatment with the proteasome inhibitor, MG132, blocked PR-induced transcription in a time-dependent fashion. MG132 treatment also inhibited transcription of beta-actin and cyclophilin, but not two proteasome subunit genes, PSMA1 and PSMC1, indicating that proteasome inhibition affects a subset of RNA polymerase II (RNAP(II))-regulated genes. Progesterone-mediated recruitment of RNAP(II) was blocked by MG132 treatment at time points later than 1 h that was not dependent on the continued presence of PR, associated cofactors, and components of the general transcription machinery, supporting the concept that proteasome-mediated degradation is needed for continued transcription. Surprisingly, progesterone-mediated acetylation of histone H4 was inhibited by MG132 with the concomitant recruitment of HDAC3, NCoR, and SMRT. We demonstrate that the steady-state protein levels of SMRT and NCoR are higher in the presence of MG132 in CAT0 cells, consistent with other reports that SMRT and NCoR are targets of the 26S proteasome. However, inhibition of histone deacetylation by trichostatin A (TSA) treatment or SMRT/NCoR knockdown by siRNA did not restore MG132-inhibited progesterone-dependent transcription. Therefore, events other than histone deacetylation and stability of SMRT and NCoR must also play a role in inhibition of PR-mediated transcription.

A case-control study on association of proteasome subunit beta 8 (PSMB8) and transporter associated with antigen processing 1 (TAP1) polymorphisms and their transcript levels in vitiligo from Gujarat.

PubMed

Jadeja, Shahnawaz D; Mansuri, Mohmmad Shoab; Singh, Mala; Dwivedi, Mitesh; Laddha, Naresh C; Begum, Rasheedunnisa

2017-01-01

Autoimmunity has been implicated in the destruction of melanocytes from vitiligo skin. Major histocompatibility complex (MHC) class-II linked genes proteasome subunit beta 8 (PSMB8) and transporter associated with antigen processing 1 (TAP1), involved in antigen processing and presentation have been reported to be associated with several autoimmune diseases including vitiligo. To explore PSMB8 rs2071464 and TAP1 rs1135216 single nucleotide polymorphisms and to estimate the expression of PSMB8 and TAP1 in patients with vitiligo and unaffected controls from Gujarat. PSMB8 rs2071464 polymorphism was genotyped using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) and TAP1 rs1135216 polymorphism was genotyped by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) in 378 patients with vitiligo and 509 controls. Transcript levels of PSMB8 and TAP1 were measured in the PBMCs of 91 patients and 96 controls by using qPCR. Protein levels of PSMB8 were also determined by Western blot analysis. The frequency of 'TT' genotype of PSMB8 polymorphism was significantly lowered in patients with generalized and active vitiligo (p = 0.019 and p = 0.005) as compared to controls suggesting its association with the activity of the disease. However, TAP1 polymorphism was not associated with vitiligo susceptibility. A significant decrease in expression of PSMB8 at both transcript level (p = 0.002) as well as protein level (p = 0.0460) was observed in vitiligo patients as compared to controls. No significant difference was observed between patients and controls for TAP1 transcripts (p = 0.553). Interestingly, individuals with the susceptible CC genotype of PSMB8 polymorphism showed significantly reduced PSMB8 transcript level as compared to that of CT and TT genotypes (p = 0.009 and p = 0.003 respectively). PSMB8 rs2071464 was associated with generalized and active vitiligo from Gujarat whereas TAP1 rs1135216 showed no association. The down-regulation of PSMB8 in patients with risk genotype 'CC' advocates the vital role of PSMB8 in the autoimmune basis of vitiligo.

A ubiquitin carboxyl extension protein secreted from a plant-parasitic nematode Globodera rostochiensis is cleaved in planta to promote plant parasitism.

PubMed

Chronis, Demosthenis; Chen, Shiyan; Lu, Shunwen; Hewezi, Tarek; Carpenter, Sara C D; Loria, Rosemary; Baum, Thomas J; Wang, Xiaohong

2013-04-01

Nematode effector proteins originating from esophageal gland cells play central roles in suppressing plant defenses and in formation of the plant feeding cells that are required for growth and development of cyst nematodes. A gene (GrUBCEP12) encoding a unique ubiquitin carboxyl extension protein (UBCEP) that consists of a signal peptide for secretion, a mono-ubiquitin domain, and a 12 amino acid carboxyl extension protein (CEP12) domain was cloned from the potato cyst nematode Globodera rostochiensis. This GrUBCEP12 gene was expressed exclusively within the nematode's dorsal esophageal gland cell, and was up-regulated in the parasitic second-stage juvenile, correlating with the time when feeding cell formation is initiated. We showed that specific GrUBCEP12 knockdown via RNA interference reduced nematode parasitic success, and that over-expression of the secreted Gr(Δ) (SP) UBCEP12 protein in potato resulted in increased nematode susceptibility, providing direct evidence that this secreted effector is involved in plant parasitism. Using transient expression assays in Nicotiana benthamiana, we found that Gr(Δ) (SP) UBCEP12 is processed into free ubiquitin and a CEP12 peptide (GrCEP12) in planta, and that GrCEP12 suppresses resistance gene-mediated cell death. A target search showed that expression of RPN2a, a gene encoding a subunit of the 26S proteasome, was dramatically suppressed in Gr(Δ) (SP) UBCEP12 but not GrCEP12 over-expression plants when compared with control plants. Together, these results suggest that, when delivered into host plant cells, Gr(Δ) (SP) UBCEP12 becomes two functional units, one acting to suppress plant immunity and the other potentially affecting the host 26S proteasome, to promote feeding cell formation. © 2013 The Authors The Plant Journal © 2013 Blackwell Publishing Ltd.

Protein complex purification from Thermoplasma acidophilum using a phage display library.

PubMed

Hubert, Agnes; Mitani, Yasuo; Tamura, Tomohiro; Boicu, Marius; Nagy, István

2014-03-01

We developed a novel protein complex isolation method using a single-chain variable fragment (scFv) based phage display library in a two-step purification procedure. We adapted the antibody-based phage display technology which has been developed for single target proteins to a protein mixture containing about 300 proteins, mostly subunits of Thermoplasma acidophilum complexes. T. acidophilum protein specific phages were selected and corresponding scFvs were expressed in Escherichia coli. E. coli cell lysate containing the expressed His-tagged scFv specific against one antigen protein and T. acidophilum crude cell lysate containing intact target protein complexes were mixed, incubated and subjected to protein purification using affinity and size exclusion chromatography steps. This method was confirmed to isolate intact particles of thermosome and proteasome suitable for electron microscopy analysis and provides a novel protein complex isolation strategy applicable to organisms where no genetic tools are available. Copyright © 2013 Elsevier B.V. All rights reserved.

Inhibiting NF-κB Activation by Small Molecules As a Therapeutic Strategy

PubMed Central

Gupta, Subash C; Sundaram, Chitra; Reuter, Simone; Aggarwal, Bharat B

2010-01-01

Because nuclear factor-κB (NF-κB) is a ubiquitously expressed proinflammatory transcription factor that regulates the expression of over 500 genes involved in cellular transformation, survival, proliferation, invasion, angiogenesis, metastasis, and inflammation, the NF-κB signaling pathway has become a potential target for pharmacological intervention. A wide variety of agents can activate NF-κB through canonical and noncanonical pathways. Canonical pathway involves various steps including the phosphorylation, ubiquitnation, and degradation of the inhibitor of NF-κB (IκBα), which leads to the nuclear translocation of the p50- p65 subunits of NF-κB followed by p65 phosphorylation, acetylation and methylation, DNA binding, and gene transcription. Thus, agents that can inhibit protein kinases, protein phosphatases, proteasomes, ubiquitnation, acetylation, methylation, and DNA binding steps have been identified as NF-κB inhibitors. Here, we review the small molecules that suppress NF-κB activation and thus may have therapeutic potential. PMID:20493977

The ubiquitin-proteasome system is required for African swine fever replication.

PubMed

Barrado-Gil, Lucía; Galindo, Inmaculada; Martínez-Alonso, Diego; Viedma, Sergio; Alonso, Covadonga

2017-01-01

Several viruses manipulate the ubiquitin-proteasome system (UPS) to initiate a productive infection. Determined viral proteins are able to change the host's ubiquitin machinery and some viruses even encode their own ubiquitinating or deubiquitinating enzymes. African swine fever virus (ASFV) encodes a gene homologous to the E2 ubiquitin conjugating (UBC) enzyme. The viral ubiquitin-conjugating enzyme (UBCv1) is expressed throughout ASFV infection and accumulates at late times post infection. UBCv is also present in the viral particle suggesting that the ubiquitin-proteasome pathway could play an important role at early ASFV infection. We determined that inhibition of the final stage of the ubiquitin-proteasome pathway blocked a post-internalization step in ASFV replication in Vero cells. Under proteasome inhibition, ASF viral genome replication, late gene expression and viral production were severely reduced. Also, ASFV enhanced proteasome activity at late times and the accumulation of polyubiquitinated proteins surrounding viral factories. Core-associated and/or viral proteins involved in DNA replication may be targets for the ubiquitin-proteasome pathway that could possibly assist virus uncoating at final core breakdown and viral DNA release. At later steps, polyubiquitinated proteins at viral factories could exert regulatory roles in cell signaling.

Architecture of human translation initiation factor 3

PubMed Central

Querol-Audi, Jordi; Sun, Chaomin; Vogan, Jacob M.; Smith, Duane; Gu, Yu; Cate, Jamie; Nogales, Eva

2013-01-01

SUMMARY Eukaryotic translation initiation factor 3 (eIF3) plays a central role in protein synthesis by organizing the formation of the 43S preinitiation complex. Using genetic tag visualization by electron microscopy, we reveal the molecular organization of ten human eIF3 subunits, including an octameric core. The structure of eIF3 bears a close resemblance to that of the proteasome lid, with a conserved spatial organization of eight core subunits containing PCI and MPN domains that coordinate functional interactions in both complexes. We further show that eIF3 subunits a and c interact with initiation factors eIF1 and eIF1A, which control the stringency of start codon selection. Finally, we find that subunit j, which modulates messenger RNA interactions with the small ribosomal subunit, makes multiple independent interactions with the eIF3 octameric core. These results highlight the conserved architecture of eIF3 and how it scaffolds key factors that control translation initiation in higher eukaryotes, including humans. PMID:23623729

The Drosophila TGF-beta/Activin-like ligands Dawdle and Myoglianin appear to modulate adult lifespan through regulation of 26S proteasome function in adult muscle

PubMed Central

Langerak, Shaughna; Kim, Myung-Jun; Lamberg, Hannah; Godinez, Michael; Main, Mackenzie; Winslow, Lindsey; O'Connor, Michael B.

2018-01-01

ABSTRACT The Drosophila Activin signaling pathway employs at least three separate ligands – Activin-β (Actβ), Dawdle (Daw), and Myoglianin (Myo) – to regulate several general aspects of fruit fly larval development, including cell proliferation, neuronal remodeling, and metabolism. Here we provide experimental evidence indicating that both Daw and Myo are anti-ageing factors in adult fruit flies. Knockdown of Myo or Daw in adult fruit flies reduced mean lifespan, while overexpression of either ligand in adult muscle tissues but not in adipose tissues enhanced mean lifespan. An examination of ubiquitinated protein aggregates in adult muscles revealed a strong inverse correlation between Myo- or Daw-initiated Activin signaling and the amount of ubiquitinated protein aggregates. We show that this correlation has important functional implications by demonstrating that the lifespan extension effect caused by overexpression of wild-type Daw or Myo in adult muscle tissues can be completely abrogated by knockdown of a 26S proteasome regulatory subunit Rpn1 in adult fly muscle, and that the prolonged lifespan caused by overexpression of Daw or Myo in adult muscle could be due to enhanced protein levels of the key subunits of 26S proteasome. Overall, our data suggest that Activin signaling initiated by Myo and Daw in adult Drosophila muscles influences lifespan, in part, by modulation of protein homeostasis through either direct or indirect regulation of the 26S proteasome levels. Since Myo is closely related to the vertebrate muscle mass regulator Myostatin (GDF8) and the Myostatin paralog GDF11, our observations may offer a new experimental model for probing the roles of GDF11/8 in ageing regulation in vertebrates. This article has an associated First Person interview with the first author of the paper. PMID:29615416

Exploring the Ubiquitin-Proteasome Protein Degradation Pathway in Yeast

ERIC Educational Resources Information Center

Will, Tamara J.; McWatters, Melissa K.; McQuade, Kristi L.

2006-01-01

This article describes an undergraduate biochemistry laboratory investigating the ubiquitin-proteasome pathway in yeast. In this exercise, the enzyme beta-galactosidase (beta-gal) is expressed in yeast under the control of a stress response promoter. Following exposure to heat stress to induce beta-gal expression, cycloheximide is added to halt…

UBXD4, a UBX-containing protein, regulates the cell surface number and stability of alpha3-containing nicotinic acetylcholine receptors.

PubMed

Rezvani, Khosrow; Teng, Yanfen; Pan, Yaping; Dani, John A; Lindstrom, Jon; García Gras, Eduardo A; McIntosh, J Michael; De Biasi, Mariella

2009-05-27

Adaptor proteins are likely to modulate spatially and temporally the trafficking of a number of membrane proteins, including neuronal nicotinic acetylcholine receptors (nAChRs). A yeast two-hybrid screen identified a novel UBX-containing protein, UBXD4, as one of the cytosolic proteins that interact directly with the alpha3 and alpha4 nAChR subunits. The function of UBX-containing proteins is largely unknown. Immunoprecipitation and confocal microscopy confirmed the interaction of UBXD4 with alpha3-containing nAChRs (alpha3* nAChRs) expressed in HEK293 cells, PC12 cells, and rat cortical neurons. Overexpression of UBXD4 in differentiated PC12 cells (dPC12) increased nAChR cell surface expression, especially that of the alpha3beta2 subtype. These findings were corroborated by electrophysiology, immunofluorescent staining, and biotinylation of surface receptors. Silencing of UBXD4 led to a significant reduction of alpha3* nAChRs in rat cortical neurons and dPC12 cells. Biochemical and immunofluorescence studies of endogenous UBXD4 showed that the protein is located in both the ER and cis-Golgi compartments. Our investigations also showed that the alpha3 subunit is ubiquitinated and that UBXD4 can interfere with its ubiquitination and consequent degradation by the proteasome. Our data suggest that UBXD4 modulates the distribution of alpha3* nAChRs between specialized intracellular compartments and the plasma membrane. This effect is achieved by controlling the stability of the alpha3 subunit and, consequently, the number of receptors at the cell surface.

UBXD4, a UBX containing protein, regulates the cell surface number and the stability of α3-containing nicotinic acetylcholine receptors

PubMed Central

Rezvani, Khosrow; Teng, Yanfen; Pan, Yaping; Dani, John A.; Lindstrom, Jon.; Gras, Eduardo A. Garcáa; McIntosh, J. Michael; De Biasi, Mariella.

2010-01-01

Adaptor proteins are likely to modulate spatially and temporally the trafficking of a number of membrane proteins, including neuronal nicotinic acetylcholine receptors (nAChRs). A yeast two-hybrid screen identified a novel UBX-containing protein, UBXD4, as one of the cytosolic proteins that interact directly with the α3 and α4 nAChR subunits. The function of UBX-containing proteins is largely unknown. Immunoprecipitation and confocal microscopy confirmed the interaction of UBXD4 with α3-containing nAChRs (α3* nAChRs) expressed in HEK293 cells, PC12 cells and rat cortical neurons. Overexpression of UBXD4 in differentiated PC12 cells (dPC12) increased nAChR cell surface expression, especially that of the α3β2 subtype. These findings were corroborated by electrophysiology, immunofluorescent staining and biotinylation of surface receptors. Silencing of UBXD4 led to a significant reduction of α3* nAChRs in rat cortical neurons and dPC12 cells. Biochemical and immunofluorescence studies of endogenous UBXD4 showed that the protein is located in both the ER and cis-Golgi compartments. Our investigations also showed that the α3 subunit is ubiquitinated and that UBXD4 can interfere with its ubiquitination and consequent degradation by the proteasome. Our data suggest that UBXD4 modulates the distribution of α3* nAChRs between specialized intracellular compartments and the plasma membrane. This effect is achieved by controlling the stability of the α3 subunit and, consequently, the number of receptors at the cell surface. PMID:19474315

Reduction of RPT6/S8 (a Proteasome Component) and Proteasome Activity in the Cortex is Associated with Cognitive Impairment in Lewy Body Dementia

PubMed Central

Alghamdi, Amani; Vallortigara, Julie; Howlett, David R.; Broadstock, Martin; Hortobágyi, Tibor; Ballard, Clive; Thomas, Alan J.; O’Brien, John T.; Aarsland, Dag; Attems, Johannes; Francis, Paul T.; Whitfield, David R.

2017-01-01

Lewy body dementia is the second most common neurodegenerative dementia and is pathologically characterized by α-synuclein positive cytoplasmic inclusions, with varying amounts of amyloid-β (Aβ) and hyperphosphorylated tau (tau) aggregates in addition to synaptic loss. A dysfunctional ubiquitin proteasome system (UPS), the major proteolytic pathway responsible for the clearance of short lived proteins, may be a mediating factor of disease progression and of the development of α-synuclein aggregates. In the present study, protein expression of a key component of the UPS, the RPT6 subunit of the 19S regulatory complex was determined. Furthermore, the main proteolytic-like (chymotrypsin- and PGPH-) activities have also been analyzed. The middle frontal (Brodmann, BA9), inferior parietal (BA40), and anterior cingulate (BA24) gyrus’ cortex were selected as regions of interest from Parkinson’s disease dementia (PDD, n = 31), dementia with Lewy bodies (DLB, n = 44), Alzheimer’s disease (AD, n = 16), and control (n = 24) brains. Clinical and pathological data available included the MMSE score. DLB, PDD, and AD were characterized by significant reductions of RPT6 (one-way ANOVA, p < 0.001; Bonferroni post hoc test) in prefrontal cortex and parietal cortex compared with controls. Strong associations were observed between RPT6 levels in prefrontal, parietal cortex, and anterior cingulate gyrus and cognitive impairment (p = 0.001, p = 0.001, and p = 0.008, respectively). These findings highlight the involvement of the UPS in Lewy body dementia and indicate that targeting the UPS may have the potential to slow down or reduce the progression of cognitive impairment in DLB and PDD. PMID:28269775

Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution

PubMed Central

McConnell, Sean C.; Hernandez, Kyle M.; Wcisel, Dustin J.; Kettleborough, Ross N.; Stemple, Derek L.; Andrade, Jorge; de Jong, Jill L. O.

2016-01-01

Antigen processing and presentation genes found within the MHC are among the most highly polymorphic genes of vertebrate genomes, providing populations with diverse immune responses to a wide array of pathogens. Here, we describe transcriptome, exome, and whole-genome sequencing of clonal zebrafish, uncovering the most extensive diversity within the antigen processing and presentation genes of any species yet examined. Our CG2 clonal zebrafish assembly provides genomic context within a remarkably divergent haplotype of the core MHC region on chromosome 19 for six expressed genes not found in the zebrafish reference genome: mhc1uga, proteasome-β 9b (psmb9b), psmb8f, and previously unknown genes psmb13b, tap2d, and tap2e. We identify ancient lineages for Psmb13 within a proteasome branch previously thought to be monomorphic and provide evidence of substantial lineage diversity within each of three major trifurcations of catalytic-type proteasome subunits in vertebrates: Psmb5/Psmb8/Psmb11, Psmb6/Psmb9/Psmb12, and Psmb7/Psmb10/Psmb13. Strikingly, nearby tap2 and MHC class I genes also retain ancient sequence lineages, indicating that alternative lineages may have been preserved throughout the entire MHC pathway since early diversification of the adaptive immune system ∼500 Mya. Furthermore, polymorphisms within the three MHC pathway steps (antigen cleavage, transport, and presentation) are each predicted to alter peptide specificity. Lastly, comparative analysis shows that antigen processing gene diversity is far more extensive than previously realized (with ancient coelacanth psmb8 lineages, shark psmb13, and tap2t and psmb10 outside the teleost MHC), implying distinct immune functions and conserved roles in shaping MHC pathway evolution throughout vertebrates. PMID:27493218

PSMC2 is up-regulated in osteosarcoma and regulates osteosarcoma cell proliferation, apoptosis and migration

PubMed Central

Song, Mingzhi; Wang, Yong

2017-01-01

Proteasome 26S subunit ATPase 2 (PSMC2) is a recently identified gene potentially associated with certain human carcinogenesis. However, the expressional correlation and functional importance of PSMC2 in osteosarcoma is still unclear. Current study was focused on elucidating the significance of PSMC2 on malignant behaviors in osteosarcoma including proliferation, apoptosis, colony formation, migration as well as invasion. The high protein levels of PSMC2 in osteosarcoma samples were identified by tissue microarrays analysis. Besides, its expression in the levels of mRNA and protein was also detected in four different osteosarcoma cell lines by real-time PCR and western blotting separately. Silencing PSMC2 by RNA interference in osteosarcoma cell lines (SaoS-2 and MG-63) would significantly suppress cell proliferation, enhance apoptosis, accelerate G2/M phase and/or S phase arrest, and decrease single cell colony formation. Similarly, pharmaceutical inhibition of proteasome with MG132 would mimic the PSMC2 depletion induced defects in cell cycle arrest, apoptosis and colonies formation. Silencing of PSMC2 was able to inhibit osteosarcoma cell motility, invasion as well as tumorigenicity in nude mice. Moreover, the gene microarray indicated knockdown of PSMC2 notably changed a number of genes, especially some cancer related genes including ITGA6, FN1, CCND1, CCNE2 and TGFβR2, and whose expression changes were further confirmed by western blotting. Our data suggested that PSMC2 may work as an oncogene for osteosarcoma and that inhibition of PSMC2 may be a therapeutic strategy for osteosarcoma treatment. PMID:27888613

The metastasis suppressor RARRES3 as an endogenous inhibitor of the immunoproteasome expression in breast cancer cells

NASA Astrophysics Data System (ADS)

Anderson, Alison M.; Kalimutho, Murugan; Harten, Sarah; Nanayakkara, Devathri M.; Khanna, Kum Kum; Ragan, Mark A.

2017-01-01

In breast cancer metastasis, the dynamic continuum involving pro- and anti-inflammatory regulators can become compromised. Over 600 genes have been implicated in metastasis to bone, lung or brain but how these genes might contribute to perturbation of immune function is poorly understood. To gain insight, we adopted a gene co-expression network approach that draws on the functional parallels between naturally occurring bone marrow-derived mesenchymal stem cells (BM-MSCs) and cancer stem cells (CSCs). Our network analyses indicate a key role for metastasis suppressor RARRES3, including potential to regulate the immunoproteasome (IP), a specialized proteasome induced under inflammatory conditions. Knockdown of RARRES3 in near-normal mammary epithelial and breast cancer cell lines increases overall transcript and protein levels of the IP subunits, but not of their constitutively expressed counterparts. RARRES3 mRNA expression is controlled by interferon regulatory factor IRF1, an inducer of the IP, and is sensitive to depletion of the retinoid-related receptor RORA that regulates various physiological processes including immunity through modulation of gene expression. Collectively, these findings identify a novel regulatory role for RARRES3 as an endogenous inhibitor of IP expression, and contribute to our evolving understanding of potential pathways underlying breast cancer driven immune modulation.

Protein oxidation and degradation during proliferative senescence of human MRC-5 fibroblasts.

PubMed

Sitte, N; Merker, K; von Zglinicki, T; Grune, T

2000-03-01

One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The aging process on a cellular level can be treated either as the ongoing proliferation until a certain number of cell divisions is reached (the Hayflick limit) or as the aging of nondividing cells, that is, the age-related changes in cells without proliferation. The present investigation was undertaken to reveal the changes in protein turnover, proteasome activity, and protein oxidation status during proliferative senescence. We were able to demonstrate that the activity of the cytosolic proteasomal system declines dramatically during the proliferative senescence of human MRC-5 fibroblasts. Regardless of the loss in activity, it could be demonstrated that there are no changes in the transcription and translation of proteasomal subunits. This decline in proteasome activity was accompanied by an increased concentration of oxidized proteins. Cells at higher proliferation stages were no longer able to respond with increased degradation of endogenous [(35)S]-Met-radiolabeled proteins after hydrogen peroxide- or quinone-induced oxidative stress. It could be demonstrated that oxidized proteins in senescent human MRC-5 fibroblasts are not as quickly removed as they are in young cells. Therefore, our study demonstrates that the accumulation of oxidized proteins and decline in protein turnover and activity of the proteasomal system are not only a process of postmitotic aging but also occur during proliferative senescence and result in an increased half-life of oxidized proteins.

Top-Down Protein Identification of Proteasome Proteins with nanoLC FT-ICR MS Employing Data-Independent Fragmentation Methods

PubMed Central

Lakshmanan, Rajeswari; Wolff, Jeremy J.; Alvarado, Rudy; Loo, Joseph A.

2014-01-01

A comparison of different data-independent fragmentation methods combined with liquid chromatography (LC) coupled to high resolution Fourier-transform ion cyclotron resonance (FT-ICR) tandem mass spectrometry (MS) is presented for top-down MS of protein mixtures. Proteins composing the 20S and 19S proteasome complex and their post-translational modifications were identified using a 15-Tesla FT-ICR mass spectrometer. The data-independent fragmentation modes with LC timescales allowed for higher duty cycle measurements that better suit on-line LC-FT-ICR-MS. Protein top-down dissociation was effected by funnel-skimmer collisionally activated dissociation (FS-CAD) and CASI (Continuous Accumulation of Selected Ions)-CAD. The N-terminus for 9 out of the 14 20S proteasome proteins were found to be modified, and the α3 protein was found to be phosphorylated; these results are consistent with previous reports. Mass measurement accuracy with the LC-FT-ICR system for the 20–30 kDa 20S proteasome proteins was 1 ppm. The intact mass of the 100 kDa Rpn1 subunit from the 19S proteasome complex regulatory particle was measured with a deviation of 17 ppm. The CASI-CAD technique is a complementary tool for intact protein fragmentation and is an effective addition to the growing inventory of dissociation methods which are compatible with on-line protein separation coupled to FT-ICR MS. PMID:24478249

Proteasomal interaction as a critical activity modulator of the human constitutive androstane receptor

PubMed Central

Chen, Tao; Laurenzana, Elizabeth M.; Coslo, Denise M.; Chen, Fengming; Omiecinski, Curtis J.

2014-01-01

The CAR (constitutive androstane receptor; NR1I3) is a critical xenobiotic sensor that regulates xenobiotic metabolism, drug clearance, energy and lipid homoeostasis, cell proliferation and development. Although constitutively active, in hepatocytes CAR is normally held quiescent through a tethering mechanism in the cytosol, anchored to a protein complex that includes several components, including heat-shock protein 90. Release and subsequent nuclear translocation of CAR is triggered through either direct binding to ligand activators such as CITCO {6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime} or through indirect chemical activation, such as with PB (phenobarbital). In the present study, we demonstrate that proteasomal inhibition markedly disrupts CAR function, repressing CAR nuclear trafficking, disrupting CAR’s interaction with nuclear co-activators and inhibiting induction of CAR target gene responses in human primary hepatocytes following treatment with either PB or CITCO. Paradoxically, these effects occur following accumulation of ubiquitinated hCAR (human CAR). Furthermore, a non-proteolytic function was indicated by its interaction with a SUG1 (suppressor for Gal1), a subunit of the 26S proteasome. Taken together, these data demonstrate that the proteasome complex functions at multiple levels to regulate the functional biology of hCAR activity. PMID:24224465

Entropic stabilization of a deubiquitinase provides conformational plasticity and slow unfolding kinetics beneficial for functioning on the proteasome

PubMed Central

Lee, Yun-Tzai Cloud; Chang, Chia-Yun; Chen, Szu-Yu; Pan, Yun-Ru; Ho, Meng-Ru; Hsu, Shang-Te Danny

2017-01-01

Human ubiquitin C-terminal hydrolyase UCH-L5 is a topologically knotted deubiquitinase that is activated upon binding to the proteasome subunit Rpn13. The length of its intrinsically disordered cross-over loop is essential for substrate recognition. Here, we showed that the catalytic domain of UCH-L5 exhibits higher equilibrium folding stability with an unfolding rate on the scale of 10−8 s−1, over four orders of magnitudes slower than its paralogs, namely UCH-L1 and -L3, which have shorter cross-over loops. NMR relaxation dynamics analysis confirmed the intrinsic disorder of the cross-over loop. Hydrogen deuterium exchange analysis further revealed a positive correlation between the length of the cross-over loop and the degree of local fluctuations, despite UCH-L5 being thermodynamically and kinetically more stable than the shorter UCHs. Considering the role of UCH-L5 in removing K48-linked ubiquitin to prevent proteasomal degradation of ubiquitinated substrates, our findings offered mechanistic insights into the evolution of UCH-L5. Compared to its paralogs, it is entropically stabilized to withstand mechanical unfolding by the proteasome while maintaining structural plasticity. It can therefore accommodate a broad range of substrate geometries at the cost of unfavourable entropic loss. PMID:28338014

Proteasomal Ubiquitin Receptor RPN-10 Controls Sex Determination in Caenorhabditis elegans

PubMed Central

Shimada, Masumi; Kanematsu, Kenji; Tanaka, Keiji; Yokosawa, Hideyoshi

2006-01-01

The ubiquitin-binding RPN-10 protein serves as a ubiquitin receptor that delivers client proteins to the 26S proteasome. Although ubiquitin recognition is an essential step for proteasomal destruction, deletion of the rpn-10 gene in yeast does not influence viability, indicating redundancy of the substrate delivery pathway. However, their specificity and biological relevance in higher eukaryotes is still enigmatic. We report herein that knockdown of the rpn-10 gene, but not any other proteasome subunit genes, sexually transforms hermaphrodites to females by eliminating hermaphrodite spermatogenesis in Caenorhabditis elegans. The feminization phenotype induced by deletion of the rpn-10 gene was rescued by knockdown of tra-2, one of sexual fate decision genes promoting female development, and its downstream target tra-1, indicating that the TRA-2–mediated sex determination pathway is crucial for the Δrpn-10–induced sterile phenotype. Intriguingly, we found that co-knockdown of rpn-10 and functionally related ubiquitin ligase ufd-2 overcomes the germline-musculinizing effect of fem-3(gf). Furthermore, TRA-2 proteins accumulated in rpn-10-defective worms. Our results show that the RPN-10–mediated ubiquitin pathway is indispensable for control of the TRA-2–mediated sex-determining pathway. PMID:17050737

Label-free Proteomic Analysis of Exosomes Derived from Inducible Hepatitis B Virus-Replicating HepAD38 Cell Line*

PubMed Central

Jia, Xiaofang; Chen, Jieliang; Megger, Dominik A.; Zhang, Xiaonan; Kozlowski, Maya; Zhang, Lijun; Fang, Zhong; Li, Jin; Chu, Qiaofang; Wu, Min; Li, Yaming; Sitek, Barbara; Yuan, Zhenghong

2017-01-01

Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that some viruses can manipulate the infection process by packing specific viral and cellular components into exosomes, small nanometer-sized (30–150 nm) vesicles secreted from various cells. However, the impact of HBV replication on the content of exosomes produced by hepatocytes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in the protein content of exosomes secreted from HepAD38 cells with or without HBV replication. Exosomes were isolated from supernantants of HepAD38 cells cultured with or without doxycycline (dox) and their purity was confirmed by transmission electron microscopy (TEM) and Western immunoblotting assays. Ion-intensity based label-free LC-MS/MS quantitation technologies were applied to analyze protein content of exosomes from HBV replicating cells [referred as HepAD38 (dox−)-exo] and from HBV nonreplicating cells [referred as HepAD38 (dox+)-exo]. A total of 1412 exosomal protein groups were identified, among which the abundance of 35 proteins was significantly changed following HBV replication. Strikingly, 5 subunit proteins from the 26S proteasome complex, including PSMC1, PSMC2, PSMD1, PSMD7 and PSMD14 were consistently enhanced in HepAD38 (dox−)-exo. Bioinformatic analysis of differential exosomal proteins confirmed the significant enrichment of components involved in the proteasomal catabolic process. Proteasome activity assays further suggested that HepAD38 (dox−)-exo had enhanced proteolytic activity compared with HepAD38 (dox+)-exo. Furthermore, human peripheral monocytes incubated with HepAD38 (dox−)-exo induced a significantly lower level of IL-6 secretion compared with IL-6 levels from HepAD38 (dox+)-exo. Irreversible inhibition of proteasomal activity within exosomes restored higher production of IL-6 by monocytes, suggesting that transmission of proteasome subunit proteins by HepAD38 (dox−)-exo might modulate the production of pro-inflammatory molecules in the recipient monocytes. These results revealed the composition and potential function of exosomes produced during HBV replication, thus providing a new perspective on the role of exosomes in HBV-host interaction. PMID:28242843

Label-free Proteomic Analysis of Exosomes Derived from Inducible Hepatitis B Virus-Replicating HepAD38 Cell Line.

PubMed

Jia, Xiaofang; Chen, Jieliang; Megger, Dominik A; Zhang, Xiaonan; Kozlowski, Maya; Zhang, Lijun; Fang, Zhong; Li, Jin; Chu, Qiaofang; Wu, Min; Li, Yaming; Sitek, Barbara; Yuan, Zhenghong

2017-04-01

Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that some viruses can manipulate the infection process by packing specific viral and cellular components into exosomes, small nanometer-sized (30-150 nm) vesicles secreted from various cells. However, the impact of HBV replication on the content of exosomes produced by hepatocytes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in the protein content of exosomes secreted from HepAD38 cells with or without HBV replication. Exosomes were isolated from supernantants of HepAD38 cells cultured with or without doxycycline (dox) and their purity was confirmed by transmission electron microscopy (TEM) and Western immunoblotting assays. Ion-intensity based label-free LC-MS/MS quantitation technologies were applied to analyze protein content of exosomes from HBV replicating cells [referred as HepAD38 (dox - )-exo] and from HBV nonreplicating cells [referred as HepAD38 (dox + )-exo]. A total of 1412 exosomal protein groups were identified, among which the abundance of 35 proteins was significantly changed following HBV replication. Strikingly, 5 subunit proteins from the 26S proteasome complex, including PSMC1, PSMC2, PSMD1, PSMD7 and PSMD14 were consistently enhanced in HepAD38 (dox - )-exo. Bioinformatic analysis of differential exosomal proteins confirmed the significant enrichment of components involved in the proteasomal catabolic process. Proteasome activity assays further suggested that HepAD38 (dox - )-exo had enhanced proteolytic activity compared with HepAD38 (dox + )-exo. Furthermore, human peripheral monocytes incubated with HepAD38 (dox - )-exo induced a significantly lower level of IL-6 secretion compared with IL-6 levels from HepAD38 (dox + )-exo. Irreversible inhibition of proteasomal activity within exosomes restored higher production of IL-6 by monocytes, suggesting that transmission of proteasome subunit proteins by HepAD38 (dox - )-exo might modulate the production of pro-inflammatory molecules in the recipient monocytes. These results revealed the composition and potential function of exosomes produced during HBV replication, thus providing a new perspective on the role of exosomes in HBV-host interaction. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Pentoxifylline and the proteasome inhibitor MG132 induce apoptosis in human leukemia U937 cells through a decrease in the expression of Bcl-2 and Bcl-XL and phosphorylation of p65.

PubMed

Bravo-Cuellar, Alejandro; Hernández-Flores, Georgina; Lerma-Díaz, José Manuel; Domínguez-Rodríguez, Jorge Ramiro; Jave-Suárez, Luis F; De Célis-Carrillo, Ruth; Aguilar-Lemarroy, Adriana; Gómez-Lomeli, Paulina; Ortiz-Lazareno, Pablo Cesar

2013-02-28

In Oncology, the resistance of the cancerous cells to chemotherapy continues to be the principal limitation. The nuclear factor-kappa B (NF-κB) transcription factor plays an important role in tumor escape and resistance to chemotherapy and this factor regulates several pathways that promote tumor survival including some antiapoptotic proteins such as Bcl-2 and Bcl-XL. In this study, we investigated, in U937 human leukemia cells, the effects of PTX and the MG132 proteasome inhibitor, drugs that can disrupt the NF-κB pathway. For this, we evaluated viability, apoptosis, cell cycle, caspases-3, -8, -9, cytochrome c release, mitochondrial membrane potential loss, p65 phosphorylation, and the modification in the expression of pro- and antiapoptotic genes, and the Bcl-2 and Bcl-XL antiapoptotic proteins. The two drugs affect the viability of the leukemia cells in a time-dependent manner. The greatest percentage of apoptosis was obtained with a combination of the drugs; likewise, PTX and MG132 induce G1 phase cell cycle arrest and cleavage of caspases -3,-8, -9 and cytochrome c release and mitochondrial membrane potential loss in U937 human leukemia cells. In these cells, PTX and the MG132 proteasome inhibitor decrease p65 (NF-κB subunit) phosphorylation and the antiapoptotic proteins Bcl-2 and Bcl-XL. We also observed, with a combination of these drugs overexpression of a group of the proapoptotic genes BAX, DIABLO, and FAS while the genes BCL-XL, MCL-1, survivin, IκB, and P65 were downregulated. The two drugs used induce apoptosis per se, this cytotoxicity was greater with combination of both drugs. These observations are related with the caspases -9, -3 cleavage and G1 phase cell cycle arrest, and a decrease in p65 phosphorylation and Bcl-2 and Bcl-XL proteins. As well as this combination of drugs promotes the upregulation of the proapoptotic genes and downregulation of antiapoptotic genes. These observations strongly confirm antileukemic potential.

AGEs-RAGE system down-regulates Sirt1 through the ubiquitin-proteasome pathway to promote FN and TGF-β1 expression in male rat glomerular mesangial cells.

PubMed

Huang, Kai-Peng; Chen, Cheng; Hao, Jie; Huang, Jun-Ying; Liu, Pei-Qing; Huang, He-Qing

2015-01-01

We previously demonstrated that advanced glycation-end products (AGEs) promote the pathological progression of diabetic nephropathy by decreasing silent information regulator 2-related protein 1 (Sirt1) expression in glomerular mesangial cells (GMCs). Here, we investigated whether AGEs-receptor for AGEs (RAGE) system down-regulated Sirt1 expression through ubiquitin-proteasome pathway and whether Sirt1 ubiquitination affected fibronectin (FN) and TGF-β1, 2 fibrotic indicators in GMCs. Sirt1 was polyubiquitinated and subsequently degraded by proteasome. AGEs increased Sirt1 ubiquitination and proteasome-mediated degradation, shortened Sirt1 half-life, and promoted FN and TGF-β1 expression. Ubiquitin-specific protease 22 (USP22) reduced Sirt1 ubiquitination and degradation and decreased FN and TGF-β1 expression in GMCs under both basal and AGEs-treated conditions. USP22 depletion enhanced Sirt1 degradation and displayed combined effects with AGEs to further promote FN and TGF-β1 expression. RAGE functioned crucial mediating roles in these processes via its C-terminal cytosolic domain. Inhibiting Sirt1 by EX-527 substantially suppressed the down-regulation of FN and TGF-β1 resulting from USP22 overexpression under both normal and AGEs-treated conditions, eventually leading to their up-regulation in GMCs. These results indicated that the AGEs-RAGE system increased the ubiquitination and subsequent proteasome-mediated degradation of Sirt1 by reducing USP22 level, and AGEs-RAGE-USP22-Sirt1 formed a cascade pathway that regulated FN and TGF-β1 level, which participated in the pathological progression of diabetic nephropathy.

An ethanolic extract of Artemisia dracunculus L. regulates gene expression of ubiquitin-proteasome system enzymes in skeletal muscle: potential role in the treatment of sarcopenic obesity.

PubMed

Kirk-Ballard, Heather; Kilroy, Gail; Day, Britton C; Wang, Zhong Q; Ribnicky, David M; Cefalu, William T; Floyd, Z Elizabeth

2014-01-01

Obesity is linked to insulin resistance, a primary component of metabolic syndrome and type 2 diabetes. The problem of obesity-related insulin resistance is compounded when age-related skeletal muscle loss, called sarcopenia, occurs with obesity. Skeletal muscle loss results from elevated levels of protein degradation and prevention of obesity-related sarcopenic muscle loss will depend on strategies that target pathways involved in protein degradation. An extract from Artemisia dracunculus, termed PMI 5011, improves insulin signaling and increases skeletal muscle myofiber size in a rodent model of obesity-related insulin resistance. The aim of this study was to examine the effect of PMI 5011 on the ubiquitin-proteasome system, a central regulator of muscle protein degradation. Gastrocnemius and vastus lateralis skeletal muscle was obtained from KK-A(y) obese diabetic mice fed a control or 1% (w/w) PMI 5011-supplemented diet. Regulation of genes encoding enzymes of the ubiquitin-proteasome system was determined using real-time quantitative reverse transcriptase polymerase chain reaction. Although MuRF-1 ubiquitin ligase gene expression is consistently down-regulated in skeletal muscle, atrogin-1, Fbxo40, and Traf6 expression is differentially regulated by PMI 5011. Genes encoding other enzymes of the ubiquitin-proteasome system ranging from ubiquitin to ubiquitin-specific proteases are also regulated by PMI 5011. Additionally, expression of the gene encoding the microtubule-associated protein-1 light chain 3 (LC3), a ubiquitin-like protein pivotal to autophagy-mediated protein degradation, is down-regulated by PMI 5011 in the vastus lateralis. PMI 5011 alters the gene expression of ubiquitin-proteasome system enzymes that are essential regulators of skeletal muscle mass. This suggests that PMI 5011 has therapeutic potential in the treatment of obesity-linked sarcopenia by regulating ubiquitin-proteasome-mediated protein degradation. Copyright © 2014 Elsevier Inc. All rights reserved.

Characterization and Quantification of Intact 26S Proteasome Proteins by Real-Time Measurement of Intrinsic Fluorescence Prior to Top-down Mass Spectrometry

PubMed Central

Russell, Jason D.; Scalf, Mark; Book, Adam J.; Ladror, Daniel T.; Vierstra, Richard D.; Smith, Lloyd M.; Coon, Joshua J.

2013-01-01

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1. PMID:23536786

Characterization and quantification of intact 26S proteasome proteins by real-time measurement of intrinsic fluorescence prior to top-down mass spectrometry.

PubMed

Russell, Jason D; Scalf, Mark; Book, Adam J; Ladror, Daniel T; Vierstra, Richard D; Smith, Lloyd M; Coon, Joshua J

2013-01-01

Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26S proteasome sample from Arabidopsis thaliana. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1.

The COP9 signalosome controls jasmonic acid synthesis and plant responses to herbivory and pathogens.

PubMed

Hind, Sarah R; Pulliam, Sarah E; Veronese, Paola; Shantharaj, Deepak; Nazir, Azka; Jacobs, Nekaiya S; Stratmann, Johannes W

2011-02-01

The COP9 signalosome (CSN) is a multi-protein complex that regulates the activities of cullin-RING E3 ubiquitin ligases (CRLs). CRLs ubiquitinate proteins in order to target them for proteasomal degradation. The CSN is required for proper plant development. Here we show that the CSN also has a profound effect on plant defense responses. Silencing of genes for CSN subunits in tomato plants resulted in a mild morphological phenotype and reduced expression of wound-responsive genes in response to mechanical wounding, attack by Manduca sexta larvae, and Prosystemin over-expression. In contrast, expression of pathogenesis-related genes was increased in a stimulus-independent manner in these plants. The reduced wound response in CSN-silenced plants corresponded with reduced synthesis of jasmonic acid (JA), but levels of salicylic acid (SA) were unaltered. As a consequence, these plants exhibited reduced resistance against herbivorous M. sexta larvae and the necrotrophic fungal pathogen Botrytis cinerea. In contrast, susceptibility to tobacco mosaic virus (TMV) was not altered in CSN-silenced plants. These data demonstrate that the CSN orchestrates not only plant development but also JA-dependent plant defense responses. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Selection of suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using quantitative real-time polymerase chain reaction.

PubMed

Zornhagen, K W; Kristensen, A T; Hansen, A E; Oxboel, J; Kjaer, A

2015-12-01

Quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) is a sensitive technique for quantifying gene expression. Stably expressed reference genes are necessary for normalization of RT-qPCR data. Only a few articles have been published on reference genes in canine tumours. The objective of this study was to demonstrate how to identify suitable reference genes for normalization of genes of interest in canine soft tissue sarcomas using RT-qPCR. Primer pairs for 17 potential reference genes were designed and tested in archival tumour biopsies from six dogs. The geNorm algorithm was used to analyse the most suitable reference genes. Eight potential reference genes were excluded from this final analysis because of their dissociation curves. β-Glucuronidase (GUSB) and proteasome subunit, beta type, 6 (PSMB6) were most stably expressed with an M value of 0.154 and a CV of 0.053 describing their average stability. We suggest that choice of reference genes should be based on specific testing in every new experimental set-up. © 2014 John Wiley & Sons Ltd.

The ubiquitin-proteasome system is necessary for long-term synaptic depression in Aplysia.

PubMed

Fioravante, Diasinou; Liu, Rong-Yu; Byrne, John H

2008-10-08

The neuropeptide Phe-Met-Arg-Phe-NH(2) (FMRFa) can induce transcription-dependent long-term synaptic depression (LTD) in Aplysia sensorimotor synapses. We investigated the role of the ubiquitin-proteasome system and the regulation of one of its components, ubiquitin C-terminal hydrolase (ap-uch), in LTD. LTD was sensitive to presynaptic inhibition of the proteasome and was associated with upregulation of ap-uch mRNA and protein. This upregulation appeared to be mediated by CREB2, which is generally regarded as a transcription repressor. Binding of CREB2 to the promoter region of ap-uch was accompanied by histone hyperacetylation, suggesting that CREB2 cannot only inhibit but also promote gene expression. CREB2 was phosphorylated after FMRFa, and blocking phospho-CREB2 blocked LTD. In addition to changes in the expression of ap-uch, the synaptic vesicle-associated protein synapsin was downregulated in LTD in a proteasome-dependent manner. These results suggest that proteasome-mediated protein degradation is engaged in LTD and that CREB2 may act as a transcription activator under certain conditions.

Cloning and characterization of the rat HIF-1 alpha prolyl-4-hydroxylase-1 gene.

PubMed

Cobb, Ronald R; McClary, John; Manzana, Warren; Finster, Silke; Larsen, Brent; Blasko, Eric; Pearson, Jennifer; Biancalana, Sara; Kauser, Katalin; Bringmann, Peter; Light, David R; Schirm, Sabine

2005-08-01

Prolyl-4-hydroxylase domain-containing enzymes (PHDs) mediate the oxygen-dependent regulation of the heterodimeric transcription factor hypoxia-inducible factor-1 (HIF-1). Under normoxic conditions, one of the subunits of HIF-1, HIF-1alpha, is hydroxylated on specific proline residues to target HIF-1alpha for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, the hydroxylation by the PHDs is attenuated by lack of the oxygen substrate, allowing HIF-1 to accumulate, translocate to the nucleus, and mediate HIF-mediated gene transcription. In several mammalian species including humans, three PHDs have been identified. We report here the cloning of a full-length rat cDNA that is highly homologous to the human and murine PHD-1 enzymes and encodes a protein that is 416 amino acids long. Both cDNA and protein are widely expressed in rat tissues and cell types. We demonstrate that purified and crude baculovirus-expressed rat PHD-1 exhibits HIF-1alpha specific prolyl hydroxylase activity with similar substrate affinities and is comparable to human PHD-1 protein.

Identification of the APC/C co-factor FZR1 as a novel therapeutic target for multiple myeloma.

PubMed

Crawford, Lisa J; Anderson, Gordon; Johnston, Cliona K; Irvine, Alexandra E

2016-10-25

Multiple Myeloma (MM) is a haematological neoplasm characterised by the clonal proliferation of malignant plasma cells in the bone marrow. The success of proteasome inhibitors in the treatment of MM has highlighted the importance of the ubiquitin proteasome system (UPS) in the pathogenesis of this disease. In this study, we analysed gene expression of UPS components to identify novel therapeutic targets within this pathway in MM. Here we demonstrate how this approach identified previously validated and novel therapeutic targets. In addition we show that FZR1 (Fzr), a cofactor of the multi-subunit E3 ligase complex anaphase-promoting complex/cyclosome (APC/C), represents a novel therapeutic target in myeloma. The APC/C associates independently with two cofactors, Fzr and Cdc20, to control cell cycle progression. We found high levels of FZR1 in MM primary cells and cell lines and demonstrate that expression is further increased on adhesion to bone marrow stromal cells (BMSCs). Specific knockdown of either FZR1 or CDC20 reduced viability and induced growth arrest of MM cell lines, and resulted in accumulation of APC/CFzr substrate Topoisomerase IIα (TOPIIα) or APC/CCdc20 substrate Cyclin B. Similar effects were observed following treatment with proTAME, an inhibitor of both APC/CFzr and APC/CCdc20. Combinations of proTAME with topoisomerase inhibitors, etoposide and doxorubicin, significantly increased cell death in MM cell lines and primary cells, particularly if TOPIIα levels were first increased through pre-treatment with proTAME. Similarly, combinations of proTAME with the microtubule inhibitor vincristine resulted in enhanced cell death. This study demonstrates the potential of targeting the APC/C and its cofactors as a therapeutic approach in MM.

The Proteasomal Rpn11 Metalloprotease Suppresses Tombusvirus RNA Recombination and Promotes Viral Replication via Facilitating Assembly of the Viral Replicase Complex

PubMed Central

Prasanth, K. Reddisiva; Barajas, Daniel

2014-01-01

ABSTRACT RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a “matchmaker” that brings the viral p92pol replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. IMPORTANCE RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in tombusvirus recombination in a yeast model host, in plants, and in vitro. We found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic recombination. PMID:25540361

Reduced Insulin/Insulin-like Growth Factor-1 Signaling and Dietary Restriction Inhibit Translation but Preserve Muscle Mass in Caenorhabditis elegans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Depuydt, Geert; Xie, Fang; Petyuk, Vladislav A.

Reduced signaling through the C. elegans insulin/IGF1 like tyrosine kinase receptor daf2 and dietary restriction via bacterial dilution are two well-characterized lifespan-extending interventions that operate in parallel or through (partially) independent mechanisms. Using accurate mass and time tag LCMS/MS quantitative proteomics we detected that the abundance of a large number of ribosomal subunits is decreased in response to dietary restriction as well as in the daf2(e1370) insulin/IGF1 receptor mutant. In addition, general protein synthesis levels in these long-lived worms are repressed. Surprisingly, ribosomal transcript levels were not correlated to actual protein abundance, suggesting that posttranscriptional regulation determines ribosome content. Proteomicsmore » also revealed increased presence of many structural muscle cell components in long-lived worms, which appears to result from prioritized preservation of muscle cell volume in nutrient-poor conditions or low insulin-like signaling. Activation of DAF16, but not diet-restriction, stimulates mRNA expression of muscle-related genes to prevent muscle atrophy. Important daf2 specific proteome changes include overexpression of aerobic metabolism enzymes and a general activation of stress responsive and immune defense systems, while increased abundance of many protein subunits of the proteasome core complex is a DR-specific characteristic.« less

A case-control study on association of proteasome subunit beta 8 (PSMB8) and transporter associated with antigen processing 1 (TAP1) polymorphisms and their transcript levels in vitiligo from Gujarat

PubMed Central

Jadeja, Shahnawaz D.; Mansuri, Mohmmad Shoab; Singh, Mala; Dwivedi, Mitesh; Laddha, Naresh C.

2017-01-01

Background Autoimmunity has been implicated in the destruction of melanocytes from vitiligo skin. Major histocompatibility complex (MHC) class-II linked genes proteasome subunit beta 8 (PSMB8) and transporter associated with antigen processing 1 (TAP1), involved in antigen processing and presentation have been reported to be associated with several autoimmune diseases including vitiligo. Objectives To explore PSMB8 rs2071464 and TAP1 rs1135216 single nucleotide polymorphisms and to estimate the expression of PSMB8 and TAP1 in patients with vitiligo and unaffected controls from Gujarat. Methods PSMB8 rs2071464 polymorphism was genotyped using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP) and TAP1 rs1135216 polymorphism was genotyped by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) in 378 patients with vitiligo and 509 controls. Transcript levels of PSMB8 and TAP1 were measured in the PBMCs of 91 patients and 96 controls by using qPCR. Protein levels of PSMB8 were also determined by Western blot analysis. Results The frequency of ‘TT’ genotype of PSMB8 polymorphism was significantly lowered in patients with generalized and active vitiligo (p = 0.019 and p = 0.005) as compared to controls suggesting its association with the activity of the disease. However, TAP1 polymorphism was not associated with vitiligo susceptibility. A significant decrease in expression of PSMB8 at both transcript level (p = 0.002) as well as protein level (p = 0.0460) was observed in vitiligo patients as compared to controls. No significant difference was observed between patients and controls for TAP1 transcripts (p = 0.553). Interestingly, individuals with the susceptible CC genotype of PSMB8 polymorphism showed significantly reduced PSMB8 transcript level as compared to that of CT and TT genotypes (p = 0.009 and p = 0.003 respectively). Conclusions PSMB8 rs2071464 was associated with generalized and active vitiligo from Gujarat whereas TAP1 rs1135216 showed no association. The down-regulation of PSMB8 in patients with risk genotype ‘CC’ advocates the vital role of PSMB8 in the autoimmune basis of vitiligo. PMID:28700671

The long N-terminus of the human monocarboxylate transporter 8 is a target of ubiquitin-dependent proteasomal degradation which regulates protein expression and oligomerization capacity.

PubMed

Zwanziger, Denise; Schmidt, Mathias; Fischer, Jana; Kleinau, Gunnar; Braun, Doreen; Schweizer, Ulrich; Moeller, Lars Christian; Biebermann, Heike; Fuehrer, Dagmar

2016-10-15

Monocarboxylate transporter 8 (MCT8) equilibrates thyroid hormones between the extra- and the intracellular sides. MCT8 exists either with a short or a long N-terminus, but potential functional differences between both variants are yet not known. We, therefore, generated MCT8 constructs which are different in N-terminal length: MCT8(1-613), MCT8(25-613), MCT8(49-613) and MCT8(75-613). The M75G substitution prevents translation of MCT8(75-613) and ensures expression of full-length MCT8 protein. The K56G substitution was made to prevent ubiquitinylation. Cell-surface expression, localization and proteasomal degradation were investigated using C-terminally GFP-tagged MCT8 constructs (HEK293 and MDCK1 cells) and oligomerization capacity was determined using N-terminally HA- and C-terminally FLAG-tagged MCT8 constructs (COS7 cells). MCT8(1-613)-GFP showed a lower protein expression than the shorter MCT8(75-613)-GFP protein. The proteasome inhibitor lactacystin increased MCT8(1-613)-GFP protein amount, suggesting proteasomal degradation of MCT8 with the long N-terminus. Ubiquitin conjugation of MCT8(1-613)-GFP was found by immuno-precipitation. A diminished ubiquitin conjugation caused by K56G substitution resulted in increased MCT8(1-613)-GFP protein expression. Sandwich ELISA was performed to investigate if the bands at higher molecular weight observed in Western blot analysis are due to MCT8 oligomerization, which was indeed shown. Our data imply a role of the long N-terminus of MCT8 as target of ubiquitin-dependent proteasomal degradation affecting MCT8 amount and subsequently oligomerization capacity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Interferon antagonist NSs of La Crosse virus triggers a DNA damage response-like degradation of transcribing RNA polymerase II.

PubMed

Verbruggen, Paul; Ruf, Marius; Blakqori, Gjon; Överby, Anna K; Heidemann, Martin; Eick, Dirk; Weber, Friedemann

2011-02-04

La Crosse encephalitis virus (LACV) is a mosquito-borne member of the negative-strand RNA virus family Bunyaviridae. We have previously shown that the virulence factor NSs of LACV is an efficient inhibitor of the antiviral type I interferon system. A recombinant virus unable to express NSs (rLACVdelNSs) strongly induced interferon transcription, whereas the corresponding wt virus (rLACV) suppressed it. Here, we show that interferon induction by rLACVdelNSs mainly occurs through the signaling pathway leading from the pattern recognition receptor RIG-I to the transcription factor IRF-3. NSs expressed by rLACV, however, acts downstream of IRF-3 by specifically blocking RNA polymerase II-dependent transcription. Further investigations revealed that NSs induces proteasomal degradation of the mammalian RNA polymerase II subunit RPB1. NSs thereby selectively targets RPB1 molecules of elongating RNA polymerase II complexes, the so-called IIo form. This phenotype has similarities to the cellular DNA damage response, and NSs was indeed found to transactivate the DNA damage response gene pak6. Moreover, NSs expressed by rLACV boosted serine 139 phosphorylation of histone H2A.X, one of the earliest cellular reactions to damaged DNA. However, other DNA damage response markers such as up-regulation and serine 15 phosphorylation of p53 or serine 1524 phosphorylation of BRCA1 were not triggered by LACV infection. Collectively, our data indicate that the strong suppression of interferon induction by LACV NSs is based on a shutdown of RNA polymerase II transcription and that NSs achieves this by exploiting parts of the cellular DNA damage response pathway to degrade IIo-borne RPB1 subunits.

Hypoxia-inducible factor-1 (HIF-1) promotes its degradation by induction of HIF-α-prolyl-4-hydroxylases

PubMed Central

2004-01-01

An important regulator involved in oxygen-dependent gene expression is the transcription factor HIF (hypoxia-inducible factor), which is composed of an oxygen-sensitive α-subunit (HIF-1α or HIF-2α) and a constitutively expressed β-subunit. In normoxia, HIF-1α is destabilized by post-translational hydroxylation of Pro-564 and Pro-402 by a family of oxygen-sensitive dioxygenases. The three HIF-modifying human enzymes have been termed prolyl hydroxylase domain containing proteins (PHD1, PHD2 and PHD3). Prolyl hydroxylation leads to pVHL (von-Hippel-Lindau protein)-dependent ubiquitination and rapid proteasomal degradation of HIF-1α. In the present study, we report that human PHD2 and PHD3 are induced by hypoxia in primary and transformed cell lines. In the human osteosarcoma cell line, U2OS, selective suppression of HIF-1α expression by RNA interference resulted in a complete loss of hypoxic induction of PHD2 and PHD3. Induction of PHD2 by hypoxia was lost in pVHL-deficient RCC4 cells. These results suggest that hypoxic induction of PHD2 and PHD3 is critically dependent on HIF-α. Using a VHL capture assay, we demonstrate that HIF-α prolyl-4-hydroxylase capacity of cytoplasmic and nuclear protein extracts was enhanced by prolonged exposure to hypoxia. Degradation of HIF-1α after reoxygenation was accelerated, which demonstrates functional relevance of the present results. We propose a direct, negative regulatory mechanism, which limits accumulation of HIF-1α in hypoxia and leads to accelerated degradation on reoxygenation after long-term hypoxia. PMID:15104534

Shotgun proteomics of Aspergillus niger microsomes upon D-xylose induction.

PubMed

Ferreira de Oliveira, José Miguel P; van Passel, Mark W J; Schaap, Peter J; de Graaff, Leo H

2010-07-01

Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these organisms. In this study, we analyzed the effects of the d-xylose induction of cellulase and hemicellulase enzyme secretion on the protein composition of secretory organelles in Aspergillus niger. We aimed to systematically identify the components involved in the secretion of these enzymes via mass spectrometry of enriched subcellular microsomal fractions. Under each condition, fractions enriched for secretory organelles were processed for tandem mass spectrometry, resulting in the identification of peptides that originate from 1,081 proteins, 254 of which-many of them hypothetical proteins-were predicted to play direct roles in the secretory pathway. d-Xylose induction led to an increase in specific small GTPases known to be associated with polarized growth, exocytosis, and endocytosis. Moreover, the endoplasmic-reticulum-associated degradation (ERAD) components Cdc48 and all 14 of the 20S proteasomal subunits were recruited to the secretory organelles. In conclusion, induction of extracellular enzymes results in specific changes in the secretory subproteome of A. niger, and the most prominent change found in this study was the recruitment of the 20S proteasomal subunits to the secretory organelles.

SCAR Mediates Light-Induced Root Elongation in Arabidopsis through Photoreceptors and Proteasomes[W][OA

PubMed Central

Dyachok, Julia; Zhu, Ling; Liao, Fuqi; He, Ji; Huq, Enamul; Blancaflor, Elison B.

2011-01-01

The ARP2/3 complex, a highly conserved nucleator of F-actin, and its activator, the SCAR complex, are essential for growth in plants and animals. In this article, we present a pathway through which roots of Arabidopsis thaliana directly perceive light to promote their elongation. The ARP2/3-SCAR complex and the maintenance of longitudinally aligned F-actin arrays are crucial components of this pathway. The involvement of the ARP2/3-SCAR complex in light-regulated root growth is supported by our finding that mutants of the SCAR complex subunit BRK1/HSPC300, or other individual subunits of the ARP2/3-SCAR complex, showed a dramatic inhibition of root elongation in the light, which mirrored reduced growth of wild-type roots in the dark. SCAR1 degradation in dark-grown wild-type roots by constitutive photomorphogenic 1 (COP1) E3 ligase and 26S proteasome accompanied the loss of longitudinal F-actin and reduced root growth. Light perceived by the root photoreceptors, cryptochrome and phytochrome, suppressed COP1-mediated SCAR1 degradation. Taken together, our data provide a biochemical explanation for light-induced promotion of root elongation by the ARP2/3-SCAR complex. PMID:21972261

Protection against murine osteoarthritis by inhibition of the 26S proteasome and lysine-48 linked ubiquitination.

PubMed

Radwan, Marta; Wilkinson, David J; Hui, Wang; Destrument, Auriane P M; Charlton, Sarah H; Barter, Matt J; Gibson, Beth; Coulombe, Josée; Gray, Douglas A; Rowan, Andrew D; Young, David A

2015-08-01

To determine whether the process of ubiquitination and/or activity of the 26S proteasome are involved in the induction of osteoarthritis (OA). Bovine cartilage resorption assays, chondrocyte cell-line SW1353 and primary human articular chondrocytes were used with the general proteasome inhibitor MG132 or vehicle to identify a role of the ubiquitin-proteasome system (UPS) in cartilage destruction and matrix metalloproteinase-13 (MMP13) expression. In vivo, MG132 or vehicle, were delivered subcutaneously to mice following destabilisation of the medial meniscus (DMM)-induced OA. Subsequently, DMM was induced in Lys-to-Arg (K48R and K63R) mutant ubiquitin (Ub) transgenic mice. Cytokine signalling in SW1353s was monitored by immunoblotting and novel ubiquitinated substrates identified using Tandem Ubiquitin Binding Entities purification followed by mass spectrometry. The ubiquitination of TRAFD1 was assessed via immunoprecipitation and immunoblotting and its role in cytokine signal-transduction determined using RNA interference and real-time RT-PCR for MMP13 and interleukin-6 (IL6). Supplementation with the proteasome inhibitor MG132 protected cartilage from cytokine-mediated resorption and degradation in vivo in mice following DMM-induced OA. Using transgenic animals only K48R-mutated Ub partially protected against OA compared to wild-type or wild-type Ub transgenic mice, and this was only evident on the medial femoral condyle. After confirming ubiquitination was vital for NF-κB signalling and MMP13 expression, a screen for novel ubiquitinated substrates involved in cytokine-signalling identified TRAFD1; the depletion of which reduced inflammatory mediator-induced MMP13 and IL6 expression. Our data for the first time identifies a role for ubiquitination and the proteasome in the induction of OA via regulation of inflammatory mediator-induced MMP13 expression. These data open avenues of research to determine whether the proteasome, or K48-linked ubiquitination, are potential therapeutic targets in OA. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Different Expression Levels of Human Mutant Ubiquitin B+1 (UBB+1) Can Modify Chronological Lifespan or Stress Resistance of Saccharomyces cerevisiae

PubMed Central

Muñoz-Arellano, Ana Joyce; Chen, Xin; Molt, Andrea; Meza, Eugenio; Petranovic, Dina

2018-01-01

The ubiquitin-proteasome system (UPS) is the main pathway responsible for the degradation of misfolded proteins, and its dysregulation has been implicated in several neurodegenerative diseases, including Alzheimer’s disease (AD). UBB+1, a mutant variant of ubiquitin B, was found to accumulate in neurons of AD patients and it has been linked to UPS dysfunction and neuronal death. Using the yeast Saccharomyces cerevisiae as a model system, we constitutively expressed UBB+1 to evaluate its effects on proteasome function and cell death, particularly under conditions of chronological aging. We showed that the expression of UBB+1 caused inhibition of the three proteasomal proteolytic activities (caspase-like (β1), trypsin-like (β2) and chymotrypsin-like (β5) activities) in yeast. Interestingly, this inhibition did not alter cell viability of growing cells. Moreover, we showed that cells expressing UBB+1 at lower level displayed an increased capacity to degrade induced misfolded proteins. When we evaluated cells during chronological aging, UBB+1 expression at lower level, prevented cells to accumulate reactive oxygen species (ROS) and avert apoptosis, dramatically increasing yeast life span. Since proteasome inhibition by UBB+1 has previously been shown to induce chaperone expression and thus protect against stress, we evaluated our UBB+1 model under heat shock and oxidative stress. Higher expression of UBB+1 caused thermotolerance in yeast due to induction of chaperones, which occurred to a lesser extent at lower expression level of UBB+1 (where we observed the phenotype of extended life span). Altering UPS capacity by differential expression of UBB+1 protects cells against several stresses during chronological aging. This system can be valuable to study the effects of UBB+1 on misfolded proteins involved in neurodegeneration and aging.

The molecular chaperone Hsp70 promotes the proteolytic removal of oxidatively damaged proteins by the proteasome

PubMed Central

Reeg, Sandra; Jung, Tobias; Castro, José P.; Davies, Kelvin J.A.; Henze, Andrea; Grune, Tilman

2016-01-01

One hallmark of aging is the accumulation of protein aggregates, promoted by the unfolding of oxidized proteins. Unraveling the mechanism by which oxidized proteins are degraded may provide a basis to delay the early onset of features, such as protein aggregate formation, that contribute to the aging phenotype. In order to prevent aggregation of oxidized proteins, cells recur to the 20S proteasome, an efficient turnover proteolysis complex. It has previously been shown that upon oxidative stress the 26S proteasome, another form, dissociates into the 20S form. A critical player implicated in its dissociation is the Heat Shock Protein 70 (Hsp70), which promotes an increase in free 20S proteasome and, therefore, an increased capability to degrade oxidized proteins. The aim of this study was to test whether or not Hsp70 is involved in cooperating with the 20S proteasome for a selective degradation of oxidatively damaged proteins. Our results demonstrate that Hsp70 expression is induced in HT22 cells as a result of mild oxidative stress conditions. Furthermore, Hsp70 prevents the accumulation of oxidized proteins and directly promotes their degradation by the 20S proteasome. In contrast the expression of the Heat shock cognate protein 70 (Hsc70) was not changed in recovery after oxidative stress and Hsc70 has no influence on the removal of oxidatively damaged proteins. We were able to demonstrate in HT22 cells, in brain homogenates from 129/SV mice and in vitro, that there is an increased interaction of Hsp70 with oxidized proteins, but also with the 20S proteasome, indicating a role of Hsp70 in mediating the interaction of oxidized proteins with the 20S proteasome. Thus, our data clearly implicate an involvement of Hsp70 oxidatively damaged protein degradation by the 20S proteasome. PMID:27498116

The molecular chaperone Hsp70 promotes the proteolytic removal of oxidatively damaged proteins by the proteasome.

PubMed

Reeg, Sandra; Jung, Tobias; Castro, José P; Davies, Kelvin J A; Henze, Andrea; Grune, Tilman

2016-10-01

One hallmark of aging is the accumulation of protein aggregates, promoted by the unfolding of oxidized proteins. Unraveling the mechanism by which oxidized proteins are degraded may provide a basis to delay the early onset of features, such as protein aggregate formation, that contribute to the aging phenotype. In order to prevent aggregation of oxidized proteins, cells recur to the 20S proteasome, an efficient turnover proteolysis complex. It has previously been shown that upon oxidative stress the 26S proteasome, another form, dissociates into the 20S form. A critical player implicated in its dissociation is the Heat Shock Protein 70 (Hsp70), which promotes an increase in free 20S proteasome and, therefore, an increased capability to degrade oxidized proteins. The aim of this study was to test whether or not Hsp70 is involved in cooperating with the 20S proteasome for a selective degradation of oxidatively damaged proteins. Our results demonstrate that Hsp70 expression is induced in HT22 cells as a result of mild oxidative stress conditions. Furthermore, Hsp70 prevents the accumulation of oxidized proteins and directly promotes their degradation by the 20S proteasome. In contrast the expression of the Heat shock cognate protein 70 (Hsc70) was not changed in recovery after oxidative stress and Hsc70 has no influence on the removal of oxidatively damaged proteins. We were able to demonstrate in HT22 cells, in brain homogenates from 129/SV mice and in vitro, that there is an increased interaction of Hsp70 with oxidized proteins, but also with the 20S proteasome, indicating a role of Hsp70 in mediating the interaction of oxidized proteins with the 20S proteasome. Thus, our data clearly implicate an involvement of Hsp70 oxidatively damaged protein degradation by the 20S proteasome. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Bortezomib partially improves laminin α2 chain-deficient muscular dystrophy.

PubMed

Körner, Zandra; Fontes-Oliveira, Cibely C; Holmberg, Johan; Carmignac, Virginie; Durbeej, Madeleine

2014-05-01

Congenital muscular dystrophy, caused by mutations in LAMA2 (the gene encoding laminin α2 chain), is a severe and incapacitating disease for which no therapy is yet available. We have recently demonstrated that proteasome activity is increased in laminin α2 chain-deficient muscle and that treatment with the nonpharmaceutical proteasome inhibitor MG-132 reduces muscle pathology in laminin α2 chain-deficient dy(3K)/dy(3K) mice. Here, we explore the use of the selective and therapeutic proteasome inhibitor bortezomib (currently used for treatment of relapsed multiple myeloma and mantle cell lymphoma) in dy(3K)/dy(3K) mice and in congenital muscular dystrophy type 1A muscle cells. Outcome measures included quantitative muscle morphology, gene and miRNA expression analyses, proteasome activity, motor activity, and survival. Bortezomib improved several histological hallmarks of disease, partially normalized miRNA expression (miR-1 and miR-133a), and enhanced body weight, locomotion, and survival of dy(3K)/dy(3K) mice. In addition, bortezomib reduced proteasome activity in congenital muscular dystrophy type 1A myoblasts and myotubes. These findings provide evidence that the proteasome inhibitor bortezomib partially reduces laminin α2 chain-deficient muscular dystrophy. Investigation of the clinical efficacy of bortezomib administration in congenital muscular dystrophy type 1A clinical trials may be warranted. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Implication of altered ubiquitin-proteasome system and ER stress in the muscle atrophy of diabetic rats.

PubMed

Reddy, S Sreenivasa; Shruthi, Karnam; Prabhakar, Y Konda; Sailaja, Gummadi; Reddy, G Bhanuprakash

2018-02-01

Skeletal muscle is adversely affected in type-1 diabetes, and excessively stimulated ubiquitin-proteasome system (UPS) was found to be a leading cause of muscle wasting or atrophy. The role of endoplasmic reticulum (ER) stress in muscle atrophy of type-1 diabetes is not known. Hence, we investigated the role of UPS and ER stress in the muscle atrophy of chronic diabetes rat model. Diabetes was induced with streptozotocin (STZ) in male Sprague-Dawley rats and were sacrificed 2- and 4-months thereafter to collect gastrocnemius muscle. In another experiment, 2-months post-STZ-injection diabetic rats were treated with MG132, a proteasome inhibitor, for the next 2-months and gastrocnemius muscle was collected. The muscle fiber cross-sectional area was diminished in diabetic rats. The expression of UPS components: E1, MURF1, TRIM72, UCHL1, UCHL5, ubiquitinated proteins, and proteasome activity were elevated in the diabetic rats indicating activated UPS. Altered expression of ER-associated degradation (ERAD) components and increased ER stress markers were detected in 4-months diabetic rats. Proteasome inhibition by MG132 alleviated alterations in the UPS and ER stress in diabetic rat muscle. Increased UPS activity and ER stress were implicated in the muscle atrophy of diabetic rats and proteasome inhibition exhibited beneficiary outcome. Copyright © 2017 Elsevier Inc. All rights reserved.

Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins.

PubMed

Bonuccelli, Gloria; Sotgia, Federica; Schubert, William; Park, David S; Frank, Philippe G; Woodman, Scott E; Insabato, Luigi; Cammer, Michael; Minetti, Carlo; Lisanti, Michael P

2003-10-01

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is absent in the skeletal muscle of DMD patients and mdx mice. At the plasma membrane of skeletal muscle fibers, dystrophin associates with a multimeric protein complex, termed the dystrophin-glycoprotein complex (DGC). Protein members of this complex are normally absent or greatly reduced in dystrophin-deficient skeletal muscle fibers, and are thought to undergo degradation through an unknown pathway. As such, we reasoned that inhibition of the proteasomal degradation pathway might rescue the expression and subcellular localization of dystrophin-associated proteins. To test this hypothesis, we treated mdx mice with the well-characterized proteasomal inhibitor MG-132. First, we locally injected MG-132 into the gastrocnemius muscle, and observed the outcome after 24 hours. Next, we performed systemic treatment using an osmotic pump that allowed us to deliver different concentrations of the proteasomal inhibitor, over an 8-day period. By immunofluorescence and Western blot analysis, we show that administration of the proteasomal inhibitor MG-132 effectively rescues the expression levels and plasma membrane localization of dystrophin, beta-dystroglycan, alpha-dystroglycan, and alpha-sarcoglycan in skeletal muscle fibers from mdx mice. Furthermore, we show that systemic treatment with the proteasomal inhibitor 1) reduces muscle membrane damage, as revealed by vital staining (with Evans blue dye) of the diaphragm and gastrocnemius muscle isolated from treated mdx mice, and 2) ameliorates the histopathological signs of muscular dystrophy, as judged by hematoxylin and eosin staining of muscle biopsies taken from treated mdx mice. Thus, the current study opens new and important avenues in our understanding of the pathogenesis of DMD. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD.

cAMP signalling decreases p300 protein levels by promoting its ubiquitin/proteasome dependent degradation via Epac and p38 MAPK in lung cancer cells.

PubMed

Jeong, Min-Jae; Kim, Eui-Jun; Cho, Eun-Ah; Ye, Sang-Kyu; Kang, Gyeong Hoon; Juhnn, Yong-Sung

2013-05-02

The transcriptional coactivator p300 functions as a histone acetyltransferase and a scaffold for transcription factors. We investigated the effect of cAMP signalling on p300 expression. The activation of cAMP signalling by the expression of constitutively active Gαs or by treatment with isoproterenol decreased the p300 protein expression in lung cancer cells. Isoproterenol promoted the ubiquitination and subsequent proteasomal degradation of p300 in an Epac-dependent manner. Epac promoted p300 degradation by inhibiting the activity of p38 MAPK. It is concluded that cAMP signalling decreases the level of the p300 protein by promoting its ubiquitin-proteasome dependent degradation, which is mediated by Epac and p38 MAPK, in lung cancer cells. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Proteomic Profiling of Radiation-Induced Skin Fibrosis in Rats: Targeting the Ubiquitin-Proteasome System

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wenjie; Cyrus Tang Hematology Center, Soochow University, Suzhou; Luo, Judong

Purpose: To investigate the molecular changes underlying the pathogenesis of radiation-induced skin fibrosis. Methods and Materials: Rat skin was irradiated to 30 or 45 Gy with an electron beam. Protein expression in fibrotic rat skin and adjacent normal tissues was quantified by label-free protein quantitation. Human skin cells HaCaT and WS-1 were treated by x-ray irradiation, and the proteasome activity was determined with a fluorescent probe. The effect of proteasome inhibitors on Transforming growth factor Beta (TGF-B) signaling was measured by Western blot and immunofluorescence. The efficacy of bortezomib in wound healing of rat skin was assessed by the skin injurymore » scale. Results: We found that irradiation induced epidermal and dermal hyperplasia in rat and human skin. One hundred ninety-six preferentially expressed and 80 unique proteins in the irradiated fibrotic skin were identified. Through bioinformatic analysis, the ubiquitin-proteasome pathway showed a significant fold change and was investigated in greater detail. In vitro experiments demonstrated that irradiation resulted in a decline in the activity of the proteasome in human skin cells. The proteasome inhibitor bortezomib suppressed profibrotic TGF-β downstream signaling but not TGF-β secretion stimulated by irradiation in HaCaT and WS-1 cells. Moreover, bortezomib ameliorated radiation-induced skin injury and attenuated epidermal hyperplasia. Conclusion: Our findings illustrate the molecular changes during radiation-induced skin fibrosis and suggest that targeting the ubiquitin-proteasome system would be an effective countermeasure.« less

Sulforaphane activates heat shock response and enhances proteasome activity through up-regulation of Hsp27.

PubMed

Gan, Nanqin; Wu, Yu-Chieh; Brunet, Mathilde; Garrido, Carmen; Chung, Fung-Lung; Dai, Chengkai; Mi, Lixin

2010-11-12

It is conceivable that stimulating proteasome activity for rapid removal of misfolded and oxidized proteins is a promising strategy to prevent and alleviate aging-related diseases. Sulforaphane (SFN), an effective cancer preventive agent derived from cruciferous vegetables, has been shown to enhance proteasome activities in mammalian cells and to reduce the level of oxidized proteins and amyloid β-induced cytotoxicity. Here, we report that SFN activates heat shock transcription factor 1-mediated heat shock response. Specifically, SFN-induced expression of heat shock protein 27 (Hsp27) underlies SFN-stimulated proteasome activity. SFN-induced proteasome activity was significantly enhanced in Hsp27-overexpressing cells but absent in Hsp27-silenced cells. The role of Hsp27 in regulating proteasome activity was further confirmed in isogenic REG cells, in which SFN-induced proteasome activation was only observed in cells stably overexpressing Hsp27, but not in the Hsp27-free parental cells. Finally, we demonstrated that phosphorylation of Hsp27 is irrelevant to SFN-induced proteasome activation. This study provides a novel mechanism underlying SFN-induced proteasome activity. This is the first report to show that heat shock response by SFN, in addition to the antioxidant response mediated by the Keap1-Nrf2 pathway, may contribute to cytoprotection.

Structural basis for translational surveillance by the large ribosomal subunit-associated protein quality control complex

PubMed Central

Lyumkis, Dmitry; Oliveira dos Passos, Dario; Tahara, Erich B.; Webb, Kristofor; Bennett, Eric J.; Vinterbo, Staal; Potter, Clinton S.; Carragher, Bridget; Joazeiro, Claudio A. P.

2014-01-01

All organisms have evolved mechanisms to manage the stalling of ribosomes upon translation of aberrant mRNA. In eukaryotes, the large ribosomal subunit-associated quality control complex (RQC), composed of the listerin/Ltn1 E3 ubiquitin ligase and cofactors, mediates the ubiquitylation and extraction of ribosome-stalled nascent polypeptide chains for proteasomal degradation. How RQC recognizes stalled ribosomes and performs its functions has not been understood. Using single-particle cryoelectron microscopy, we have determined the structure of the RQC complex bound to stalled 60S ribosomal subunits. The structure establishes how Ltn1 associates with the large ribosomal subunit and properly positions its E3-catalytic RING domain to mediate nascent chain ubiquitylation. The structure also reveals that a distinguishing feature of stalled 60S particles is an exposed, nascent chain-conjugated tRNA, and that the Tae2 subunit of RQC, which facilitates Ltn1 binding, is responsible for selective recognition of stalled 60S subunits. RQC components are engaged in interactions across a large span of the 60S subunit surface, connecting the tRNA in the peptidyl transferase center to the distally located nascent chain tunnel exit. This work provides insights into a mechanism linking translation and protein degradation that targets defective proteins immediately after synthesis, while ignoring nascent chains in normally translating ribosomes. PMID:25349383

Radiosensitizing effect of PSMC5, a 19S proteasome ATPase, in H460 lung cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yim, Ji-Hye; Yun, Hong Shik; Department of Life Science, College of Natural Sciences, Hanyang University, Seoul 133-791

2016-01-01

The function of PSMC5 (proteasome 26S subunit, ATPase 5) in tumors, particularly with respect to cancer radioresistance, is not known. Here, we identified PSMC5 as a novel radiosensitivity biomarker, demonstrating that radiosensitive H460 cells were converted to a radioresistance phenotype by PSMC5 depletion. Exposure of H460 cells to radiation induced a marked accumulation of cell death-promoting reactive oxygen species, but this effect was blocked in radiation-treated H460 PSMC5-knockdown cells through downregulation of the p53-p21 pathway. Interestingly, PSMC5 depletion in H460 cells enhanced both AKT activation and MDM2 transcription, thereby promoting the degradation of p53 and p21 proteins. Furthermore, specific inhibitionmore » of AKT with triciribine or knockdown of MDM2 with small interfering RNA largely restored p21 expression in PSMC5-knockdown H460 cells. Our data suggest that PSMC5 facilitates the damaging effects of radiation in radiation-responsive H460 cancer cells and therefore may serve as a prognostic indicator for radiotherapy and molecular targeted therapy in lung cancer patients. - Highlights: • PSMC5 is a radiation-sensitive biomarker in H460 cells. • PSMC5 depletion inhibits radiation-induced apoptosis in H460 cells. • PSMC5 knockdown blocks ROS generation through inhibition of the p53-p21 pathway. • PSMC5 knockdown enhances p21 degradation via AKT-dependent MDM2 stabilization.« less

Proteomics Analysis of Human Skeletal Muscle Reveals Novel Abnormalities in Obesity and Type 2 Diabetes

PubMed Central

Hwang, Hyonson; Bowen, Benjamin P.; Lefort, Natalie; Flynn, Charles R.; De Filippis, Elena A.; Roberts, Christine; Smoke, Christopher C.; Meyer, Christian; Højlund, Kurt; Yi, Zhengping; Mandarino, Lawrence J.

2010-01-01

OBJECTIVE Insulin resistance in skeletal muscle is an early phenomenon in the pathogenesis of type 2 diabetes. Studies of insulin resistance usually are highly focused. However, approaches that give a more global picture of abnormalities in insulin resistance are useful in pointing out new directions for research. In previous studies, gene expression analyses show a coordinated pattern of reduction in nuclear-encoded mitochondrial gene expression in insulin resistance. However, changes in mRNA levels may not predict changes in protein abundance. An approach to identify global protein abundance changes involving the use of proteomics was used here. RESEARCH DESIGN AND METHODS Muscle biopsies were obtained basally from lean, obese, and type 2 diabetic volunteers (n = 8 each); glucose clamps were used to assess insulin sensitivity. Muscle protein was subjected to mass spectrometry–based quantification using normalized spectral abundance factors. RESULTS Of 1,218 proteins assigned, 400 were present in at least half of all subjects. Of these, 92 were altered by a factor of 2 in insulin resistance, and of those, 15 were significantly increased or decreased by ANOVA (P < 0.05). Analysis of protein sets revealed patterns of decreased abundance in mitochondrial proteins and altered abundance of proteins involved with cytoskeletal structure (desmin and alpha actinin-2 both decreased), chaperone function (TCP-1 subunits increased), and proteasome subunits (increased). CONCLUSIONS The results confirm the reduction in mitochondrial proteins in insulin-resistant muscle and suggest that changes in muscle structure, protein degradation, and folding also characterize insulin resistance. PMID:19833877

Effects of proteasome inhibitor MG-132 on the parasite Schistosoma mansoni

PubMed Central

de Paula, Renato G.; Ornelas, Alice M. M.; Moreira, Érika B. C.; Badoco, Fernanda Rafacho; Magalhães, Lizandra G.; Verjovski-Almeida, Sergio; Rodrigues, Vanderlei

2017-01-01

Proteasome is a proteolytic complex responsible for intracellular protein turnover in eukaryotes, archaea and in some actinobacteria species. Previous work has demonstrated that in Schistosoma mansoni parasites, the proteasome inhibitor MG-132 affects parasite development. However, the molecular targets affected by MG-132 in S. mansoni are not entirely known. Here, we used expression microarrays to measure the genome-wide changes in gene expression of S. mansoni adult worms exposed in vitro to MG-132, followed by in silico functional analyses of the affected genes using Ingenuity Pathway Analysis (IPA). Scanning electron microscopy was used to document changes in the parasites’ tegument. We identified 1,919 genes with a statistically significant (q-value ≤ 0.025) differential expression in parasites treated for 24 h with MG-132, when compared with control. Of these, a total of 1,130 genes were up-regulated and 790 genes were down-regulated. A functional gene interaction network comprised of MG-132 and its target genes, known from the literature to be affected by the compound in humans, was identified here as affected by MG-132. While MG-132 activated the expression of the 26S proteasome genes, it also decreased the expression of 19S chaperones assembly, 20S proteasome maturation, ubiquitin-like NEDD8 and its partner cullin-3 ubiquitin ligase genes. Interestingly, genes that encode proteins related to potassium ion binding, integral membrane component, ATPase and potassium channel activities were significantly down-regulated, whereas genes encoding proteins related to actin binding and microtubule motor activity were significantly up-regulated. MG-132 caused important changes in the worm tegument; peeling, outbreaks and swelling in the tegument tubercles could be observed, which is consistent with interference on the ionic homeostasis in S. mansoni. Finally, we showed the down-regulation of Bax pro-apoptotic gene, as well as up-regulation of two apoptosis inhibitor genes, IAP1 and BRE1, and in contrast, down-regulation of Apaf-1 apoptotic activator, thus suggesting that apoptosis is deregulated in S. mansoni exposed to MG-132. A considerable insight has been gained concerning the potential of MG-132 as a gene expression modulator, and overall the data suggest that the proteasome might be an important molecular target for the design of new drugs against schistosomiasis. PMID:28898250

Mouse models of two missense mutations in actin-binding domain 1 of dystrophin associated with Duchenne or Becker muscular dystrophy.

PubMed

McCourt, Jackie L; Talsness, Dana M; Lindsay, Angus; Arpke, Robert W; Chatterton, Paul D; Nelson, D'anna M; Chamberlain, Christopher M; Olthoff, John T; Belanto, Joseph J; McCourt, Preston M; Kyba, Michael; Lowe, Dawn A; Ervasti, James M

2018-02-01

Missense mutations in the dystrophin protein can cause Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) through an undefined pathomechanism. In vitro studies suggest that missense mutations in the N-terminal actin-binding domain (ABD1) cause protein instability, and cultured myoblast studies reveal decreased expression levels that can be restored to wild-type with proteasome inhibitors. To further elucidate the pathophysiology of missense dystrophin in vivo, we generated two transgenic mdx mouse lines expressing L54R or L172H mutant dystrophin, which correspond to missense mutations identified in human patients with DMD or BMD, respectively. Our biochemical, histologic and physiologic analysis of the L54R and L172H mice show decreased levels of dystrophin which are proportional to the phenotypic severity. Proteasome inhibitors were ineffective in both the L54R and L172H mice, yet mice homozygous for the L172H transgene were able to express even higher levels of dystrophin which caused further improvements in muscle histology and physiology. Given that missense dystrophin is likely being degraded by the proteasome but whole body proteasome inhibition was not possible, we screened for ubiquitin-conjugating enzymes involved in targeting dystrophin to the proteasome. A myoblast cell line expressing L54R mutant dystrophin was screened with an siRNA library targeting E1, E2 and E3 ligases which identified Amn1, FBXO33, Zfand5 and Trim75. Our study establishes new mouse models of dystrophinopathy and identifies candidate E3 ligases that may specifically regulate dystrophin protein turnover in vivo. © The Author(s) 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Palmitoylation of the β4-Subunit Regulates Surface Expression of Large Conductance Calcium-activated Potassium Channel Splice Variants*

PubMed Central

Chen, Lie; Bi, Danlei; Tian, Lijun; McClafferty, Heather; Steeb, Franziska; Ruth, Peter; Knaus, Hans Guenther; Shipston, Michael J.

2013-01-01

Regulatory β-subunits of large conductance calcium- and voltage-activated potassium (BK) channels play an important role in generating functional diversity and control of cell surface expression of the pore forming α-subunits. However, in contrast to α-subunits, the role of reversible post-translational modification of intracellular residues on β-subunit function is largely unknown. Here we demonstrate that the human β4-subunit is S-acylated (palmitoylated) on a juxtamembrane cysteine residue (Cys-193) in the intracellular C terminus of the regulatory β-subunit. β4-Subunit palmitoylation is important for cell surface expression and endoplasmic reticulum (ER) exit of the β4-subunit alone. Importantly, palmitoylated β4-subunits promote the ER exit and surface expression of the pore-forming α-subunit, whereas β4-subunits that cannot be palmitoylated do not increase ER exit or surface expression of α-subunits. Strikingly, however, this palmitoylation- and β4-dependent enhancement of α-subunit surface expression was only observed in α-subunits that contain a putative trafficking motif (… REVEDEC) at the very C terminus of the α-subunit. Engineering this trafficking motif to other C-terminal α-subunit splice variants results in α-subunits with reduced surface expression that can be rescued by palmitoylated, but not depalmitoylated, β4-subunits. Our data reveal a novel mechanism by which palmitoylated β4-subunit controls surface expression of BK channels through masking of a trafficking motif in the C terminus of the α-subunit. As palmitoylation is dynamic, this mechanism would allow precise control of specific splice variants to the cell surface. Our data provide new insights into how complex interplay between the repertoire of post-transcriptional and post-translational mechanisms controls cell surface expression of BK channels. PMID:23504458

Epigenetics of proteasome inhibition in the liver of rats fed ethanol chronically

PubMed Central

Oliva, Joan; Dedes, Jennifer; Li, Jun; French, Samuel W; Bardag-Gorce, Fawzia

2009-01-01

AIM: To examine the effects of ethanol-induced proteasome inhibition, and the effects of proteasome inhibition in the regulation of epigenetic mechanisms. METHODS: Rats were fed ethanol for 1 mo using the Tsukamoto-French model and were compared to rats given the proteasome inhibitor PS-341 (Bortezomib, Velcade™) by intraperitoneal injection. Microarray analysis and real time PCR were performed and proteasome activity assays and Western blot analysis were performed using isolated nuclei. RESULTS: Chronic ethanol feeding caused a significant inhibition of the ubiquitin proteasome pathway in the nucleus, which led to changes in the turnover of transcriptional factors, histone-modifying enzymes, and, therefore, affected epigenetic mechanisms. Chronic ethanol feeding was related to an increase in histone acetylation, and it is hypothesized that the proteasome proteolytic activity regulated histone modifications by controlling the stability of histone modifying enzymes, and, therefore, regulated the chromatin structure, allowing easy access to chromatin by RNA polymerase, and, thus, proper gene expression. Proteasome inhibition by PS-341 increased histone acetylation similar to chronic ethanol feeding. In addition, proteasome inhibition caused dramatic changes in hepatic remethylation reactions as there was a significant decrease in the enzymes responsible for the regeneration of S-adenosylmethionine, and, in particular, a significant decrease in the betaine-homocysteine methyltransferase enzyme. This suggested that hypomethylation was associated with proteasome inhibition, as indicated by the decrease in histone methylation. CONCLUSION: The role of proteasome inhibition in regulating epigenetic mechanisms, and its link to liver injury in alcoholic liver disease, is thus a promising approach to study liver injury due to chronic ethanol consumption. PMID:19222094

Evidence for the Existence in Arabidopsis thaliana of the Proteasome Proteolytic Pathway

PubMed Central

Polge, Cécile; Jaquinod, Michel; Holzer, Frances; Bourguignon, Jacques; Walling, Linda; Brouquisse, Renaud

2009-01-01

Heavy metals are known to generate reactive oxygen species that lead to the oxidation and fragmentation of proteins, which become toxic when accumulated in the cell. In this study, we investigated the role of the proteasome during cadmium stress in the leaves of Arabidopsis thaliana plants. Using biochemical and proteomics approaches, we present the first evidence of an active proteasome pathway in plants. We identified and characterized the peptidases acting sequentially downstream from the proteasome in animal cells as follows: tripeptidyl-peptidase II, thimet oligopeptidase, and leucine aminopeptidase. We investigated the proteasome proteolytic pathway response in the leaves of 6-week-old A. thaliana plants grown hydroponically for 24, 48, and 144 h in the presence or absence of 50 μm cadmium. The gene expression and proteolytic activity of the proteasome and the different proteases of the pathway were found to be up-regulated in response to cadmium. In an in vitro assay, oxidized bovine serum albumin and lysozyme were more readily degraded in the presence of 20 S proteasome and tripeptidyl-peptidase II than their nonoxidized form, suggesting that oxidized proteins are preferentially degraded by the Arabidopsis 20 S proteasome pathway. These results show that, in response to cadmium, the 20 S proteasome proteolytic pathway is up-regulated at both RNA and activity levels in Arabidopsis leaves and may play a role in degrading oxidized proteins generated by the stress. PMID:19822524

Shotgun Proteomics of Aspergillus niger Microsomes upon d-Xylose Induction▿ †

PubMed Central

de Oliveira, José Miguel P. Ferreira; van Passel, Mark W. J.; Schaap, Peter J.; de Graaff, Leo H.

2010-01-01

Protein secretion plays an eminent role in cell maintenance and adaptation to the extracellular environment of microorganisms. Although protein secretion is an extremely efficient process in filamentous fungi, the mechanisms underlying protein secretion have remained largely uncharacterized in these organisms. In this study, we analyzed the effects of the d-xylose induction of cellulase and hemicellulase enzyme secretion on the protein composition of secretory organelles in Aspergillus niger. We aimed to systematically identify the components involved in the secretion of these enzymes via mass spectrometry of enriched subcellular microsomal fractions. Under each condition, fractions enriched for secretory organelles were processed for tandem mass spectrometry, resulting in the identification of peptides that originate from 1,081 proteins, 254 of which—many of them hypothetical proteins—were predicted to play direct roles in the secretory pathway. d-Xylose induction led to an increase in specific small GTPases known to be associated with polarized growth, exocytosis, and endocytosis. Moreover, the endoplasmic-reticulum-associated degradation (ERAD) components Cdc48 and all 14 of the 20S proteasomal subunits were recruited to the secretory organelles. In conclusion, induction of extracellular enzymes results in specific changes in the secretory subproteome of A. niger, and the most prominent change found in this study was the recruitment of the 20S proteasomal subunits to the secretory organelles. PMID:20453123

The proteasomal Rpn11 metalloprotease suppresses tombusvirus RNA recombination and promotes viral replication via facilitating assembly of the viral replicase complex.

PubMed

Prasanth, K Reddisiva; Barajas, Daniel; Nagy, Peter D

2015-03-01

RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92(pol) replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in tombusvirus recombination in a yeast model host, in plants, and in vitro. We found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic recombination. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The Role of Proteasome Inhibitor MG132 in 2,4-Dinitrofluorobenzene-Induced Atopic Dermatitis in NC/Nga Mice.

PubMed

Ohkusu-Tsukada, Kozo; Ito, Daiki; Takahashi, Kimimasa

2018-01-01

Although immunosuppressants for therapy of atopic dermatitis (AD) are still being sought, proteasome inhibitors are also potential candidates for the treatment of AD. Proteasome inhibitors exert various effects by blocking proteasomal degradation and help regulate processes such as apoptosis induction via caspase-9, cell cycle progression via cyclins, NF-κB inactivation via IκB, and downregulation of antigen cross-presentation. The cells targeted by proteasome inhibitors are therefore activated cells undergoing proliferation or differentiation, and antigen-presenting cells carrying out protein degradation. This study investigated the therapeutic effects and side effects of a proteasome inhibitor, MG132, on the treatment of AD. AD-like disease in NC/Nga mice housed under specific pathogen-free conditions was induced by repeated application of 2,4-dinitrofluorobenzene (DNFB). Disease progression was evaluated by inflammation score, histopathology, and serum IgE level, and the effects of systemic MG132 administration were investigated. The percentages and absolute numbers for each population of Th1, Th2, and Th17 cells in the axillary lymph nodes were analyzed by flow cytometry. DNFB application increased the expression of a unique major histocompatibility complex class I mutant molecule D/Ldm7 in dendritic cells (DCs), and increased Th1 and Th17 cells in NC/Nga mice. In vivo MG132 administration to NC/Nga mice with DNFB-induced dermatitis reduced Th17 cells but maintained the level of Th1 cells, resulting in the alleviation of dermatitis lesions by decreasing both serum IgE hyperproduction and mast cell migration. To understand the mechanisms maintaining Th1 cell levels following in vivo MG132-administration, we focused on the role of proteasomes regulating D/Ldm7 expression. Interestingly, 20S proteasome activity was higher in NC/Nga DCs than in BALB/c DCs. In vitro MG132 administration partially increased D/Ldm7 expression in a dose-dependent manner during DC maturation, and induced IFN-γ production from autoreactive CD8+ T cells but not from CD4+ T cells following coculturing with D/Ldm7-upregulated DCs. Although MG132 administration temporarily alleviated AD pathogenesis in NC/Nga mice, prolonged MG132 treatment may result in immunopathogenesis leading to chronic AD due to its side effect of maintaining Th1 levels via autoreactive CD8+ T cells. © 2018 S. Karger AG, Basel.

PSD-95 stabilizes NMDA receptors by inducing the degradation of STEP61.

PubMed

Won, Sehoon; Incontro, Salvatore; Nicoll, Roger A; Roche, Katherine W

2016-08-09

Phosphorylation regulates surface and synaptic expression of NMDA receptors (NMDARs). Both the tyrosine kinase Fyn and the tyrosine phosphatase striatal-enriched protein tyrosine phosphatase (STEP) are known to target the NMDA receptor subunit GluN2B on tyrosine 1472, which is a critical residue that mediates NMDAR endocytosis. STEP reduces the surface expression of NMDARs by promoting dephosphorylation of GluN2B Y1472, whereas the synaptic scaffolding protein postsynaptic density protein 95 (PSD-95) stabilizes the surface expression of NMDARs. However, nothing is known about a potential functional interaction between STEP and PSD-95. We now report that STEP61 binds to PSD-95 but not to other PSD-95 family members. We find that PSD-95 expression destabilizes STEP61 via ubiquitination and degradation by the proteasome. Using subcellular fractionation, we detect low amounts of STEP61 in the PSD fraction. However, STEP61 expression in the PSD is increased upon knockdown of PSD-95 or in vivo as detected in PSD-95-KO mice, demonstrating that PSD-95 excludes STEP61 from the PSD. Importantly, only extrasynaptic NMDAR expression and currents were increased upon STEP knockdown, as is consistent with low STEP61 localization in the PSD. Our findings support a dual role for PSD-95 in stabilizing synaptic NMDARs by binding directly to GluN2B but also by promoting synaptic exclusion and degradation of the negative regulator STEP61.

PSD-95 stabilizes NMDA receptors by inducing the degradation of STEP61

PubMed Central

Won, Sehoon; Incontro, Salvatore; Nicoll, Roger A.; Roche, Katherine W.

2016-01-01

Phosphorylation regulates surface and synaptic expression of NMDA receptors (NMDARs). Both the tyrosine kinase Fyn and the tyrosine phosphatase striatal-enriched protein tyrosine phosphatase (STEP) are known to target the NMDA receptor subunit GluN2B on tyrosine 1472, which is a critical residue that mediates NMDAR endocytosis. STEP reduces the surface expression of NMDARs by promoting dephosphorylation of GluN2B Y1472, whereas the synaptic scaffolding protein postsynaptic density protein 95 (PSD-95) stabilizes the surface expression of NMDARs. However, nothing is known about a potential functional interaction between STEP and PSD-95. We now report that STEP61 binds to PSD-95 but not to other PSD-95 family members. We find that PSD-95 expression destabilizes STEP61 via ubiquitination and degradation by the proteasome. Using subcellular fractionation, we detect low amounts of STEP61 in the PSD fraction. However, STEP61 expression in the PSD is increased upon knockdown of PSD-95 or in vivo as detected in PSD-95–KO mice, demonstrating that PSD-95 excludes STEP61 from the PSD. Importantly, only extrasynaptic NMDAR expression and currents were increased upon STEP knockdown, as is consistent with low STEP61 localization in the PSD. Our findings support a dual role for PSD-95 in stabilizing synaptic NMDARs by binding directly to GluN2B but also by promoting synaptic exclusion and degradation of the negative regulator STEP61. PMID:27457929

Proteolytic turnover of the Gal4 transcription factor is not required for function in vivo.

PubMed

Nalley, Kip; Johnston, Stephen Albert; Kodadek, Thomas

2006-08-31

Transactivator-promoter complexes are essential intermediates in the activation of eukaryotic gene expression. Recent studies of these complexes have shown that some are quite dynamic in living cells owing to rapid and reversible disruption of activator-promoter complexes by molecular chaperones, or a slower, ubiquitin-proteasome-pathway-mediated turnover of DNA-bound activator. These mechanisms may act to ensure continued responsiveness of activators to signalling cascades by limiting the lifetime of the active protein-DNA complex. Furthermore, the potency of some activators is compromised by proteasome inhibition, leading to the suggestion that periodic clearance of activators from a promoter is essential for high-level expression. Here we describe a variant of the chromatin immunoprecipitation assay that has allowed direct observation of the kinetic stability of native Gal4-promoter complexes in yeast. Under non-inducing conditions, the complex is dynamic, but on induction the Gal4-promoter complexes 'lock in' and exhibit long half-lives. Inhibition of proteasome-mediated proteolysis had little or no effect on Gal4-mediated gene expression. These studies, combined with earlier data, show that the lifetimes of different transactivator-promoter complexes in vivo can vary widely and that proteasome-mediated turnover is not a general requirement for transactivator function.

Proinflammatory cytokines cause FAT10 upregulation in cancers of liver and colon.

PubMed

Lukasiak, S; Schiller, C; Oehlschlaeger, P; Schmidtke, G; Krause, P; Legler, D F; Autschbach, F; Schirmacher, P; Breuhahn, K; Groettrup, M

2008-10-09

The mRNA of the ubiquitin-like modifier FAT10 has been reported to be overexpressed in 90% of hepatocellular carcinoma (HCC) and in over 80% of colon, ovary and uterus carcinomas. Elevated FAT10 expression in malignancies was attributed to transcriptional upregulation upon the loss of p53. Moreover, FAT10 induced chromosome instability in long-term in vitro culture, which led to the hypothesis that FAT10 might be involved in carcinogenesis. In this study we show that interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha synergistically upregulated FAT10 expression in liver and colon cancer cells 10- to 100-fold. Real-time RT-PCR revealed that FAT10 mRNA was significantly overexpressed in 37 of 51 (72%) of human HCC samples and in 8 of 15 (53%) of human colon carcinomas. The FAT10 cDNA sequences in HCC samples were not mutated and intact FAT10 protein was detectable. FAT10 expression in both cancer tissues correlated with expression of the IFN-gamma- and TNF-alpha-dependent proteasome subunit LMP2 strongly suggesting that proinflammatory cytokines caused the joint overexpression of FAT10 and LMP2. NIH3T3 transformation assays revealed that FAT10 had no transforming capability. Taken together, FAT10 qualifies as a marker for an interferon response in HCC and colon carcinoma but is not significantly overexpressed in cancers lacking a proinflammatory environment.

The multicatalytic proteinase complex (proteasome): structure and conformational changes associated with changes in proteolytic activity.

PubMed Central

Djaballah, H; Rowe, A J; Harding, S E; Rivett, A J

1993-01-01

The multicatalytic proteinase complex or proteasome is a high-molecular-mass multisubunit proteinase which is found in the nucleus and cytoplasm of eukaryotic cells. Electron microscopy of negatively stained rat liver proteinase preparations suggests that the particle has a hollow cylindrical shape (approximate width 11 nm and height 17 nm using methylamine tungstate as the negative stain) with a pseudo-helical arrangement of subunits rather than the directly stacked arrangement suggested previously. The side-on view has a 2-fold rotational symmetry, while end-on there appears to be six or seven subunits around the ring. This model is very different from that proposed by others for the proteinase from rat liver but resembles the structure of the simpler archaebacterial proteasome. The possibility of conformational changes associated with the addition of effectors of proteolytic activity has been investigated by sedimentation velocity analysis and dynamic light-scattering measurements. The results provide the first direct evidence for conformational changes associated with the observed positive co-operativity in one component of the peptidylglutamylpeptide hydrolase activity as well as with the stimulation of peptidylglutamylpeptide hydrolase activities by MnCl2. In the latter case, there appears to be a correlation between changes in the shape of the molecule and the effect on activity. KCl and low concentrations of SDS may also act by inducing conformational changes within the complex. Sedimentation-velocity measurements also provide evidence for the formation of intermediates during dissociation of the complex by urea, guanidinium chloride or sodium thiocyanate. Dissociation of the complex either by these agents or by treatment at low pH leads to inactivation of its proteolytic components. The results suggest that activation and inhibition of the various proteolytic activities may be mediated by measurable changes in size and shape of the molecules. Images Figure 1 Figure 2 PMID:8318014

Accumulation of polyubiquitylated proteins in response to Ala-Ala-Phe-chloromethylketone is independent of the inhibition of Tripeptidyl peptidase II.

PubMed

Villasevil, Eugenia M; Guil, Sara; López-Ferreras, Lorena; Sánchez, Carlos; Del Val, Margarita; Antón, Luis C

2010-09-01

In the present study we have addressed the issue of proteasome independent cytosolic protein degradation. Tripeptidyl peptidase II (TPPII) has been suggested to compensate for a reduced proteasome activity, partly based on evidence using the inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk). Here we show that AAF-cmk induces the formation of polyubiquitin-containing accumulations in osteosarcoma and Burkitt's lymphoma cell lines. These accumulations meet many of the landmarks of the aggresomes that form after proteasome inhibition. Using a combination of experiments with chemical inhibitors and interference of gene expression, we show that TPPII inhibition is not responsible for these accumulations. Our evidence suggests that the relevant target(s) is/are in the ubiquitin-proteasome pathway, most likely upstream the proteasome. We obtained evidence supporting this model by inhibition of Hsp90, which also acts upstream the proteasome. Although our data suggest that Hsp90 is not a target of AAF-cmk, its inhibition resulted in accumulations similar to those obtained with AAF-cmk. Therefore, our results question the proposed role for TPPII as a prominent alternative to the proteasome in cellular proteolysis. Copyright 2010 Elsevier B.V. All rights reserved.

The combination of ionizing radiation and proteasomal inhibition by bortezomib enhances the expression of NKG2D ligands in multiple myeloma cells.

PubMed

Lee, Young Shin; Heo, Woong; Nam, Jiho; Jeung, Young Hwa; Bae, Jaeho

2018-05-01

Bortezomib, which is a potent proteasome inhibitor, has been used as a first-line drugs to treat multiple myeloma for a few decades, and radiotherapy has frequently been applied to manage acute bone lesions in the patients. Therefore, it was necessary to investigate what the benefits might be if the two therapies were applied simultaneously in the treatment of multiple myeloma. Since it was known that radiotherapy and proteasome inhibitors could increase the expression of NKG2D ligands through induction of protein synthesis and suppression of protein degradation of NKG2D ligands, respectively, we supposed that the combined treatment might further enhance the expression of NKG2D ligands. In this study, we analyzed the expression level of NKG2D ligands using multiplex PCR and flow cytometry after treatment of IM-9 and RPMI-8226 myeloma cells with bortezomib and ionizing radiation; we then assayed the susceptibility to NK-92 cells. Although the expression of only some kinds of NKG2D ligands were increased by treatment with bortezomib alone, five kinds of NKG2D ligands that we assayed were further induced at the surface protein level after combined treatment with ionizing radiation and bortezomib. Furthermore, combined treatment made myeloma cells more susceptible to NK-92 cells, compared with treatment with bortezomib alone. In conclusion, the combination therapy of ionizing radiation plus the proteasome inhibitor bortezomib is a promising therapeutical strategy for enhancing NK cell-mediated anticancer immune responses.

Dynamic Interactions between Bombyx mori Nucleopolyhedrovirus and Its Host Cells Revealed by Transcriptome Analysis

PubMed Central

Xue, Jian; Qiao, Nan; Zhang, Wei; Cheng, Ruo-Lin; Zhang, Xiao-Qin; Bao, Yan-Yuan; Xu, Yi-Peng; Gu, Lin-Zhu

2012-01-01

Although microarray and expressed sequence tag (EST)-based approaches have been used to profile gene expression during baculovirus infection, the response of host genes to baculovirus infection and the interaction between baculovirus and its host remain largely unknown. To determine the host response to Bombyx mori nucleopolyhedrovirus infection and the dynamic interaction between the virus and its host, eight digital gene expression libraries were examined in a Bm5 cell line before infection and at 1.5, 3, 6, 12, 24, 48, and 96 h postinfection. Gene set enrichment analysis of differentially expressed genes at each time point following infection showed that gene sets including cytoskeleton, transcription, translation, energy metabolism, iron ion metabolism, and the ubiquitin-proteasome pathway were altered after viral infection. In addition, a time course depicting protein-protein interaction networks between the baculovirus and the host were constructed and revealed that viral proteins interact with a multitude of cellular machineries, such as the proteasome, cytoskeleton, and spliceosome. Several viral proteins, including IE2, CG30, PE38, and PK-1/2, were predicted to play key roles in mediating virus-host interactions. Based on these results, we tested the role of the ubiquitin-proteasome pathway and iron ion metabolism in the viral infection cycle. Treatment with a proteasome inhibitor and deferoxamine mesylate in vitro and in vivo confirmed that these pathways regulate viral infection. Taken together, these findings provide new insights into the interaction between the baculovirus and its host and identify molecular mechanisms that can be used to block viral infection and improve baculovirus expression systems. PMID:22532689

Newborn mouse lens proteome and its alteration by lysine 6 mutant ubiquitin

USDA-ARS?s Scientific Manuscript database

Ubiquitin is a tag that often initiates degradation of proteins by the proteasome in the ubiquitin proteasome system. Targeted expression of K6W mutant ubiquitin (K6W-Ub) in the lens results in defects in lens development and cataract formation, suggesting critical functions for ubiquitin in lens. T...

Berberine Suppresses Cyclin D1 Expression through Proteasomal Degradation in Human Hepatoma Cells.

PubMed

Wang, Ning; Wang, Xuanbin; Tan, Hor-Yue; Li, Sha; Tsang, Chi Man; Tsao, Sai-Wah; Feng, Yibin

2016-11-15

The aim of this study is to explore the underlying mechanism on berberine-induced Cyclin D1 degradation in human hepatic carcinoma. We observed that berberine could suppress both in vitro and in vivo expression of Cyclin D1 in hepatoma cells. Berberine exhibits dose- and time-dependent inhibition on Cyclin D1 expression in human hepatoma cell HepG2. Berberine increases the phosphorylation of Cyclin D1 at Thr286 site and potentiates Cyclin D1 nuclear export to cytoplasm for proteasomal degradation. In addition, berberine recruits the Skp, Cullin, F-box containing complex-β-Transducin Repeat Containing Protein (SCF β-TrCP ) complex to facilitate Cyclin D1 ubiquitin-proteasome dependent proteolysis. Knockdown of β-TrCP blocks Cyclin D1 turnover induced by berberine; blocking the protein degradation induced by berberine in HepG2 cells increases tumor cell resistance to berberine. Our results shed light on berberine's potential as an anti-tumor agent for clinical cancer therapy.

Berberine Suppresses Cyclin D1 Expression through Proteasomal Degradation in Human Hepatoma Cells

PubMed Central

Wang, Ning; Wang, Xuanbin; Tan, Hor-Yue; Li, Sha; Tsang, Chi Man; Tsao, Sai-Wah; Feng, Yibin

2016-01-01

The aim of this study is to explore the underlying mechanism on berberine-induced Cyclin D1 degradation in human hepatic carcinoma. We observed that berberine could suppress both in vitro and in vivo expression of Cyclin D1 in hepatoma cells. Berberine exhibits dose- and time-dependent inhibition on Cyclin D1 expression in human hepatoma cell HepG2. Berberine increases the phosphorylation of Cyclin D1 at Thr286 site and potentiates Cyclin D1 nuclear export to cytoplasm for proteasomal degradation. In addition, berberine recruits the Skp, Cullin, F-box containing complex-β-Transducin Repeat Containing Protein (SCFβ-TrCP) complex to facilitate Cyclin D1 ubiquitin-proteasome dependent proteolysis. Knockdown of β-TrCP blocks Cyclin D1 turnover induced by berberine; blocking the protein degradation induced by berberine in HepG2 cells increases tumor cell resistance to berberine. Our results shed light on berberine′s potential as an anti-tumor agent for clinical cancer therapy. PMID:27854312

Targeted Delivery of Proteasome Inhibitors to Somatostatin-Receptor-Expressing Cancer Cells by Octreotide Conjugation.

PubMed

Beck, Philipp; Cui, Haissi; Hegemann, Julian D; Marahiel, Mohammed A; Krüger, Achim; Groll, Michael

2015-12-01

Clinical application of proteasome inhibitors (PIs) is so far limited to peripheral blood cancers due to the pronounced cytotoxicity towards all cell types. Targeted delivery of PIs could permit the treatment of other cancers along with decreasing side effects. Herein we describe the first small-molecule proteasome inhibitor conjugate for targeted delivery, created by fusing PIs to a synthetic ligand of somatostatin receptors, which are highly expressed in a variety of tumors. X-ray crystallographic studies and in vitro IC50 measurements demonstrated that addition of the cyclopeptide octreotide as a targeting vehicle does not affect the PI's binding mode. The cytotoxicity of the conjugate against somatostatin-receptor-expressing cells was up to 11-fold higher than that of a non-targeting surrogate. We have therefore established PIs as a new payload for drug conjugates and have shown that targeted delivery thereof could be a promising approach for the broader application of this FDA-approved class of compounds. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inhibition of autophagy induced by proteasome inhibition increases cell death in human SHG-44 glioma cells.

PubMed

Ge, Peng-Fei; Zhang, Ji-Zhou; Wang, Xiao-Fei; Meng, Fan-Kai; Li, Wen-Chen; Luan, Yong-Xin; Ling, Feng; Luo, Yi-Nan

2009-07-01

The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Recent studies suggest that proteasome inhibitors may reduce tumor growth and activate autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the effect of autophagy on the destiny of glioma cells remains unclear. In this study, we sought to investigate whether inhibition of the proteasome can induce autophagy and the effects of autophagy on the fate of human SHG-44 glioma cells. The proteasome inhibitor MG-132 was used to induce autophagy in SHG-44 glioma cells, and the effect of autophagy on the survival of SHG-44 glioma cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by MTT assay. Apoptosis and cell cycle were detected by flow cytometry. The expression of autophagy related proteins was determined by Western blot. MG-132 inhibited cell proliferation, induced cell death and cell cycle arrest at G(2)/M phase, and activated autophagy in SHG-44 glioma cells. The expression of autophagy-related Beclin-1 and LC3-I was significantly up-regulated and part of LC3-I was converted into LC3-II. However, when SHG-44 glioma cells were co-treated with MG-132 and 3-MA, the cells became less viable, but cell death and cell numbers at G(2)/M phase increased. Moreover, the accumulation of acidic vesicular organelles was decreased, the expression of Beclin-1 and LC3 was significantly down-regulated and the conversion of LC3-II from LC3-I was also inhibited. Inhibition of the proteasome can induce autophagy in human SHG-44 glioma cells, and inhibition of autophagy increases cell death. This discovery may shed new light on the effect of autophagy on modulating the fate of SHG-44 glioma cells.Acta Pharmacologica Sinica (2009) 30: 1046-1052; doi: 10.1038/aps.2009.71.

Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility

PubMed Central

Young, Barry P.; Loewen, Christopher J.; Mayor, Thibault

2016-01-01

Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations. PMID:27448207

Association of Rpn10 with high molecular weight complex is enhanced during retinoic acid-induced differentiation of neuroblastoma cells.

PubMed

Tayama, Yoko; Kawahara, Hiroyuki; Minami, Ryosuke; Shimada, Masumi; Yokosawa, Hideyoshi

2007-12-01

The ubiquitin-binding Rpn10 protein serves as an ubiquitin receptor that delivers client proteins to the 26S proteasome, the protein degradation complex. It has been suggested that the ubiquitin-dependent protein degradation is critical for neuronal differentiation and for preventing neurodegenerative diseases. Our previous study indicated the importance of Rpn10 in control of cellular differentiation (Shimada et al., Mol Biol Cell 17:5356-5371, 2006), though the functional relevance of Rpn10 in neuronal cell differentiation remains a mystery to be uncovered. In the present study, we have examined the level of Rpn10 in a proteasome-containing high molecular weight (HMW) protein fraction prepared from the mouse neuroblastoma cell line Neuro2a. We here report that the protein level of Rpn10 in HMW fraction from un-differentiated Neuro2a cells was significantly lower than that of other cultured cell lines. We have found that retinoic acid-induced neural differentiation of Neuro2a cells significantly stimulates the incorporation of Rpn10 into HMW fractions, although the amounts of 26S proteasome subunits were not changed. Our findings provide the first evidence that the modulation of Rpn10 is linked to the control of retinoic acid-induced differentiation of neuroblastoma cells.

The Evolution of COP9 Signalosome in Unicellular and Multicellular Organisms.

PubMed

Barth, Emanuel; Hübler, Ron; Baniahmad, Aria; Marz, Manja

2016-05-02

The COP9 signalosome (CSN) is a highly conserved protein complex, recently being crystallized for human. In mammals and plants the COP9 complex consists of nine subunits, CSN 1-8 and CSNAP. The CSN regulates the activity of culling ring E3 ubiquitin and plays central roles in pleiotropy, cell cycle, and defense of pathogens. Despite the interesting and essential functions, a thorough analysis of the CSN subunits in evolutionary comparative perspective is missing. Here we compared 61 eukaryotic genomes including plants, animals, and yeasts genomes and show that the most conserved subunits of eukaryotes among the nine subunits are CSN2 and CSN5. This may indicate a strong evolutionary selection for these two subunits. Despite the strong conservation of the protein sequence, the genomic structures of the intron/exon boundaries indicate no conservation at genomic level. This suggests that the gene structure is exposed to a much less selection compared with the protein sequence. We also show the conservation of important active domains, such as PCI (proteasome lid-CSN-initiation factor) and MPN (MPR1/PAD1 amino-terminal). We identified novel exons and alternative splicing variants for all CSN subunits. This indicates another level of complexity of the CSN. Notably, most COP9-subunits were identified in all multicellular and unicellular eukaryotic organisms analyzed, but not in prokaryotes or archaeas. Thus, genes encoding CSN subunits present in all analyzed eukaryotes indicate the invention of the signalosome at the root of eukaryotes. The identification of alternative splice variants indicates possible "mini-complexes" or COP9 complexes with independent subunits containing potentially novel and not yet identified functions. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

The Evolution of COP9 Signalosome in Unicellular and Multicellular Organisms

PubMed Central

Barth, Emanuel; Hübler, Ron; Baniahmad, Aria; Marz, Manja

2016-01-01

The COP9 signalosome (CSN) is a highly conserved protein complex, recently being crystallized for human. In mammals and plants the COP9 complex consists of nine subunits, CSN 1–8 and CSNAP. The CSN regulates the activity of culling ring E3 ubiquitin and plays central roles in pleiotropy, cell cycle, and defense of pathogens. Despite the interesting and essential functions, a thorough analysis of the CSN subunits in evolutionary comparative perspective is missing. Here we compared 61 eukaryotic genomes including plants, animals, and yeasts genomes and show that the most conserved subunits of eukaryotes among the nine subunits are CSN2 and CSN5. This may indicate a strong evolutionary selection for these two subunits. Despite the strong conservation of the protein sequence, the genomic structures of the intron/exon boundaries indicate no conservation at genomic level. This suggests that the gene structure is exposed to a much less selection compared with the protein sequence. We also show the conservation of important active domains, such as PCI (proteasome lid-CSN-initiation factor) and MPN (MPR1/PAD1 amino-terminal). We identified novel exons and alternative splicing variants for all CSN subunits. This indicates another level of complexity of the CSN. Notably, most COP9-subunits were identified in all multicellular and unicellular eukaryotic organisms analyzed, but not in prokaryotes or archaeas. Thus, genes encoding CSN subunits present in all analyzed eukaryotes indicate the invention of the signalosome at the root of eukaryotes. The identification of alternative splice variants indicates possible “mini-complexes” or COP9 complexes with independent subunits containing potentially novel and not yet identified functions. PMID:27044515

REGγ regulates ERα degradation via ubiquitin–proteasome pathway in breast cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chai, Fan; Liang, Yan; Bi, Jiong

2015-01-02

Highlights: • High expression of REGγ is correlated with ERα status and poor clinical features. • Cell growth, mobility and invasion are significantly impaired by REGγ knockdown. • REGγ indirectly regulates ERα protein expression. - Abstract: REGγ is a proteasome coactivator which regulates proteolytic activity in eukaryotic cells. Abundant lines of evidence have showed that REGγ is over expressed in a number of human carcinomas. However, its precise role in the pathogenesis of cancer is still unclear. In this study, by examining 200 human breast cancer specimens, we demonstrated that REGγ was highly expressed in breast cancers, and the expressionmore » of REGγ was positively correlated with breast cancer patient estrogen receptor alpha (ERα) status. Moreover, the expression of REGγ was found positively associated with poor clinical features and low survival rates in ERα positive breast cancer patients. Further cell culture studies using MCF7 and BT474 breast cancer cell lines showed that cell proliferation, motility, and invasion capacities were decreased significantly by REGγ knockdown. Lastly, we demonstrated that REGγ indirectly regulates the degradation of ERα protein via ubiquitin–proteasome pathway. In conclusion, our findings provide the evidence that REGγ expression was positively correlated with ERα status and poor clinical prognosis in ERα positive breast cancer patients. As well, we disclose a new connection between the two molecules that are both highly expressed in most breast cancer cases.« less

Stress-induced NQO1 controls stability of C/EBPα against 20S proteasomal degradation to regulate p63 expression with implications in protection against chemical-induced skin cancer.

PubMed

Patrick, B A; Jaiswal, A K

2012-10-04

Previously, we have shown a role of cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) in the stabilization of p63 against 20S proteasomal degradation resulting in thinning of the epithelium and chemical-induced skin cancer (Oncogene (2011) 30, 1098-1107). Current studies have demonstrated that NQO1 control of CCAAT-enhancer binding protein (C/EBPα) against 20S proteasomal degradation also contributes to the upregulation of p63 expression and protection. Western and immunohistochemistry analysis revealed that disruption of the NQO1 gene in mice and mouse keratinocytes led to degradation of C/EBPα and loss of p63 gene expression. p63 promoter mutagenesis, transfection and chromatin immunoprecipitation assays identified a C/EBPα-binding site between nucleotide position -185 and -174 that bound to C/EBPα and upregulated p63 gene expression. Co-immunoprecipitation and immunoblot analysis demonstrated that 20S proteasomes directly interacted and degraded C/EBPα. NQO1 direct interaction with C/EBPα led to stabilization of C/EBPα against 20S proteasomal degradation. NQO1 protection of C/EBPα required binding of NADH with NQO1. Exposure of skin and keratinocytes to the chemical stress agent benzo(a)pyrene led to induction of NQO1 and stabilization of C/EBPα protein, resulting in an increase in p63 RNA and protein in wild-type but not in NQO1-/- mice. Collectively, the current data combined with previous data suggest that stress induction of NQO1 through both stabilization of C/EBPα and increase in p63 and direct stabilization of p63 controls keratinocyte differentiation, leading to protection against chemical-induced skin carcinogenesis. The studies are significant as 2-4% human individuals are homozygous and 23% are heterozygous for the NQO1P187S mutation and might be susceptible to stress-induced skin diseases.

Efficient Identification of Novel Hla-A*0201–Presented Cytotoxic T Lymphocyte Epitopes in the Widely Expressed Tumor Antigen Prame by Proteasome-Mediated Digestion Analysis

PubMed Central

Kessler, Jan H.; Beekman, Nico J.; Bres-Vloemans, Sandra A.; Verdijk, Pauline; van Veelen, Peter A.; Kloosterman-Joosten, Antoinette M.; Vissers, Debby C.J.; ten Bosch, George J.A.; Kester, Michel G.D.; Sijts, Alice; Drijfhout, Jan Wouter; Ossendorp, Ferry; Offringa, Rienk; Melief, Cornelis J.M.

2001-01-01

We report the efficient identification of four human histocompatibility leukocyte antigen (HLA)-A*0201–presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an improved “reverse immunology” strategy. Next to motif-based HLA-A*0201 binding prediction and actual binding and stability assays, analysis of in vitro proteasome-mediated digestions of polypeptides encompassing candidate epitopes was incorporated in the epitope prediction procedure. Proteasome cleavage pattern analysis, in particular determination of correct COOH-terminal cleavage of the putative epitope, allows a far more accurate and selective prediction of CTL epitopes. Only 4 of 19 high affinity HLA-A*0201 binding peptides (21%) were found to be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides that are not processed and limits the number of peptides to be assayed for binding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA100–108; SLYSFPEPEA, PRA142–151; ALYVDSLFFL, PRA300–309; and SLLQHLIGL, PRA425–433) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A*0201. This indicates that these epitopes are expressed on cancer cells of diverse histologic origin, making them attractive targets for immunotherapy of cancer. PMID:11136822

Eupatolide inhibits lipopolysaccharide-induced COX-2 and iNOS expression in RAW264.7 cells by inducing proteasomal degradation of TRAF6.

PubMed

Lee, Jongkyu; Tae, Nara; Lee, Jung Joon; Kim, Taeho; Lee, Jeong-Hyung

2010-06-25

Inula britannica is a traditional medicinal plant used to treat bronchitis, digestive disorders, and inflammation in Eastern Asia. Here, we identified eupatolide, a sesquiterpene lactone from I. britannica, as an inhibitor of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression. Eupatolide inhibited the production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) as well as iNOS and COX-2 protein expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Eupatolide dose-dependently decreased the mRNA levels and the promoter activities of COX-2 and iNOS in LPS-stimulated RAW264.7 cells. Moreover, eupatolide significantly suppressed the LPS-induced expression of nuclear factor-kappa B (NF-kappaB) and activator protein-1 (AP-1) reporter genes. Pretreatment of eupatolide inhibited LPS-induced phosphorylation and degradation of I kappaB alpha, and phosphorylation of RelA/p65 on Ser-536 as well as the activation of mitogen-activated protein kinases (MAPKs) and Akt in LPS-stimulated RAW264.7 cells. Eupatolide induced proteasomal degradation of tumor necrosis factor receptor-associated factor-6 (TRAF6), and subsequently inhibited LPS-induced TRAF6 polyubiquitination. These results suggest that eupatolide blocks LPS-induced COX-2 and iNOS expression at the transcriptional level through inhibiting the signaling pathways such as NF-kappaB and MAPKs via proteasomal degradation of TRAF6. Taken together, eupatolide may be a novel anti-inflammatory agent that induces proteasomal degradation of TRAF6, and a valuable compound for modulating inflammatory conditions. (c) 2010 Elsevier B.V. All rights reserved.

Phosphorylation Regulates the Ubiquitin-independent Degradation of Yeast Pah1 Phosphatidate Phosphatase by the 20S Proteasome*

PubMed Central

Hsieh, Lu-Sheng; Su, Wen-Min; Han, Gil-Soo; Carman, George M.

2015-01-01

Saccharomyces cerevisiae Pah1 phosphatidate phosphatase, which catalyzes the conversion of phosphatidate to diacylglycerol for triacylglycerol synthesis and simultaneously controls phosphatidate levels for phospholipid synthesis, is subject to the proteasome-mediated degradation in the stationary phase of growth. In this study, we examined the mechanism for its degradation using purified Pah1 and isolated proteasomes. Pah1 expressed in S. cerevisiae or Escherichia coli was not degraded by the 26S proteasome, but by its catalytic 20S core particle, indicating that its degradation is ubiquitin-independent. The degradation of Pah1 by the 20S proteasome was dependent on time and proteasome concentration at the pH optimum of 7.0. The 20S proteasomal degradation was conserved for human lipin 1 phosphatidate phosphatase. The degradation analysis using Pah1 truncations and its fusion with GFP indicated that proteolysis initiates at the N- and C-terminal unfolded regions. The folded region of Pah1, in particular the haloacid dehalogenase-like domain containing the DIDGT catalytic sequence, was resistant to the proteasomal degradation. The structural change of Pah1, as reflected by electrophoretic mobility shift, occurs through its phosphorylation by Pho85-Pho80, and the phosphorylation sites are located within its N- and C-terminal unfolded regions. Phosphorylation of Pah1 by Pho85-Pho80 inhibited its degradation, extending its half-life by ∼2-fold. The dephosphorylation of endogenously phosphorylated Pah1 by the Nem1-Spo7 protein phosphatase, which is highly specific for the sites phosphorylated by Pho85-Pho80, stimulated the 20S proteasomal degradation and reduced its half-life by 2.6-fold. These results indicate that the proteolysis of Pah1 by the 20S proteasome is controlled by its phosphorylation state. PMID:25809482

Novel copper complexes as potential proteasome inhibitors for cancer treatment (Review).

PubMed

Zhang, Zhen; Wang, Huiyun; Yan, Maocai; Wang, Huannan; Zhang, Chunyan

2017-01-01

The use of metal complexes in the pharmaceutical industry has recently increased and as a result, novel metal‑based complexes have initiated an interest as potential anticancer agents. Copper (Cu), which is an essential trace element in all living organisms, is important in maintaining the function of numerous proteins and enzymes. It has recently been demonstrated that Cu complexes may be used as tumor‑specific proteasome inhibitors and apoptosis inducers, by targeting the ubiquitin‑proteasome pathway (UPP). Cu complexes have demonstrated promising results in preclinical studies. The UPP is important in controlling the expression, activity and location of various proteins. Therefore, selective proteasome inhibition and apoptotic induction in cancer cells have been regarded as potential anticancer strategies. The present short review discusses recent progress in the development of Cu complexes, including clioquinol, dithiocarbamates and Schiff bases, as proteasome inhibitors for cancer treatment. A discussion of recent research regarding the understanding of metal inhibitors based on Cu and ligand platforms is presented.

Proteomic characterization of Withaferin A-targeted protein networks for the treatment of monoclonal myeloma gammopathies.

PubMed

Dom, Martin; Offner, Fritz; Vanden Berghe, Wim; Van Ostade, Xaveer

2018-05-15

Withaferin A (WA), a natural steroid lactone from the plant Withania somnifera, is often studied because of its antitumor properties. Although many in vitro and in vivo studies have been performed, the identification of Withaferin A protein targets and its mechanism of antitumor action remain incomplete. We used quantitative chemoproteomics and differential protein expression analysis to characterize the WA antitumor effects on a multiple myeloma cell model. Identified relevant targets were further validated by Ingenuity Pathway Analysis and Western blot and indicate that WA targets protein networks that are specific for monoclonal gammopathy of undetermined significance (MGUS) and other closely related disorders, such as multiple myeloma (MM) and Waldenström macroglobulinemia (WM). By blocking the PSMB10 proteasome subunit, downregulation of ANXA4, potential association with HDAC6 and upregulation of HMOX1, WA puts a massive blockage on both proteotoxic and oxidative stress responses pathways, leaving cancer cells defenseless against WA induced stresses. These results indicate that WA mediated apoptosis is preceded by simultaneous targeting of cellular stress response pathways like proteasome degradation, autophagy and unfolded protein stress response and thus suggests that WA can be used as an effective treatment for MGUS and other closely related disorders. Multifunctional antitumor compounds are of great potential since they reduce the risk of multidrug resistance in chemotherapy. Unfortunately, characterization of all protein targets of a multifunctional compound is lacking. Therefore, we optimized an SILAC quantitative chemoproteomics workflow to identify the potential protein targets of Withaferin A (WA), a natural multifunctional compound with promising antitumor properties. To further understand the antitumor mechanisms of WA, we performed a differential protein expression analysis and combined the altered expression data with chemoproteome WA target data in the highly curated Ingenuity Pathway database. We provide a first global overview on how WA kills multiple myeloma cancer cells and serve as a starting point for further in depth experiments. Furthermore, the combined approach can be used for other types of cancer and/or other promising multifunctional compounds, thereby increasing the potential development of new antitumor therapies. Copyright © 2018 Elsevier B.V. All rights reserved.

HIV-1 Envelope Resistance to Proteasomal Cleavage: Implications for Vaccine Induced Immune Responses

PubMed Central

Steers, Nicholas J.; Ratto-Kim, Silvia; de Souza, Mark S.; Currier, Jeffrey R.; Kim, Jerome H.; Michael, Nelson L.; Alving, Carl R.; Rao, Mangala

2012-01-01

Background Antigen processing involves many proteolytic enzymes such as proteasomes and cathepsins. The processed antigen is then presented on the cell surface bound to either MHC class I or class II molecules and induces/interacts with antigen-specific CD8+ and CD4+ T-cells, respectively. Preliminary immunological data from the RV144 phase III trial indicated that the immune responses were biased towards the Env antigen with a dominant CD4+ T-cell response. Methods In this study, we examined the susceptibility of HIV-1 Env-A244 gp120 protein, one of the protein boost subunits of the RV144 Phase III vaccine trial, to proteasomes and cathepsins and identified the generated peptide epitope repertoire by mass spectrometry. The peptide fragments were tested for cytokine production in CD4+ T-cell lines derived from RV144 volunteers. Results Env-A244 was resistant to proteasomes, thus diminishing the possibility of the generation of class I epitopes by the classical MHC class I pathway. However, Env-A244 was efficiently cleaved by cathepsins generating peptide arrays identified by mass spectrometry that contained both MHC class I and class II epitopes as reported in the Los Alamos database. Each of the cathepsins generated distinct degradation patterns containing regions of light and dense epitope clusters. The sequence DKKQKVHALF that is part of the V2 loop of gp120 produced by cathepsins induced a polyfunctional cytokine response including the generation of IFN-γ from CD4+ T-cell lines-derived from RV144 vaccinees. This sequence is significant since antibodies to the V1/V2-loop region correlated inversely with HIV-1 infection in the RV144 trial. Conclusions Based on our results, the susceptibility of Env-A244 to cathepsins and not to proteasomes suggests a possible mechanism for the generation of Env-specific CD4+T cell and antibody responses in the RV144 vaccinees. PMID:22880042

Experimental hyperthyroidism in rats increases the expression of the ubiquitin ligases atrogin-1 and MuRF1 and stimulates multiple proteolytic pathways in skeletal muscle.

PubMed

O'Neal, Patrick; Alamdari, Nima; Smith, Ira; Poylin, Vitaliy; Menconi, Michael; Hasselgren, Per-Olof

2009-11-01

Muscle wasting is commonly seen in patients with hyperthyroidism and is mainly caused by stimulated muscle proteolysis. Loss of muscle mass in several catabolic conditions is associated with increased expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF1 but it is not known if atrogin-1 and MuRF1 are upregulated in hyperthyroidism. In addition, it is not known if thyroid hormone increases the activity of proteolytic mechanisms other than the ubiquitin-proteasome pathway. We tested the hypotheses that experimental hyperthyroidism in rats, induced by daily intraperitoneal injections of 100 microg/100 g body weight of triiodothyronine (T3), upregulates the expression of atrogin-1 and MuRF1 in skeletal muscle and stimulates lysosomal, including cathepsin L, calpain-, and caspase-3-dependent protein breakdown in addition to proteasome-dependent protein breakdown. Treatment of rats with T3 for 3 days resulted in an approximately twofold increase in atrogin-1 and MuRF1 mRNA levels. The same treatment increased proteasome-, cathepsin L-, and calpain-dependent proteolytic rates by approximately 40% but did not influence caspase-3-dependent proteolysis. The expression of atrogin-1 and MuRF1 remained elevated during a more prolonged period (7 days) of T3 treatment. The results provide support for a role of the ubiquitin-proteasome pathway in muscle wasting during hyperthyroidism and suggest that other proteolytic pathways as well may be activated in the hyperthyroid state. (c) 2009 Wiley-Liss, Inc.

Silica-coated magnetic nanoparticles impair proteasome activity and increase the formation of cytoplasmic inclusion bodies in vitro

PubMed Central

Phukan, Geetika; Shin, Tae Hwan; Shim, Jeom Soon; Paik, Man Jeong; Lee, Jin-Kyu; Choi, Sangdun; Kim, Yong Man; Kang, Seong Ho; Kim, Hyung Sik; Kang, Yup; Lee, Soo Hwan; Mouradian, M. Maral; Lee, Gwang

2016-01-01

The potential toxicity of nanoparticles, particularly to neurons, is a major concern. In this study, we assessed the cytotoxicity of silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye (MNPs@SiO2(RITC)) in HEK293 cells, SH-SY5Y cells, and rat primary cortical and dopaminergic neurons. In cells treated with 1.0 μg/μl MNPs@SiO2(RITC), the expression of several genes related to the proteasome pathway was altered, and proteasome activity was significantly reduced, compared with control and with 0.1 μg/μl MNPs@SiO2(RITC)-treated cells. Due to the reduction of proteasome activity, formation of cytoplasmic inclusions increased significantly in HEK293 cells over-expressing the α–synuclein interacting protein synphilin-1 as well as in primary cortical and dopaminergic neurons. Primary neurons, particularly dopaminergic neurons, were more vulnerable to MNPs@SiO2(RITC) than SH-SY5Y cells. Cellular polyamines, which are associated with protein aggregation, were significantly altered in SH-SY5Y cells treated with MNPs@SiO2(RITC). These findings highlight the mechanisms of neurotoxicity incurred by nanoparticles. PMID:27378605

MTA1 regulation of ERβ pathway in salivary gland carcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohshiro, Kazufumi, E-mail: bcmkxo@gwu.edu; Kumar, Rakesh

Abstracts: Although Metastatic-tumor antigen 1 (MTA1) is differentially expressed in metastatic cancer and coregulates the status and activity of nuclear receptors, its role upon estrogen receptor β (ERβ) – a potent tumor suppressor, remains poorly understood. Here we investigated whether MTA1 regulates the expression and functions of ERβ, an ER isoform predominantly expressed in salivary gland cancer cells. We found that the depletion of the endogenous MTA1 in the HSG and HSY salivary duct carcinoma cell lines enhances the expression of ERβ while MTA1 overexpression augmented the expression of ERβ in salivary duct carcinoma cells. Furthermore, MTA1 knockdown inhibited themore » proliferations and invasion of HSG and HSY cells. The noted ERβ downregulation by MTA1 overexpression involves the process of proteasomal degradation, as a proteasome inhibitor could block it. In addition, both MTA1 knockdown and ERβ overexpression attenuated the cell migration and inhibited the ERK1/2 signaling in the both cell lines. These findings imply that MTA1 dysregulation in a subset of salivary gland cancer might promote aggressive phenotypes by compromising the tumor suppressor activity of ERβ, and hence, MTA1-ERβ axis might serve a new therapeutic target for the salivary gland cancer. - Highlights: • MTA1 silencing upregulates ERβ expression in salivary gland carcinoma cells. • MTA1 overexpression downregulates ERβ expression via proteasomal degradation. • Upregulation of ERβ expression inhibits cell migration and ERK signaling. • MTA1 knockdown inhibits cell proliferation and invasion.« less

BCL11B is frequently downregulated in HTLV-1-infected T-cells through Tax-mediated proteasomal degradation.

PubMed

Permatasari, Happy Kurnia; Nakahata, Shingo; Ichikawa, Tomonaga; Morishita, Kazuhiro

2017-08-26

Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia-lymphoma (ATLL). The HTLV-1-encoded protein Tax plays important roles in the proliferation of HTLV-1-infected T-cells by affecting cellular proteins. In this study, we showed that Tax transcriptionally and post-transcriptionally downregulates the expression of the tumor suppressor gene B-cell leukemia/lymphoma 11B (BCL11B), which encodes a lymphoid-related transcription factor. BCL11B expression was downregulated in HTLV-1-infected T-cell lines at the mRNA and protein levels, and forced expression of BCL11B suppressed the proliferation of these cells. The proteasomal inhibitor MG132 increased BCL11B expression in HTLV-1-infected cell lines, and colocalization of Tax with BCL11B was detected in the cytoplasm of HTLV-1-infected T-cells following MG132 treatment. shRNA knock-down of Tax expression also increased the expression of BCL11B in HTLV-1-infected cells. Moreover, we found that Tax physically binds to BCL11B protein and induces the polyubiquitination of BCL11B and proteasome-dependent degradation of BCL11B. Thus, inactivation of BCL11B by Tax protein may play an important role in the Tax-mediated leukemogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Combination of quercetin and tannic acid in inhibiting 26S proteasome affects S5a and 20S expression, and accumulation of ubiquitin resulted in apoptosis in cancer chemoprevention.

PubMed

Chang, Tsui-Ling; Wang, Chi-Hsien

2013-04-01

To look for oral proteasome inhibitors, daily injested food is the best source for cancer chemoprevention. A combination of active components from vegetables, coffee, tea, and fruit could be more efficient to inhibit 26S proteasome activities for preventing cancer diseases. Tannic acid and quercetin have been shown to strongly inhibit 26S proteasome activity, but the molecular target involved remains unknown. Overlay assay, peptide assay, Western blot, and 2-D gels were used to assess the combination of quercetin and tannic acid as a potential inhibitor. Here, we demonstrated that the combination of quercetin and tannic acid (1) synergistically suppresses chymotrypsin-, caspase-, and trypsin-like proteolytic activities, (2) are tightly binding substrates, (3) do not perturb the proteasome structure, (4) inhibit the 26S proteasome affected by ubiquitin, ATP, or β-casein, and (5) inhibit β-casein degradation by the 26S proteasome in vitro. Finally, the inhibition of the proteasome by a combination of quercetin plus tannic acid in Hep-2 cells resulted in the induction of S5a at low dose, accumulation of ubiquitin, and the cleavage of pro-caspase-3, followed by the induction of apoptotic cell death. Evaluating the combination of quercetin and tannic acid as an oral drug to prevent cancer may provide a pharmacological rationale to pursue preclinical trials of this combination.

Identification of Autophagosome-associated Proteins and Regulators by Quantitative Proteomic Analysis and Genetic Screens*

PubMed Central

Dengjel, Jörn; Høyer-Hansen, Maria; Nielsen, Maria O.; Eisenberg, Tobias; Harder, Lea M.; Schandorff, Søren; Farkas, Thomas; Kirkegaard, Thomas; Becker, Andrea C.; Schroeder, Sabrina; Vanselow, Katja; Lundberg, Emma; Nielsen, Mogens M.; Kristensen, Anders R.; Akimov, Vyacheslav; Bunkenborg, Jakob; Madeo, Frank; Jäättelä, Marja; Andersen, Jens S.

2012-01-01

Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection. PMID:22311637

Modulation of intracellular protein degradation by SSB1-SIS1 chaperon system in yeast S. cerevisiae.

PubMed

Ohba, M

1997-06-09

In prokaryotes, DnaK-DnaJ chaperon is involved in the protein degradation catalyzed by proteases La and ClpA/B complex as shown in E. coli. To extend this into eukaryotic cells, we examined the effects of hsp70 genes, SSA1 and SSB1, and DnaJ genes, SIS1 and YDJ1, on the growth of proteasome subunit mutants of the yeast S. cerevisiae. The results identified SSB1 and SIS1 as a pair of chaperon genes specifically involved in efficient protein turnover in the yeast, whose overexpression suppressed the growth defects caused by the proteasome mutations. Moreover, a single amino acid substitution in the putative peptide-binding site of SSB1 protein profoundly enhanced the suppression activity, indicating that the activity is mediated by the peptide-binding activity of this chaperon. Thus SSB1, with its partner DnaJ, SIS1, modulates the efficiency of protein turnover through its chaperon activity.

Proteasome inhibition mediates p53 reactivation and anti-cancer activity of 6-gingerol in cervical cancer cells.

PubMed

Rastogi, Namrata; Duggal, Shivali; Singh, Shailendra Kumar; Porwal, Konica; Srivastava, Vikas Kumar; Maurya, Rakesh; Bhatt, M L B; Mishra, Durga Prasad

2015-12-22

Human papilloma virus (HPV) expressing E6 and E7 oncoproteins, is known to inactivate the tumor suppressor p53 through proteasomal degradation in cervical cancers. Therefore, use of small molecules for inhibition of proteasome function and induction of p53 reactivation is a promising strategy for induction of apoptosis in cervical cancer cells. The polyphenolic alkanone, 6-Gingerol (6G), present in the pungent extracts of ginger (Zingiber officinale Roscoe) has shown potent anti-tumorigenic and pro-apoptotic activities against a variety of cancers. In this study we explored the molecular mechanism of action of 6G in human cervical cancer cells in vitro and in vivo. 6G potently inhibited proliferation of the HPV positive cervical cancer cells. 6G was found to: (i) inhibit the chymotrypsin activity of proteasomes, (ii) induce reactivation of p53, (iii) increase levels of p21, (iv) induce DNA damage and G2/M cell cycle arrest, (v) alter expression levels of p53-associated apoptotic markers like, cleaved caspase-3 and PARP, and (vi) potentiate the cytotoxicity of cisplatin. 6G treatment induced significant reduction of tumor volume, tumor weight, proteasome inhibition and p53 accumulation in HeLa xenograft tumor cells in vivo. The 6G treatment was devoid of toxic effects as it did not affect body weights, hematological and osteogenic parameters. Taken together, our data underscores the therapeutic and chemosensitizing effects of 6G in the management and treatment of cervical cancer.

Carnosic acid protects SH-SY5Y cells against 6-hydroxydopamine-induced cell death through upregulation of parkin pathway.

PubMed

Lin, Chia-Yuan; Tsai, Chia-Wen; Tsai, Chia-Wen

2016-11-01

Parkin is a Parkinson's disease (PD)-linked gene that plays an important role in the ubiquitin-proteasome system (UPS). This study explored whether carnosic acid (CA) from rosemary protects against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity via upregulation of parkin in vivo and in vitro. We found that the reduction in proteasomal activity by 6-OHDA was attenuated in SH-SY5Y cells pretreated with 1 μM CA. Immunoblots showed that CA reversed the induction of ubiquitinated protein and the reduction of PTEN-induced putative kinase 1 (PINK1) and parkin protein in 6-OHDA-treated SH-SY5Y cells and rats. Moreover, in a transgenic OW13 Caenorhabditis elegans model of PD that expresses human α-synuclein in muscle cells, CA reduced α-synuclein accumulation in a dose-dependent manner. In cells pretreated with the proteasome inhibitor MG132, CA no longer reversed the 6-OHDA-mediated induction of cleavage of caspase 3 and poly(ADP)-ribose polymerase and no longer reversed the suppression of proteasome activity. When parkin expression was silenced by use of small interfering RNA, the ability of CA to inhibit apoptosis and induce proteasomal activity was significantly reduced. The reduction in 6-OHDA-induced neurotoxicity by CA was associated with the induction of parkin, which in turn upregulated the UPS and then decreased cell death. Copyright © 2016. Published by Elsevier Ltd.

Proteasome inhibition mediates p53 reactivation and anti-cancer activity of 6-Gingerol in cervical cancer cells

PubMed Central

Rastogi, Namrata; Duggal, Shivali; Singh, Shailendra Kumar; Porwal, Konica; Srivastava, Vikas Kumar; Maurya, Rakesh; Bhatt, Madan L.B.; Mishra, Durga Prasad

2015-01-01

Human papilloma virus (HPV) expressing E6 and E7 oncoproteins, is known to inactivate the tumor suppressor p53 through proteasomal degradation in cervical cancers. Therefore, use of small molecules for inhibition of proteasome function and induction of p53 reactivation is a promising strategy for induction of apoptosis in cervical cancer cells. The polyphenolic alkanone, 6-Gingerol (6G), present in the pungent extracts of ginger (Zingiber officinale Roscoe) has shown potent anti-tumorigenic and pro-apoptotic activities against a variety of cancers. In this study we explored the molecular mechanism of action of 6G in human cervical cancer cells in vitro and in vivo. 6G potently inhibited proliferation of the HPV positive cervical cancer cells. 6G was found to: (i) inhibit the chymotrypsin activity of proteasomes, (ii) induce reactivation of p53, (iii) increase levels of p21, (iv) induce DNA damage and G2/M cell cycle arrest, (v) alter expression levels of p53-associated apoptotic markers like, cleaved caspase-3 and PARP, and (vi) potentiate the cytotoxicity of cisplatin. 6G treatment induced significant reduction of tumor volume, tumor weight, proteasome inhibition and p53 accumulation in HeLa xenograft tumor cells in vivo. The 6G treatment was devoid of toxic effects as it did not affect body weights, hematological and osteogenic parameters. Taken together, our data underscores the therapeutic and chemosensitizing effects of 6G in the management and treatment of cervical cancer. PMID:26621832

Proteomic analysis of papaya (Carica papaya L.) displaying typical sticky disease symptoms.

PubMed

Rodrigues, Silas P; Ventura, José A; Aguilar, Clemente; Nakayasu, Ernesto S; Almeida, Igor C; Fernandes, Patricia M B; Zingali, Russolina B

2011-07-01

Papaya (Carica papaya L.) hosts the only described laticifer-infecting virus (Papaya meleira virus, PMeV), which is the causal agent of papaya sticky disease. To understand the systemic effects of PMeV in papaya, we conducted a comprehensive proteomic analysis of leaf samples from healthy and diseased plants grown under field conditions. First, a reference 2-DE map was established for proteins from healthy samples. A total of 486 reproducible spots were identified, and MALDI-TOF-MS/MS data identified 275 proteins accounting for 159 distinct proteins from 231 spots that were annotated. Second, the differential expression of proteins from healthy and diseased leaves was determined through parallel experiments, using 2-DE and DIGE followed by MALDI-TOF-MS/MS and LC-IonTrap-MS/MS, respectively. Conventional 2-DE analysis revealed 75 differentially expressed proteins. Of those, 48 proteins were identified, with 26 being upregulated (U) and 22 downregulated (D). In general, metabolism-related proteins were downregulated, and stress-responsive proteins were upregulated. This expression pattern was corroborated by the results of the DIGE analysis, which identified 79 differentially expressed proteins, with 23 identified (17 U and 6 D). Calreticulin and the proteasome subunits 20S and RPT5a were shown to be upregulated during infection by both 2-DE and DIGE analyses. These data may help shed light on plant responses against stresses and viral infections. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteolytic activities in cortex of apical parts of Vicia faba ssp. minor seedling roots during kinetin-induced programmed cell death.

PubMed

Kaźmierczak, Andrzej; Doniak, Magdalena; Kunikowska, Anita

2017-11-01

Programmed cell death (PCD) is a crucial process in plant development. In this paper, proteolytically related aspects of kinetin-induced PCD in cortex cells of Vicia faba ssp. minor seedlings were examined using morphological, fluorometric, spectrophotometric, and fluorescence microscopic analyses. Cell viability estimation after 46 μM kinetin treatment of seedling roots showed that the number of dying cortex cells increased with treatment duration, reaching maximum after 72 h. Weight of the apical root segments increased with time and was about 2.5-fold greater after 96 h, while the protein content remained unchanged, compared to the control. The total and cysteine-dependent proteolytic activities fluctuated during 1-96-h treatment, which was not accompanied by the changes in the protein amount, indicating that the absolute protein amounts decreased during kinetin-induced PCD. N-ethylmaleimide (NEM), phenylmethylsulfonyl fluoride (PMSF), and Z-Leu-Leu-Nva-H (MG115), the respective cysteine, serine, and proteasome inhibitors, suppressed kinetin-induced PCD. PMSF significantly decreased serine-dependent proteolytic activities without changing the amount of proteins, unlike NEM and MG115. More pronounced effect of PMSF over NEM indicated that in the root apical segments, the most important proteolytic activity during kinetin-induced PCD was that of serine proteases, while that of cysteine proteases may be important for protein degradation in the last phase of the process. Both NEM and PMSF inhibited apoptotic-like structure formation during kinetin-induced PCD. The level of caspase-3-like activity of β1 proteasome subunit increased after kinetin treatment. Addition of proteasome inhibitor MG-115 reduced the number of dying cells, suggesting that proteasomes might play an important role during kinetin-induced PCD.

Role of PARP activity in lung cancer-induced cachexia: Effects on muscle oxidative stress, proteolysis, anabolic markers, and phenotype.

PubMed

Chacon-Cabrera, Alba; Mateu-Jimenez, Mercè; Langohr, Klaus; Fermoselle, Clara; García-Arumí, Elena; Andreu, Antoni L; Yelamos, Jose; Barreiro, Esther

2017-12-01

Strategies to treat cachexia are still at its infancy. Enhanced muscle protein breakdown and ubiquitin-proteasome system are common features of cachexia associated with chronic conditions including lung cancer (LC). Poly(ADP-ribose) polymerases (PARP), which play a major role in chromatin structure regulation, also underlie maintenance of muscle metabolism and body composition. We hypothesized that protein catabolism, proteolytic markers, muscle fiber phenotype, and muscle anabolism may improve in respiratory and limb muscles of LC-cachectic Parp-1-deficient (Parp-1 -/- ) and Parp-2 -/- mice. In diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) bearing mice (wild type, Parp-1 -/- , and Parp-2 -/- ), PARP activity (ADP-ribose polymers, pADPr), redox balance, muscle fiber phenotype, apoptotic nuclei, tyrosine release, protein ubiquitination, muscle-specific E3 ligases, NF-κB signaling pathway, markers of muscle anabolism (Akt, mTOR, p70S6K, and mitochondrial DNA) were evaluated along with body and muscle weights, and limb muscle force. Compared to wild type cachectic animals, in both respiratory and limb muscles of Parp-1 -/- and Parp-2 -/- cachectic mice: cancer induced-muscle wasting characterized by increased PARP activity, protein oxidation, tyrosine release, and ubiquitin-proteasome system (total protein ubiquitination, atrogin-1, and 20S proteasome C8 subunit) were blunted, the reduction in contractile myosin and atrophy of the fibers was attenuated, while no effects were seen in other structural features (inflammatory cells, internal or apoptotic nuclei), and markers of muscle anabolism partly improved. Activation of either PARP-1 or -2 is likely to play a role in muscle protein catabolism via oxidative stress, NF-κB signaling, and enhanced proteasomal degradation in cancer-induced cachexia. Therapeutic potential of PARP activity inhibition deserves attention. © 2017 Wiley Periodicals, Inc.

Effect of cellular ubiquitin levels on the regulation of oxidative stress response and proteasome function via Nrf1.

PubMed

Lee, Donghee; Ryu, Kwon-Yul

2017-04-01

The polyubiquitin genes Ubb and Ubc are upregulated under oxidative stress induced by arsenite [As(III)]. However, the role of ubiquitin (Ub) under As(III) exposure is not known in detail. In a previous study, we showed that the reduced viability observed in Ubc -/- mouse embryonic fibroblasts under As(III) exposure was not due to dysregulation of the Nrf2-Keap1 pathway, which prompted us to investigate another NFE2 family protein, nuclear factor erythroid 2-related factor 1 (Nrf1). In this study, we found that Ub deficiency due to Ubc knockdown in N2a cells reduced cell viability and proteasome activity under As(III) exposure. Furthermore, mRNA levels of the proteasome subunit Psma1 were also reduced. In addition, Ub deficiency led to the nuclear accumulation of the p65 isoform of Nrf1 under As(III) exposure. Interestingly, the overexpression of p65-Nrf1 recapitulated the phenotypes of Ub-deficient N2a cells under As(III) exposure. On the other hand, Nrf1 knockdown suppressed the death of Ub-deficient N2a cells upon exposure to As(III). Therefore, the levels of p65-Nrf1 may play an important role in the maintenance of cell viability under oxidative stress induced by As(III). Copyright © 2017 Elsevier Inc. All rights reserved.

Distal Renal Tubules Are Deficient in Aggresome Formation and Autophagy upon Aldosterone Administration

PubMed Central

Cheema, Muhammad Umar; Damkier, Helle Hasager; Nielsen, Jakob; Poulsen, Ebbe Toftgaard; Enghild, Jan J.; Fenton, Robert A.; Praetorius, Jeppe

2014-01-01

Prolonged elevations of plasma aldosterone levels are associated with renal pathogenesis. We hypothesized that renal distress could be imposed by an augmented aldosterone-induced protein turnover challenging cellular protein degradation systems of the renal tubular cells. Cellular accumulation of specific protein aggregates in rat kidneys was assessed after 7 days of aldosterone administration. Aldosterone induced intracellular accumulation of 60 s ribosomal protein L22 in protein aggregates, specifically in the distal convoluted tubules. The mineralocorticoid receptor inhibitor spironolactone abolished aldosterone-induced accumulation of these aggregates. The aldosterone-induced protein aggregates also contained proteasome 20 s subunits. The partial de-ubiquitinase ataxin-3 was not localized to the distal renal tubule protein aggregates, and the aggregates only modestly colocalized with aggresome transfer proteins dynactin p62 and histone deacetylase 6. Intracellular protein aggregation in distal renal tubules did not lead to development of classical juxta-nuclear aggresomes or to autophagosome formation. Finally, aldosterone treatment induced foci in renal cortex of epithelial vimentin expression and a loss of E-cadherin expression, as signs of cellular stress. The cellular changes occurred within high, but physiological aldosterone concentrations. We conclude that aldosterone induces protein accumulation in distal renal tubules; these aggregates are not cleared by autophagy that may lead to early renal tubular damage. PMID:25000288

Lopinavir up-regulates expression of the antiviral protein ribonuclease L in human papillomavirus-positive cervical carcinoma cells.

PubMed

Batman, Gavin; Oliver, Anthony W; Zehbe, Ingeborg; Richard, Christina; Hampson, Lynne; Hampson, Ian N

2011-01-01

We have previously shown that the HIV protease inhibitor lopinavir has selective toxicity against human papillomavirus (HPV)-positive cervical carcinoma cells via an unknown mechanism. SiHa cervical carcinoma cells were stably transfected with the proteasome sensor vector pZsProSensor-1 to confirm lopinavir inhibits the proteasome in these cells. The Panorama Xpress profiler 725 antibody array was then used to analyse specific changes in protein expression in lopinavir-treated versus control untreated SiHa cells followed by PCR and western blotting. Colorimetric growth assays of lopinavir-treated E6/E7 immortalised versus control human keratinocytes were performed. Targeted small interfering RNA gene silencing followed by growth assay comparison of lopinavir-treated/untreated SiHa cells was also used. Lopinavir induced an increase in the fluorescence of pZsProSensor-1 transfected SiHa cells, indicative of proteasomal inhibition. Ribonuclease L (RNASEL) protein was shown to be up-regulated in lopinavir-treated SiHa cells, which was confirmed by PCR and western blot. Targeted silencing of RNASEL reduced the sensitivity of SiHa cells to lopinavir. Selective toxicity against E6/E7 immortalised keratinocytes versus control cells was also seen with lopinavir and was associated with up-regulated RNASEL expression. These data are consistent with the toxicity of lopinavir against HPV-positive cervical carcinoma cells being related to its ability to block viral proteasome activation and induce an up-regulation of the antiviral protein RNASEL. This is supported by the drug's selective toxicity and up-regulation of RNASEL in E6/E7 immortalised keratinocytes combined with the increased resistance to lopinavir observed in SiHa cells following silencing of RNASEL gene expression.

A heart that beats for 500 years: age-related changes in cardiac proteasome activity, oxidative protein damage and expression of heat shock proteins, inflammatory factors, and mitochondrial complexes in Arctica islandica, the longest-living noncolonial animal.

PubMed

Sosnowska, Danuta; Richardson, Chris; Sonntag, William E; Csiszar, Anna; Ungvari, Zoltan; Ridgway, Iain

2014-12-01

Study of negligibly senescent animals may provide clues that lead to better understanding of the cardiac aging process. To elucidate mechanisms of successful cardiac aging, we investigated age-related changes in proteasome activity, oxidative protein damage and expression of heat shock proteins, inflammatory factors, and mitochondrial complexes in the heart of the ocean quahog Arctica islandica, the longest-lived noncolonial animal (maximum life span potential: 508 years). We found that in the heart of A. islandica the level of oxidatively damaged proteins did not change significantly up to 120 years of age. No significant aging-induced changes were observed in caspase-like and trypsin-like proteasome activity. Chymotrypsin-like proteasome activity showed a significant early-life decline, then it remained stable for up to 182 years. No significant relationship was observed between the extent of protein ubiquitination and age. In the heart of A. islandica, an early-life decline in expression of HSP90 and five mitochondrial electron transport chain complexes was observed. We found significant age-related increases in the expression of three cytokine-like mediators (interleukin-6, interleukin-1β, and tumor necrosis factor-α) in the heart of A. islandica. Collectively, in extremely long-lived molluscs, maintenance of protein homeostasis likely contributes to the preservation of cardiac function. Our data also support the concept that low-grade chronic inflammation in the cardiovascular system is a universal feature of the aging process, which is also manifest in invertebrates. © The Author 2013. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

An anti-HIV-1 compound that increases steady-state expression of apoplipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G.

PubMed

Ejima, Tomohiko; Hirota, Mayuko; Mizukami, Tamio; Otsuka, Masami; Fujita, Mikako

2011-10-01

Human apoplipoprotein B mRNA-editing enzyme-catalytic polypeptide-like (APOBEC) 3G (A3G) is an antiviral protein that blocks HIV-1 replication. However, the antiviral activity of A3G is overcome by the HIV-1 protein Vif. This inhibitory function of Vif is related to its ability to degrade A3G in the proteasome. This finding prompted us to examine the activities of 4-(dimethylamino)-2,6-bis[(N-(2-[(2-nitrophenyl)dithio]ethyl)amino)methyl]pyridine (SN-2) and SN-3. We found that 5 µM SN-2 increases the expression of A3G to a level much higher than that observed in the absence of Vif, without affecting the level of Vif expression. The proteasome inhibitor MG-132 increased the level of both A3G and Vif expression. These results demonstrate that A3G is ubiquitinated and degraded in the proteasome by a factor other than Vif, and that SN-2 selectively inhibits these processes. Furthermore, 5 µM SN-2 significantly inhibited the MAGI cell infectivity of wild-type HIV-1. These findings may contribute to the development of a novel anti-HIV-1 drug.

Modulation of apoptosis sensitivity through the interplay with autophagic and proteasomal degradation pathways

PubMed Central

Delgado, M E; Dyck, L; Laussmann, M A; Rehm, M

2014-01-01

Autophagic and proteasomal degradation constitute the major cellular proteolysis pathways. Their physiological and pathophysiological adaptation and perturbation modulates the relative abundance of apoptosis-transducing proteins and thereby can positively or negatively adjust cell death susceptibility. In addition to balancing protein expression amounts, components of the autophagic and proteasomal degradation machineries directly interact with and co-regulate apoptosis signal transduction. The influence of autophagic and proteasomal activity on apoptosis susceptibility is now rapidly gaining more attention as a significant modulator of cell death signalling in the context of human health and disease. Here we present a concise and critical overview of the latest knowledge on the molecular interplay between apoptosis signalling, autophagy and proteasomal protein degradation. We highlight that these three pathways constitute an intricate signalling triangle that can govern and modulate cell fate decisions between death and survival. Owing to rapid research progress in recent years, it is now possible to provide detailed insight into the mechanisms of pathway crosstalk, common signalling nodes and the role of multi-functional proteins in co-regulating both protein degradation and cell death. PMID:24457955

Therapeutic proteasome inhibition in experimental acute pancreatitis

PubMed Central

Letoha, Tamás; Fehér, Liliána Z; Pecze, László; Somlai, Csaba; Varga, Ilona; Kaszaki, József; Tóth, Gábor; Vizler, Csaba; Tiszlavicz, László; Takács, Tamás

2007-01-01

AIM: To establish the therapeutic potential of proteasome inhibition, we examined the therapeutic effects of MG132 (Z-Leu-Leu-Leu-aldehyde) in an experimental model of acute pancreatitis. METHODS: Pancreatitis was induced in rats by two hourly intraperitoneal (ip) injections of cholecystokinin octapeptide (CCK; 2 × 100 μg/kg) and the proteasome inhibitor MG132 (10 mg/kg ip) was administered 30 min after the second CCK injection. Animals were sacrificed 4 h after the first injection of CCK. RESULTS: Administering the proteasome inhibitor MG132 (at a dose of 10 mg/kg, ip) 90 min after the onset of pancreatic inflammation induced the expression of cell-protective 72 kDa heat shock protein (HSP72) and decreased DNA-binding of nuclear factor-κB (NF-κB). Furthermore MG132 treatment resulted in milder inflammatory response and cellular damage, as revealed by improved laboratory and histological parameters of pancreatitis and associated oxidative stress. CONCLUSION: Our findings suggest that proteasome inhibition might be beneficial not only for the prevention, but also for the therapy of acute pancreatitis. PMID:17724800

Cigarette smoking during pregnancy regulates the expression of specific nicotinic acetylcholine receptor (nAChR) subunits in the human placenta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Machaalani, R., E-mail: rita.machaalani@sydney.edu.au; Bosch Institute, The University of Sydney, NSW 2006; The Children's Hospital at Westmead, NSW 2145

Smoking during pregnancy is associated with low birth weight, premature delivery, and neonatal morbidity and mortality. Nicotine, a major pathogenic compound of cigarette smoke, binds to the nicotinic acetylcholine receptors (nAChRs). A total of 16 nAChR subunits have been identified in mammals (9 α, 4 β, and 1 δ, γ and ε subunits). The effect of cigarette smoking on the expression of these subunits in the placenta has not yet been determined, thus constituting the aim of this study. Using RT-qPCR and western blotting, this study investigated all 16 mammalian nAChR subunits in the normal healthy human placenta, and comparedmore » mRNA and protein expressions in the placentas from smokers (n = 8) to controls (n = 8). Our data show that all 16 subunit mRNAs are expressed in the normal, non-diseased human placenta and that the expression of α2, α3, α4, α9, β2 and β4 subunits is greater than the other subunits. For mRNA, cigarette smoke exposure was associated with increased expression of the α9 subunit, and decreased expression of the δ subunit. At the protein level, expression of both α9 and δ was increased. Thus, cigarette smoking in pregnancy is sufficient to regulate nAChR subunits in the placenta, specifically α9 and δ subunits, and could contribute to the adverse effects of vasoconstriction and decreased re-epithelialisation (α9), and increased calcification and apoptosis (δ), seen in the placentas of smoking women. - Highlights: • All 16 mammalian nAChR subunits are expressed in the human placenta. • Cigarette smoking increases α9 mRNA and protein in the placenta. • Cigarette smoking decreases δ mRNA but increases δ protein in the placenta.« less

Large conductance Ca(2+)-activated K(+) channel (BKCa) activating properties of a series of novel N-arylbenzamides: Channel subunit dependent effects.

PubMed

Kirby, R W; Martelli, A; Calderone, V; McKay, N G; Lawson, K

2013-07-15

Large conductance calcium activated potassium channels (BKCa) are fundamental in the control of cellular excitability. Thus, compounds that activate BKCa channels could provide potential therapies in the treatment of pathologies of the cardiovascular and central nervous system. A series of novel N-arylbenzamide compounds, and the reference compound NS1619, were evaluated for BKCa channel opener properties in Human Embryonic Kidney (HEK293) cells expressing the human BKCa channel α-subunit alone or α+β1-subunit complex. Channel activity was determined using a non-radioactive Rb(+) efflux assay to construct concentration effect curves for each compound. All N-arylbenzamide compounds and NS1619 evoked significant (p <0.05) concentration related increases in Rb(+) efflux both in cells expressing α-subunit alone or α+β1-subunits. Co-expression of the β1-subunit modified the Rb(+) efflux responses, relative to that obtained in cells expressing the α-subunit alone, for most of the N-arylbenzamide compounds, in contrast to NS1619. The EC40 values of NS1619, BKMe1 and BKOEt1 were not significantly affected by the co-expression of the BKCa channel α+β1-subunits. In contrast, 5 other N-arylbenzamides (BKPr2, BKPr3, BKPr4, BKH1 and BKVV) showed a significant (p <0.05) 2- to 10-fold increase in EC40 values when tested on the BKCa α+β1-subunit expressing cells compared to BKCa α-subunit expressing cells. Further, the Emax values for BKPr4, BKVV and BKH1 were lower in the BKCa channel α+β1-subunit expressing cells. In conclusion, the N-arylbenzamides studied, like NS1619, were able to activate BKCa channels formed of the α-subunit only. The co-expression of the β1-subunit, however, modified the ability of certain compounds to active the channel leading to differentiated pharmacodynamic profiles. Copyright © 2013 Elsevier Ltd. All rights reserved.

Skeletal muscle myotubes of the severely obese exhibit altered ubiquitin-proteasome and autophagic/lysosomal proteolytic flux

PubMed Central

Bollinger, Lance M.; Powell, Jonathan J. S.; Houmard, Joseph A.; Witczak, Carol A.; Brault, Jeffrey J.

2015-01-01

Objective Whole-body protein metabolism is dysregulated with obesity. Our goal was to determine if activity and expression of major protein degradation pathways are compromised specifically in human skeletal muscle with obesity. Methods We utilized primary Human Skeletal Muscle cell (HSkM) cultures since cellular mechanisms can be studied absent of hormones and contractile activity that could independently influence metabolism. HSkM from 10 lean (BMI ≤ 26.0 kg/m2) and 8 severely obese (BMI ≥ 39.0) women were examined basally and when stimulated to atrophy (serum and amino acid starvation). Results HSkM from obese donors had a lower proportion of type I myosin heavy chain and slower flux through the autophagic/lysosomal pathway. During starvation, flux through the ubiquitin-proteasome system diverged according to obesity status, with a decrease in the lean and an increase in HSkM from obese subjects. HSkMC from the obese also displayed elevated proteasome activity despite no difference in proteasome content. Atrophy-related gene expression and myotube area were similar in myotubes derived from lean and obese individuals under basal and starved conditions. Conclusions Our data indicate that muscle cells of the lean and severely obese have innate differences in management of protein degradation, which may explain their metabolic differences. PMID:26010327

Toll-like receptor 4 mediates lipopolysaccharide-induced muscle catabolism via coordinate activation of ubiquitin-proteasome and autophagy-lysosome pathways

PubMed Central

Doyle, Alexander; Zhang, Guohua; Abdel Fattah, Elmoataz A.; Eissa, N. Tony; Li, Yi-Ping

2011-01-01

Cachectic muscle wasting is a frequent complication of many inflammatory conditions, due primarily to excessive muscle catabolism. However, the pathogenesis and intervention strategies against it remain to be established. Here, we tested the hypothesis that Toll-like receptor 4 (TLR4) is a master regulator of inflammatory muscle catabolism. We demonstrate that TLR4 activation by lipopolysaccharide (LPS) induces C2C12 myotube atrophy via up-regulating autophagosome formation and the expression of ubiquitin ligase atrogin-1/MAFbx and MuRF1. TLR4-mediated activation of p38 MAPK is necessary and sufficient for the up-regulation of atrogin1/MAFbx and autophagosomes, resulting in myotube atrophy. Similarly, LPS up-regulates muscle autophagosome formation and ubiquitin ligase expression in mice. Importantly, autophagy inhibitor 3-methyladenine completely abolishes LPS-induced muscle proteolysis, while proteasome inhibitor lactacystin partially blocks it. Furthermore, TLR4 knockout or p38 MAPK inhibition abolishes LPS-induced muscle proteolysis. Thus, TLR4 mediates LPS-induced muscle catabolism via coordinate activation of the ubiquitin-proteasome and the autophagy-lysosomal pathways.—Doyle, A., Zhang, G., Abdel Fattah, E. A., Eissa, N. T., Li, Y.-P. Toll-like receptor 4 mediates lipopolysaccharide-induced muscle catabolism via coordinate activation of ubiquitin-proteasome and autophagy-lysosome pathways. PMID:20826541

Enhanced Polyubiquitination of Shank3 and NMDA receptor in a mouse model of Autism

PubMed Central

Bangash, M Ali; Park, Joo Min; Melnikova, Tatiana; Wang, Dehua; Jeon, Soo Kyeong; Lee, Deidre; Syeda, Sbaa; Kim, Juno; Kouser, Mehreen; Schwartz, Joshua; Cui, Yiyuan; Zhao, Xia; Speed, Haley E.; Kee, Sara E.; Tu, Jian Cheng; Hu, Jia-Hua; Petralia, Ronald S.; Linden, David J.; Powell, Craig M.; Savonenko, Alena; Xiao, Bo; Worley, Paul F.

2011-01-01

Summary We have created a mouse genetic model that mimics a human mutation of Shank3 that deletes the C-terminus and is associated with autism. Expressed as a single copy [Shank3(+/ΔC) mice], Shank3ΔC protein interacts with the WT gene product and results in >90 % reduction of Shank3 at synapses. This “gain of function” phenotype is linked to increased polyubiquitination of WT Shank3 and its redistribution into proteasomes. Similarly, the NR1 subunit of the NMDA receptor is reduced at synapses with increased polyubiquitination. Assays of post-synaptic density proteins, spine morphology and synapse number are unchanged in Shank3(+/ΔC) mice, but the amplitude of NMDAR responses is reduced together with reduced NMDAR-dependent LTP and LTD. Reciprocally, mGluR-dependent LTD is markedly enhanced. Shank3(+/ΔC) mice show behavioral deficits suggestive of autism and reduced NMDA receptor function. These studies reveal a mechanism distinct from haploinsufficiency by which mutations of Shank3 can evoke an autism-like disorder. PMID:21565394

Enhanced polyubiquitination of Shank3 and NMDA receptor in a mouse model of autism.

PubMed

Bangash, M Ali; Park, Joo Min; Melnikova, Tatiana; Wang, Dehua; Jeon, Soo Kyeong; Lee, Deidre; Syeda, Sbaa; Kim, Juno; Kouser, Mehreen; Schwartz, Joshua; Cui, Yiyuan; Zhao, Xia; Speed, Haley E; Kee, Sara E; Tu, Jian Cheng; Hu, Jia-Hua; Petralia, Ronald S; Linden, David J; Powell, Craig M; Savonenko, Alena; Xiao, Bo; Worley, Paul F

2011-05-27

We have created a mouse genetic model that mimics a human mutation of Shank3 that deletes the C terminus and is associated with autism. Expressed as a single copy [Shank3(+/ΔC) mice], Shank3ΔC protein interacts with the wild-type (WT) gene product and results in >90% reduction of Shank3 at synapses. This "gain-of-function" phenotype is linked to increased polyubiquitination of WT Shank3 and its redistribution into proteasomes. Similarly, the NR1 subunit of the NMDA receptor is reduced at synapses with increased polyubiquitination. Assays of postsynaptic density proteins, spine morphology, and synapse number are unchanged in Shank3(+/ΔC) mice, but the amplitude of NMDAR responses is reduced together with reduced NMDAR-dependent LTP and LTD. Reciprocally, mGluR-dependent LTD is markedly enhanced. Shank3(+/ΔC) mice show behavioral deficits suggestive of autism and reduced NMDA receptor function. These studies reveal a mechanism distinct from haploinsufficiency by which mutations of Shank3 can evoke an autism-like disorder. Copyright © 2011 Elsevier Inc. All rights reserved.

Screen for mitochondrial DNA copy number maintenance genes reveals essential role for ATP synthase

PubMed Central

Fukuoh, Atsushi; Cannino, Giuseppe; Gerards, Mike; Buckley, Suzanne; Kazancioglu, Selena; Scialo, Filippo; Lihavainen, Eero; Ribeiro, Andre; Dufour, Eric; Jacobs, Howard T

2014-01-01

The machinery of mitochondrial DNA (mtDNA) maintenance is only partially characterized and is of wide interest due to its involvement in disease. To identify novel components of this machinery, plus other cellular pathways required for mtDNA viability, we implemented a genome-wide RNAi screen in Drosophila S2 cells, assaying for loss of fluorescence of mtDNA nucleoids stained with the DNA-intercalating agent PicoGreen. In addition to previously characterized components of the mtDNA replication and transcription machineries, positives included many proteins of the cytosolic proteasome and ribosome (but not the mitoribosome), three proteins involved in vesicle transport, some other factors involved in mitochondrial biogenesis or nuclear gene expression, > 30 mainly uncharacterized proteins and most subunits of ATP synthase (but no other OXPHOS complex). ATP synthase knockdown precipitated a burst of mitochondrial ROS production, followed by copy number depletion involving increased mitochondrial turnover, not dependent on the canonical autophagy machinery. Our findings will inform future studies of the apparatus and regulation of mtDNA maintenance, and the role of mitochondrial bioenergetics and signaling in modulating mtDNA copy number. PMID:24952591

Genome-scale analyses of butanol tolerance in Saccharomyces cerevisiae reveal an essential role of protein degradation

PubMed Central

2013-01-01

Background n-Butanol and isobutanol produced from biomass-derived sugars are promising renewable transport fuels and solvents. Saccharomyces cerevisiae has been engineered for butanol production, but its high butanol sensitivity poses an upper limit to product titers that can be reached by further pathway engineering. A better understanding of the molecular basis of butanol stress and tolerance of S. cerevisiae is important for achieving improved tolerance. Results By combining a screening of the haploid S. cerevisiae knock-out library, gene overexpression, and genome analysis of evolutionary engineered n-butanol-tolerant strains, we established that protein degradation plays an essential role in tolerance. Strains deleted in genes involved in the ubiquitin-proteasome system and in vacuolar degradation of damaged proteins showed hypersensitivity to n-butanol. Overexpression of YLR224W, encoding the subunit responsible for the recognition of damaged proteins of an ubiquitin ligase complex, resulted in a strain with a higher n-butanol tolerance. Two independently evolved n-butanol-tolerant strains carried different mutations in both RPN4 and RTG1, which encode transcription factors involved in the expression of proteasome and peroxisomal genes, respectively. Introduction of these mutated alleles in the reference strain increased butanol tolerance, confirming their relevance in the higher tolerance phenotype. The evolved strains, in addition to n-butanol, were also more tolerant to 2-butanol, isobutanol and 1-propanol, indicating a common molecular basis for sensitivity and tolerance to C3 and C4 alcohols. Conclusions This study shows that maintenance of protein integrity plays an essential role in butanol tolerance and demonstrates new promising targets to engineer S. cerevisiae for improved tolerance. PMID:23552365

Terminating protein ubiquitination: Hasta la vista, ubiquitin.

PubMed

Stringer, Daniel K; Piper, Robert C

2011-09-15

Ubiquitination is a post-translational modification that generally directs proteins for degradation by the proteasome or by lysosomes. However, ubiquitination has been implicated in many other cellular processes, including transcriptional regulation, DNA repair, regulation of protein-protein interactions and association with ubiquitin-binding scaffolds. Ubiquitination is a dynamic process. Ubiquitin is added to proteins by E3 ubiquitin ligases as a covalent modification to one or multiple lysine residues as well as non-lysine amino acids. Ubiquitin itself contains seven lysines, each of which can also be ubiquitinated, leading to polyubiquitin chains that are best characterized for linkages occurring through K48 and K63. Ubiquitination can also be reversed by the action of deubiquitination enzymes (DUbs). Like E3 ligases, DUbs play diverse and critical roles in cells. ( 1) Ubiquitin is expressed as a fusion protein, as a linear repeat or as a fusion to ribosomal subunits, and DUbs are necessary to liberate free ubiquitin, making them the first enzyme of the ubiquitin cascade. Proteins destined for degradation by the proteasome or by lysosomes are deubiquitinated prior to their degradation, which allows ubiquitin to be recycled by the cell, contributing to the steady-state pool of free ubiquitin. Proteins destined for degradation by lysosomes are also acted upon by both ligases and DUbs. Deubiquitination can also act as a means to prevent protein degradation, and many proteins are thought to undergo rounds of ubiquitination and deubiquitination, ultimately resulting in either the degradation or stabilization of those proteins. Despite years of study, examining the effects of the ubiquitination of proteins remains quite challenging. This is because the methods that are currently being employed to study ubiquitination are limiting. Here, we briefly examine current strategies to study the effects of ubiquitination and describe an additional novel approach that we have developed.

Characterizing the effect of Bortezomib on Rift Valley Fever Virus multiplication.

PubMed

Keck, Forrest; Amaya, Moushimi; Kehn-Hall, Kylene; Roberts, Brian; Bailey, Charles; Narayanan, Aarthi

2015-08-01

Rift Valley Fever Virus (RVFV) belongs to the family Bunyaviridae and is a known cause of epizootics and epidemics in Africa and the Middle East. With no FDA approved therapeutics available to treat RVFV infection, understanding the interactions between the virus and the infected host is crucial to developing novel therapeutic strategies. Here, we investigated the requirement of the ubiquitin-proteasome system (UPS) for the establishment of a productive RVFV infection. It was previously shown that the UPS plays a central role in RVFV multiplication involving degradation of PKR and p62 subunit of TFIIH. Using the FDA-approved proteasome inhibitor Bortezomib, we observed robust inhibition of intracellular and extracellular viral loads. Bortezomib treatment did not affect the nuclear/cytoplasmic distribution of the non-structural S-segment protein (NSs); however, the ability of NSs to form nuclear filaments was abolished as a result of Bortezomib treatment. In silico ubiquitination prediction analysis predicted that known NSs interactors (SAP30, YY1, and mSin3A) have multiple putative ubiquitination sites, while NSs itself was not predicted to be ubiquitinated. Immunoprecipitation studies indicated a decrease in interaction between SAP30 - NSs, and mSin3A - NSs in the context of Bortezomib treatment. This decrease in association between SAP30 - NSs also correlated with a decrease in the ubiquitination status of SAP30 with Bortezomib treatment. Bortezomib treatment, however, resulted in increased ubiquitination of mSin3A, suggesting that Bortezomib dynamically affects the ubiquitination status of host proteins that interact with NSs. Finally, we observed that expression of interferon beta (IFN-β) was increased in Bortezomib treated cells which indicated that the cellular antiviral mechanism was revived as a result of treatment and may contribute to control of viral multiplication. Copyright © 2015 Elsevier B.V. All rights reserved.

Expression of accessory colonization factor subunit A (ACFA) of Vibrio cholerae and ACFA fused to cholera toxin B subunit in transgenic tomato (Solanum lycopersicum).

PubMed

Sharma, Manoj Kumar; Jani, Dewal; Thungapathra, M; Gautam, J K; Meena, L S; Singh, Yogendra; Ghosh, Amit; Tyagi, Akhilesh Kumar; Sharma, Arun Kumar

2008-05-20

In earlier study from our group, cholera toxin B subunit had been expressed in tomato for developing a plant-based vaccine against cholera. In the present investigation, gene for accessory colonization factor (acf) subunit A, earlier reported to be essential for efficient colonization in the intestine, has been expressed in Escherichia coli as well as tomato plants. Gene encoding for a chimeric protein having a fusion of cholera toxin B subunit and accessory colonization factor A was also expressed in tomato to generate more potent combinatorial antigen. CaMV35S promoter with a duplicated enhancer sequence was used for expression of these genes in tomato. Integration of transgenes into tomato genome was confirmed by PCR and Southern hybridization. Expression of the genes was confirmed at transcript and protein levels. Accessory colonization factor A and cholera toxin B subunit fused to this protein accumulated up to 0.25% and 0.08% of total soluble protein, respectively, in the fruits of transgenic plants. Whereas protein purified from E. coli, in combination with cholera toxin B subunit can be used for development of conventional subunit vaccine, tomato fruits expressing these proteins can be used together with tomato plants expressing cholera toxin B subunit for development of oral vaccine against cholera.

Yeast Pah1p Phosphatidate Phosphatase Is Regulated by Proteasome-mediated Degradation*

PubMed Central

Pascual, Florencia; Hsieh, Lu-Sheng; Soto-Cardalda, Aníbal; Carman, George M.

2014-01-01

Yeast PAH1-encoded phosphatidate phosphatase is the enzyme responsible for the production of the diacylglycerol used for the synthesis of triacylglycerol that accumulates in the stationary phase of growth. Paradoxically, the growth phase-mediated inductions of PAH1 and phosphatidate phosphatase activity do not correlate with the amount of Pah1p; enzyme abundance declined in a growth phase-dependent manner. Pah1p from exponential phase cells was a relatively stable protein, and its abundance was not affected by incubation with an extract from stationary phase cells. Recombinant Pah1p was degraded upon incubation with the 100,000 × g pellet fraction of stationary phase cells, although the enzyme was stable when incubated with the same fraction of exponential phase cells. MG132, an inhibitor of proteasome function, prevented degradation of the recombinant enzyme. Endogenously expressed and plasmid-mediated overexpressed levels of Pah1p were more abundant in the stationary phase of cells treated with MG132. Pah1p was stabilized in mutants with impaired proteasome (rpn4Δ, blm10Δ, ump1Δ, and pre1 pre2) and ubiquitination (hrd1Δ, ubc4Δ, ubc7Δ, ubc8Δ, and doa4Δ) functions. The pre1 pre2 mutations that eliminate nearly all chymotrypsin-like activity of the 20 S proteasome had the greatest stabilizing effect on enzyme levels. Taken together, these results supported the conclusion that Pah1p is subject to proteasome-mediated degradation in the stationary phase. That Pah1p abundance was stabilized in pah1Δ mutant cells expressing catalytically inactive forms of Pah1p and dgk1Δ mutant cells with induced expression of DGK1-encoded diacylglycerol kinase indicated that alteration in phosphatidate and/or diacylglycerol levels might be the signal that triggers Pah1p degradation. PMID:24563465

The cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cho, Eun-Ah; Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr

2012-06-01

Highlights: Black-Right-Pointing-Pointer cAMP signaling system inhibits repair of {gamma}-ray-induced DNA damage. Black-Right-Pointing-Pointer cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. Black-Right-Pointing-Pointer cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. Black-Right-Pointing-Pointer The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNAmore » repair activity, and we investigated the effects of the cAMP signaling system on {gamma}-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (G{alpha}sQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of G{alpha}sQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after {gamma}-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2 Prime -O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2 Prime -O-Me-cAMP and restored XRCC1 protein level following {gamma}-ray irradiation. From these results, we conclude that the cAMP signaling system inhibits the repair of {gamma}-ray-induced DNA damage by promoting the ubiquitin-proteasome dependent degradation of XRCC1 in an Epac-dependent pathway in lung cancer cells.« less

Nonproteolytic Roles of 19S ATPases in Transcription of CIITApIV Genes

PubMed Central

Maganti, Nagini; Moody, Tomika D.; Truax, Agnieszka D.; Thakkar, Meghna; Spring, Alexander M.; Germann, Markus W.; Greer, Susanna F.

2014-01-01

Accumulating evidence shows the 26S proteasome is involved in the regulation of gene expression. We and others have demonstrated that proteasome components bind to sites of gene transcription, regulate covalent modifications to histones, and are involved in the assembly of activator complexes in mammalian cells. The mechanisms by which the proteasome influences transcription remain unclear, although prior observations suggest both proteolytic and non-proteolytic activities. Here, we define novel, non-proteolytic, roles for each of the three 19S heterodimers, represented by the 19S ATPases Sug1, S7, and S6a, in mammalian gene expression using the inflammatory gene CIITApIV. These 19S ATPases are recruited to induced CIITApIV promoters and also associate with CIITA coding regions. Additionally, these ATPases interact with elongation factor PTEFb complex members CDK9 and Hexim-1 and with Ser5 phosphorylated RNA Pol II. Both the generation of transcripts from CIITApIV and efficient recruitment of RNA Pol II to CIITApIV are negatively impacted by siRNA mediated knockdown of these 19S ATPases. Together, these results define novel roles for 19S ATPases in mammalian gene expression and indicate roles for these ATPases in promoting transcription processes. PMID:24625964

MURF2B, a Novel LC3-Binding Protein, Participates with MURF2A in the Switch between Autophagy and Ubiquitin Proteasome System during Differentiation of C2C12 Muscle Cells

PubMed Central

Pizon, Véronique; Rybina, Sofia; Gerbal, Fabien; Delort, Florence; Vicart, Patrick; Baldacci, Giuseppe; Karsenti, Eric

2013-01-01

The ubiquitin proteasome system and macroautophagy are proteolytic pathways essential in the maintenance of cellular homeostasis during differentiation and remodelling of skeletal muscle. In both pathways, proteins to be degraded are tagged with polyubiquitin. In skeletal muscles, the MURF2 proteins display E3 ubiquitin ligase structure suggesting that they may covalently attach ubiquitin polypeptides to still unknown target proteins. So far only MURF2A isoforms were studied and shown to interact with p62/SQSTM1, a protein implicated in macroautophagic and ubiquitin proteasome system degradations. Here, we analyzed the MURF2B and MURF2A proteins and show that the ratio of the isoforms changes during differentiation of muscle C2C12 cells and that the shift of the isoforms expression follows the sequential activation of autophagic or proteasomal degradation. We also show that MURF2B has a functional domain needed for its interaction with LC3, a protein needed for autophagic vesicles formation. Using specific MURF2 RNAi cells we observed that MURF2A and MURF2B are both needed for the formation of autophagosomes and that in the absence of MURF2B, the cells expressing MURF2A display an activated ubiquitin proteasome system implicated in the degradation of p62/SQSTM1 by UPS. Altogether, our results indicate that MURF2A and MURF2B proteins could participate in the molecular switch between the two ubiquitin degradative pathways. PMID:24124537

Effects of inhibition of ubiquitin-proteasome pathway on human primary leukemic cells.

PubMed

Lan, Yu; Zhang, Xuemin; Yang, Pingdi; Hu, Meiru; Yu, Ming; Yang, Yi; Shen, Beifen

2002-12-01

Though there were a lot of reports about the totally different responses to the inhibition of ubiquitin-proteasome pathway in different kinds of cell lines, much less has been known about the responses in primary human leukemic cells. In this study, the effects of inhibition of ubiquitin-proteasome pathway on human bone marrow (BM) mononuclear cells (MNCs) obtained from 10 normal persons and 8 leukemia patients were examined. The results showed that the responses obviously varied individually. Among them, BM MNCs in 3 cases of leukemic patients were extremely sensitive, demonstrated by that > 90% cells were induced to undergo apoptosis within 24 h, but MNCs in 10 cases of normal persons showed resistance to the inhibition and no apoptosis was observed. Furthermore, Western blots revealed that the Bcl-2 expression was relatively high in the sensitive primary leukemia cells, and especially the cleavage of 26 ku Bcl-2 into a 22 ku fragment occurred during the induction of apoptosis. In contrast, the Bcl-2 expression was either undetectable or detectable but no cleavage of that above was observed in the cells insensitive to the inhibition of the pathway (including BM MNCs in normal persons). Together with the observations on the leukemic cell lines, these findings suggested the correlation of the specific cleavage of Bcl-2 into a shortened fragment with the sensitivity of cells to the inhibition of ubiquitin-proteasome pathway, which provides clues to the further understanding of the mechanisms of that dramatically different responses existing in different kinds of cells to the inhibition of ubiquitin-proteasome pathway.

Proteasome inhibitors promote the sequestration of PrPSc into aggresomes within the cytosol of prion-infected CAD neuronal cells.

PubMed

Dron, Michel; Dandoy-Dron, Françoise; Farooq Salamat, Muhammad Khalid; Laude, Hubert

2009-08-01

Dysfunction of the endoplasmic reticulum associated protein degradation/proteasome system is believed to contribute to the initiation or aggravation of neurodegenerative disorders associated with protein misfolding, and there is some evidence to suggest that proteasome dysfunctions might be implicated in prion disease. This study investigated the effect of proteasome inhibitors on the biogenesis of both the cellular (PrP(C)) and abnormal (PrP(Sc)) forms of prion protein in CAD neuronal cells, a newly introduced prion cell system. In uninfected cells, proteasome impairment altered the intracellular distribution of PrP(C), leading to a strong accumulation in the Golgi apparatus. Moreover, a detergent-insoluble and weakly protease-resistant PrP species of 26 kDa, termed PrP(26K), accumulated in the cells, whether they were prion-infected or not. However, no evidence was found that, in infected cells, this PrP(26K) species converts into the highly proteinase K-resistant PrP(Sc). In the infected cultures, proteasome inhibition caused an increased intracellular aggregation of PrP(Sc) that was deposited into large aggresomes. These findings strengthen the view that, in neuronal cells expressing wild-type PrP(C) from the natural promoter, proteasomal impairment may affect both the process of PrP(C) biosynthesis and the subcellular sites of PrP(Sc) accumulation, despite the fact that these two effects could essentially be disconnected.

Experimental and Computational Analysis of Polyglutamine-Mediated Cytotoxicity

PubMed Central

Tang, Matthew Y.; Proctor, Carole J.; Woulfe, John; Gray, Douglas A.

2010-01-01

Expanded polyglutamine (polyQ) proteins are known to be the causative agents of a number of human neurodegenerative diseases but the molecular basis of their cytoxicity is still poorly understood. PolyQ tracts may impede the activity of the proteasome, and evidence from single cell imaging suggests that the sequestration of polyQ into inclusion bodies can reduce the proteasomal burden and promote cell survival, at least in the short term. The presence of misfolded protein also leads to activation of stress kinases such as p38MAPK, which can be cytotoxic. The relationships of these systems are not well understood. We have used fluorescent reporter systems imaged in living cells, and stochastic computer modeling to explore the relationships of polyQ, p38MAPK activation, generation of reactive oxygen species (ROS), proteasome inhibition, and inclusion body formation. In cells expressing a polyQ protein inclusion, body formation was preceded by proteasome inhibition but cytotoxicity was greatly reduced by administration of a p38MAPK inhibitor. Computer simulations suggested that without the generation of ROS, the proteasome inhibition and activation of p38MAPK would have significantly reduced toxicity. Our data suggest a vicious cycle of stress kinase activation and proteasome inhibition that is ultimately lethal to cells. There was close agreement between experimental data and the predictions of a stochastic computer model, supporting a central role for proteasome inhibition and p38MAPK activation in inclusion body formation and ROS-mediated cell death. PMID:20885783

Developmental regulation of N-methyl-D-aspartate- and kainate-type glutamate receptor expression in the rat spinal cord

NASA Technical Reports Server (NTRS)

Stegenga, S. L.; Kalb, R. G.

2001-01-01

Spinal motor neurons undergo experience-dependent development during a critical period in early postnatal life. It has been suggested that the repertoire of glutamate receptor subunits differs between young and mature motor neurons and contributes to this activity-dependent development. In the present study we examined the expression patterns of N-methyl-D-aspartate- and kainate-type glutamate receptor subunits during the postnatal maturation of the spinal cord. Young motor neurons express much higher levels of the N-methyl-D-aspartate receptor subunit NR1 than do adult motor neurons. Although there are eight potential splice variants of NR1, only a subgroup is expressed by motor neurons. With respect to NR2 receptor subunits, young motor neurons express NR2A and C, while adult motor neurons express only NR2A. Young motor neurons express kainate receptor subunits GluR5, 6 and KA2 but we are unable to detect these or any other kainate receptor subunits in the adult spinal cord. Other spinal cord regions display a distinct pattern of developmental regulation of N-methyl-D-aspartate and kainate receptor subunit expression in comparison to motor neurons. Our findings indicate a precise spatio-temporal regulation of individual subunit expression in the developing spinal cord. Specific combinations of subunits in developing neurons influence their excitable properties and could participate in the emergence of adult neuronal form and function.

Aerobic exercise training improves oxidative stress and ubiquitin proteasome system activity in heart of spontaneously hypertensive rats.

PubMed

de Andrade, Luiz Henrique Soares; de Moraes, Wilson Max Almeida Monteiro; Matsuo Junior, Eduardo Hiroshi; de Orleans Carvalho de Moura, Elizabeth; Antunes, Hanna Karen Moreira; Montemor, Jairo; Antonio, Ednei Luiz; Bocalini, Danilo Sales; Serra, Andrey Jorge; Tucci, Paulo José Ferreira; Brum, Patricia Chakur; Medeiros, Alessandra

2015-04-01

The activity of the ubiquitin proteasome system (UPS) and the level of oxidative stress contribute to the transition from compensated cardiac hypertrophy to heart failure in hypertension. Moreover, aerobic exercise training (AET) is an important therapy for the treatment of hypertension, but its effects on the UPS are not completely known. The aim of this study was to evaluate the effect of AET on UPS's activity and oxidative stress level in heart of spontaneously hypertensive rats (SHR). A total of 53 Wistar and SHR rats were randomly divided into sedentary and trained groups. The AET protocol was 5×/week in treadmill for 13 weeks. Exercise tolerance test, non-invasive blood pressure measurement, echocardiographic analyses, and left ventricle hemodynamics were performed during experimental period. The expression of ubiquitinated proteins, 4-hydroxynonenal (4-HNE), Akt, phospho-Akt(ser473), GSK3β, and phospho-GSK3β(ser9) were analyzed by western blotting. The evaluation of lipid hydroperoxide concentration was performed using the xylenol orange method, and the proteasomal chymotrypsin-like activity was measured by fluorimetric assay. Sedentary hypertensive group presented cardiac hypertrophy, unaltered expression of total Akt, phospho-Akt, total GSK3β and phospho-GSK3β, UPS hyperactivity, increased lipid hydroperoxidation as well as elevated expression of 4-HNE but normal cardiac function. In contrast, AET significantly increased exercise tolerance, decreased resting systolic blood pressure and heart rate in hypertensive animals. In addition, the AET increased phospho-Akt expression, decreased phospho-GSK3β, and did not alter the expression of total Akt, total GSK3β, and ubiquitinated proteins, however, significantly attenuated 4-HNE levels, lipid hydroperoxidation, and UPS's activity toward normotensive group levels. Our results provide evidence for the main effect of AET on attenuating cardiac ubiquitin proteasome hyperactivity and oxidative stress in SHR rats.

Proteasomal degradation of sphingosine kinase 1 and inhibition of dihydroceramide desaturase by the sphingosine kinase inhibitors, SKi or ABC294640, induces growth arrest in androgen-independent LNCaP-AI prostate cancer cells.

PubMed

McNaughton, Melissa; Pitman, Melissa; Pitson, Stuart M; Pyne, Nigel J; Pyne, Susan

2016-03-29

Sphingosine kinases (two isoforms termed SK1 and SK2) catalyse the formation of the bioactive lipid sphingosine 1-phosphate. We demonstrate here that the SK2 inhibitor, ABC294640 (3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide) or the SK1/SK2 inhibitor, SKi (2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole)) induce the proteasomal degradation of SK1a (Mr = 42 kDa) and inhibit DNA synthesis in androgen-independent LNCaP-AI prostate cancer cells. These effects are recapitulated by the dihydroceramide desaturase (Des1) inhibitor, fenretinide. Moreover, SKi or ABC294640 reduce Des1 activity in Jurkat cells and ABC294640 induces the proteasomal degradation of Des1 (Mr = 38 kDa) in LNCaP-AI prostate cancer cells. Furthermore, SKi or ABC294640 or fenretinide increase the expression of the senescence markers, p53 and p21 in LNCaP-AI prostate cancer cells. The siRNA knockdown of SK1 or SK2 failed to increase p53 and p21 expression, but the former did reduce DNA synthesis in LNCaP-AI prostate cancer cells. Moreover, N-acetylcysteine (reactive oxygen species scavenger) blocked the SK inhibitor-induced increase in p21 and p53 expression but had no effect on the proteasomal degradation of SK1a. In addition, siRNA knockdown of Des1 increased p53 expression while a combination of Des1/SK1 siRNA increased the expression of p21. Therefore, Des1 and SK1 participate in regulating LNCaP-AI prostate cancer cell growth and this involves p53/p21-dependent and -independent pathways. Therefore, we propose targeting androgen-independent prostate cancer cells with compounds that affect Des1/SK1 to modulate both de novo and sphingolipid rheostat pathways in order to induce growth arrest.

Proteasome inhibition blocks NF-κB and ERK1/2 pathways, restores antigen expression and sensitizes resistant human melanoma to TCR-engineered CTLs

PubMed Central

Jazirehi, Ali R.; Economou, James S.

2012-01-01

Adoptive cell transfer (ACT) of ex vivo engineered autologous lymphocytes encoding high-affinity MART-1/HLA-A*0201-specific T-cell receptor (TCR) α/β chains (F5 CTL), densely infiltrate into sites of metastatic disease, mediating dramatic but partial clinical responses in melanoma patients. We hypothesized that MART-1 down-modulation in addition to aberrant apoptotic/survival signaling could confer resistance to death signals delivered by transgenic CTLs. To explore this hypothesis, we established an in vitro model of resistant (R) lines from MART-1+/HLA-A*0201+ F5 CTL-sensitive parental (P) lines under serial F5 CTL-selective pressure. We have recently reported that several melanoma R lines, while retaining MART-1 expression, exhibited constitutive NF-κB activation and over-expression of NF-κB-dependent resistance factors. Another established melanoma cell line M244, otherwise sensitive to F5 CTL, yielded R lines after serial F5 CTL selective pressure which had both reduced MART-1 expression levels, thus, could not be recognized, and were resistant to CTL-delivered apoptotic death signals. The proteasome inhibitor bortezomib blocked NF-κB activity, decreased phopspho-ERK1/2, increased phospho-JNK levels, reduced expression of resistance-factors, restored MART-1 expression to sufficient levels, which in combination allowed M244R lines be sensitized to F5 CTL-killing. These findings suggest that proteasome inhibition in immune resistant tumors can restore proapoptotic signaling and improve tumor antigen expression. PMID:22532603

Rat nicotinic ACh receptor α7 and β2 subunits co-assemble to form functional heteromeric nicotinic receptor channels

PubMed Central

Khiroug, Serguei S; Harkness, Patricia C; Lamb, Patricia W; Sudweeks, Sterling N; Khiroug, Leonard; Millar, Neil S; Yakel, Jerrel L

2002-01-01

Rat hippocampal interneurons express diverse subtypes of functional nicotinic acetylcholine receptors (nAChRs), including α7-containing receptors that have properties unlike those expected for homomeric α7 nAChRs. We previously reported a strong correlation between expression of the α7 and of the β2 subunits in individual neurons. To explore whether co-assembly of the α7 and β2 subunits might occur, these subunits were co-expressed in Xenopus oocytes and the functional properties of heterologously expressed nAChRs were characterized by two-electrode voltage clamp. Co-expression of the β2 subunit, both wild-type and mutant forms, with the α7 subunit significantly slowed the rate of nAChR desensitization and altered the pharmacological properties. Whereas ACh, carbachol and choline were full or near-full agonists for homomeric α7 receptor channels, both carbachol and choline were only partial agonists in oocytes expressing both α7 and β2 subunits. In addition the EC50 values for all three agonists significantly increased when the β2 subunit was co-expressed with the α7 subunit. Co-expression with the β2 subunit did not result in any significant change in the current-voltage curve. Biochemical evidence for the co-assembly of the α7 and β2 subunits was obtained by co-immunoprecipitation of these subunits from transiently transfected human embryonic kidney (TSA201) cells. These data provide direct biophysical and molecular evidence that the nAChR α7 and β2 subunits co-assemble to form a functional heteromeric nAChR with functional and pharmacological properties different from those of homomeric α7 channels. This co-assembly may help to explain nAChR channel diversity in rat hippocampal interneurons, and perhaps in other areas of the nervous system. PMID:11956333

Cellular degradation activity is maintained during aging in long-living queen bees.

PubMed

Hsu, Chin-Yuan; Qiu, Jiantai Timothy; Chan, Yu-Pei

2016-11-01

Queen honeybees (Apis mellifera) have a much longer lifespan than worker bees. Whether cellular degradation activity is involved in the longevity of queen bees is unknown. In the present study, cellular degradation activity was evaluated in the trophocytes and oenocytes of young and old queen bees. The results indicated that (i) 20S proteasome activity and the size of autophagic vacuoles decreased with aging, and (ii) there were no significant differences between young and old queen bees with regard to 20S proteasome expression or efficiency, polyubiquitin aggregate expression, microtubule-associated protein 1 light chain 3-II (LC3-II) expression, 70 kDa heat shock cognate protein (Hsc70) expression, the density of autophagic vacuoles, p62/SQSTM1 expression, the activity or density of lysosomes, or molecular target of rapamycin expression. These results indicate that cellular degradation activity maintains a youthful status in the trophocytes and oenocytes of queen bees during aging and that cellular degradation activity is involved in maintaining the longevity of queen bees.

Celastrol, a triterpene extracted from the Chinese "Thunder of God Vine," is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice.

PubMed

Yang, Huanjie; Chen, Di; Cui, Qiuzhi Cindy; Yuan, Xiao; Dou, Q Ping

2006-05-01

Interest in the use of traditional medicines for cancer prevention and treatment is increasing. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as novel anticancer drugs. Celastrol, an active compound extracted from the root bark of the Chinese medicine "Thunder of God Vine" (Tripterygium wilfordii Hook F.), was used for years as a natural remedy for inflammatory conditions. Although Celastrol has been shown to induce leukemia cell apoptosis, the molecular target involved has not been identified. Furthermore, whether Celastrol has antitumor activity in vivo has never been conclusively shown. Here, we report, for the first time, that Celastrol potently and preferentially inhibits the chymotrypsin-like activity of a purified 20S proteasome (IC(50) = 2.5 micromol/L) and human prostate cancer cellular 26S proteasome (at 1-5 micromol/L). Inhibition of the proteasome activity by Celastrol in PC-3 (androgen receptor- or AR-negative) or LNCaP (AR-positive) cells results in the accumulation of ubiquitinated proteins and three natural proteasome substrates (IkappaB-alpha, Bax, and p27), accompanied by suppression of AR protein expression (in LNCaP cells) and induction of apoptosis. Treatment of PC-3 tumor-bearing nude mice with Celastrol (1-3 mg/kg/d, i.p., 1-31 days) resulted in significant inhibition (65-93%) of the tumor growth. Multiple assays using the animal tumor tissue samples from both early and end time points showed in vivo inhibition of the proteasomal activity and induction of apoptosis after Celastrol treatment. Our results show that Celastrol is a natural proteasome inhibitor that has a great potential for cancer prevention and treatment.

Changes in protein expression in testes of L2 strain Taiwan country chickens in response to acute heat stress.

PubMed

Wang, Shih-Han; Cheng, Chuen-Yu; Chen, Chao-Jung; Chen, Hsin-Hsin; Tang, Pin-Chi; Chen, Chih-Feng; Lee, Yen-Pai; Huang, San-Yuan

2014-07-01

Heat stress causes a decrease of fertility in roosters. Yet, the way acute heat stress affects protein expression remains poorly understood. This study investigated differential protein expression in testes of the L2 strain of Taiwan country chickens following acute heat stress. Twelve 45-week-old roosters were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2 hours of recovery, and with 6 hours of recovery. Testis samples were collected for morphologic assay and protein analysis. Some of the differentially expressed proteins were validated by Western blot and immunohistochemistry. Abnormal and apoptotic spermatogenic cells were observed at 2 hours of recovery after acute heat stress, especially among the spermatocytes. Two-dimensional difference gel electrophoresis revealed that 119 protein spots were differentially expressed in chicken testes following heat stress, and peptide mass fingerprinting revealed that these spots contained 92 distinct proteins. In the heat-stressed samples, the heat shock proteins, chaperonin containing t-complex, and proteasome subunits were downregulated, and glutathione S-transferase, transgelin, and DJ-1 were upregulated. Our results demonstrate that acute heat stress impairs the processes of translation, protein folding, and protein degradation, and thus results in apoptosis and interferes with spermatogenesis. On the other hand, the increased expression of antioxidant enzymes, including glutathione S-transferase and DJ-1, may attenuate heat-induced damage. These findings may have implications for breeding chickens that can tolerate more extreme conditions. Copyright © 2014 Elsevier Inc. All rights reserved.

Bromodomain and Extraterminal (BET) Protein Inhibition Suppresses Human T Cell Leukemia Virus 1 (HTLV-1) Tax Protein-mediated Tumorigenesis by Inhibiting Nuclear Factor κB (NF-κB) Signaling*

PubMed Central

Wu, Xuewei; Qi, Jun; Bradner, James E.; Xiao, Gutian; Chen, Lin-Feng

2013-01-01

The etiology of human T cell leukemia virus 1 (HTLV-1)-mediated adult T cell leukemia is associated with the ability of viral oncoprotein Tax to induce sustained NF-κB activation and the expression of many NF-κB target genes. Acetylation of the RelA subunit of NF-κB and the subsequent recruitment of bromodomain-containing factor Brd4 are important for the expression of NF-κB target genes in response to various stimuli. However, their contributions to Tax-mediated NF-κB target gene expression and tumorigenesis remain unclear. Here we report that Tax induced the acetylation of lysine 310 of RelA and the binding of Brd4 to acetylated RelA to facilitate Tax-mediated transcriptional activation of NF-κB. Depletion of Brd4 down-regulated Tax-mediated NF-κB target gene expression and cell proliferation. Inhibiting the interaction of Brd4 and acetylated RelA with the bromodomain extraterminal protein inhibitor JQ1 suppressed the proliferation of Tax-expressing rat fibroblasts and Tax-positive HTLV-1-infected cells and Tax-mediated cell transformation and tumorigenesis. Moreover, JQ1 attenuated the Tax-mediated transcriptional activation of NF-κB, triggering the polyubiquitination and proteasome-mediated degradation of constitutively active nuclear RelA. Our results identify Brd4 as a key regulator for Tax-mediated NF-κB gene expression and suggest that targeting epigenetic regulators such as Brd4 with the bromodomain extraterminal protein inhibitor might be a potential therapeutic strategy for cancers and other diseases associated with HTLV-1 infection. PMID:24189064

Phase I/II study of the novel proteasome inhibitor delanzomib (CEP-18770) for relapsed and refractory multiple myeloma.

PubMed

Vogl, Dan T; Martin, Thomas G; Vij, Ravi; Hari, Parameswaran; Mikhael, Joseph R; Siegel, David; Wu, Ka Lung; Delforge, Michel; Gasparetto, Cristina

2017-08-01

Delanzomib (CEP-18770), a reversible P2 threonine boronic acid proteasome (β5/β1 subunits) inhibitor that showed promising anti-myeloma effects in preclinical studies, was investigated in a single-agent multicenter phase I/II study in patients with relapsed/refractory myeloma. Sixty-one patients (17 during dose escalation; 44 in the expansion cohort) received delanzomib on days 1, 8, and 15 in 28-d cycles; 47 received the maximum tolerated dose (MTD), 2.1 mg/m 2 . Dose-limiting toxicities (DLTs) at 2.4 mg/m 2 were rash and thrombocytopenia. At the MTD, the most prominent adverse events were nausea, vomiting, anorexia, fatigue, and pyrexia; grade 3/4 thrombocytopenia and neutropenia occurred in 53 and 23% of patients, respectively. Peripheral neuropathy (21%) was limited to grades 1/2. At the MTD, 26 patients (55%) had stable disease and four (9%) had a partial response (PR). Median time to progression (TTP) was 2.5 months across the cohort. Based upon the efficacy results, development of delanzomib for myeloma was discontinued.

The Enigma of Tripeptidyl-Peptidase II: Dual Roles in Housekeeping and Stress

PubMed Central

Preta, Giulio; de Klark, Rainier; Gavioli, Riccardo; Glas, Rickard

2010-01-01

The tripeptidyl-peptidase II complex consists of repeated 138 kDa subunits, assembled into two twisted strands that form a high molecular weight complex (>5 MDa). TPPII, like many other cytosolic peptidases, plays a role in the ubiquitin-proteasome pathway downstream of the proteasome as well as in the production and destruction of MHC class I antigens and degradation of neuropeptides. Tripeptidyl-peptidase II activity is increased in cells with an increased demand for protein degradation, but whether degradation of cytosolic peptides is the only cell biological role for TPPII has remained unclear. Recent data indicated that TPPII translocates into the nucleus to control DNA damage responses in malignant cells, supporting that cytosolic “housekeeping peptidases” may have additional roles in cell biology, besides their contribution to protein turnover. Overall, TPPII has an emerging importance in several cancer-related fields, such as metabolism, cell death control, and control of genome integrity; roles that are not understood in detail. The present paper reviews the cell biology of TPPII and discusses distinct roles for TPPII in the nucleus and cytosol. PMID:20847939

High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources.

PubMed

Ausseil, Frederic; Samson, Arnaud; Aussagues, Yannick; Vandenberghe, Isabelle; Creancier, Laurent; Pouny, Isabelle; Kruczynski, Anna; Massiot, Georges; Bailly, Christian

2007-02-01

To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.

BAG3 down-modulation reduces anaplastic thyroid tumor growth by enhancing proteasome-mediated degradation of BRAF protein.

PubMed

Chiappetta, Gennaro; Basile, Anna; Arra, Claudio; Califano, Daniela; Pasquinelli, Rosa; Barbieri, Antonio; De Simone, Veronica; Rea, Domenica; Giudice, Aldo; Pezzullo, Luciano; De Laurenzi, Vincenzo; Botti, Gerardo; Losito, Simona; Conforti, Daniela; Turco, Maria Caterina

2012-01-01

Anaplastic thyroid tumors (ATC) express high levels of BAG3, a member of the BAG family of cochaperone proteins that is involved in regulating cell apoptosis through multiple mechanisms. The objective of the study was the investigation of the influence of B-cell lymphoma-2-associated athanogene 3 (BAG3) on ATC growth. We investigated the effects of BAG3 down-modulation, obtained by using a specific small interfering RNA, on in vitro and in vivo growth of the human ATC cell line 8505C. Because BRAF protein plays an important role in ATC cell growth, we analyzed the effects of BAG3 down-modulation on BRAF protein levels. Furthermore, by using a proteasome inhibitor, we verified whether BAG3-mediated regulation of BRAF levels involved a proteasome-dependent mechanism. BAG3 down-modulation significantly inhibits ATC growth in vitro and in vivo. BAG3 coimmunoprecipitates with BRAF protein, and its down-modulation results in a significant reduction of BRAF protein levels, which can be reverted by incubation with the proteasome inhibitor MG132. BAG3 protein sustains ATC growth in vitro and in vivo. The underlying molecular mechanism appears to rely on BAG3 binding to BRAF, thus protecting it from proteasome-dependent degradation. These results are in line with the reported ability of BAG3 to interfere with the proteasomal delivery of a number of other client proteins.

Placing a Disrupted Degradation Motif at the C Terminus of Proteasome Substrates Attenuates Degradation without Impairing Ubiquitylation*

PubMed Central

Alfassy, Omri S.; Cohen, Itamar; Reiss, Yuval; Tirosh, Boaz; Ravid, Tommer

2013-01-01

Protein elimination by the ubiquitin-proteasome system requires the presence of a cis-acting degradation signal. Efforts to discern degradation signals of misfolded proteasome substrates thus far revealed a general mechanism whereby the exposure of cryptic hydrophobic motifs provides a degradation determinant. We have previously characterized such a determinant, employing the yeast kinetochore protein Ndc10 as a model substrate. Ndc10 is essentially a stable protein that is rapidly degraded upon exposure of a hydrophobic motif located at the C-terminal region. The degradation motif comprises two distinct and essential elements: DegA, encompassing two amphipathic helices, and DegB, a hydrophobic sequence within the loosely structured C-terminal tail of Ndc10. Here we show that the hydrophobic nature of DegB is irrelevant for the ubiquitylation of substrates containing the Ndc10 degradation motif, but is essential for proteasomal degradation. Mutant DegB, in which the hydrophobic sequence was disrupted, acted as a dominant degradation inhibitory element when expressed at the C-terminal regions of ubiquitin-dependent and -independent substrates of the 26S proteasome. This mutant stabilized substrates in both yeast and mammalian cells, indicative of a modular recognition moiety. The dominant function of the mutant DegB provides a powerful experimental tool for evaluating the physiological implications of stabilization of specific proteasome substrates in intact cells and for studying the associated pathological effects. PMID:23519465

Ursolic acid facilitates apoptosis in rheumatoid arthritis synovial fibroblasts by inducing SP1-mediated Noxa expression and proteasomal degradation of Mcl-1.

PubMed

Kim, Eugene Y; Sudini, Kuladeep; Singh, Anil K; Haque, Mahamudul; Leaman, Douglas; Khuder, Sadik; Ahmed, Salahuddin

2018-05-25

Rheumatoid arthritis (RA) is characterized by hyperplastic pannus formation mediated by activated synovial fibroblasts (RASFs) that cause joint destruction. We have shown earlier that RASFs exhibit resistance to apoptosis, primarily as a result of enhanced expression of myeloid cell leukemia-1 (Mcl-1). In this study, we discovered that ursolic acid (UA), a plant-derived pentacyclic triterpenoid, selectively induces B-cell lymphoma 2 homology 3-only protein Noxa in human RASFs. We observed that UA-induced Noxa expression was followed by a consequent decrease in Mcl-1 expression in a dose-dependent manner. Subsequent evaluation of the signaling pathways showed that UA-induced Noxa is primarily mediated by the JNK pathway in human RASFs. Chromatin immunoprecipitation (IP) studies into the promoter region of Noxa indicated the role of transcription factor specificity protein 1 in JNK-mediated Noxa expression. Furthermore, the results from IP studies and proximity ligation assays indicated that UA-induced Noxa colocalizes and associates with Mcl-1 to prime it for proteasomal degradation through K 48 -linked ubiquitination by the selective recruitment of Mcl-1 ubiquitin ligase E3, a homologous to E6-associated protein C terminus domain-containing E3 ubiquitin ligase. These findings unveil a novel mechanism of inducing apoptosis in RASFs and a potential adjunct therapeutic strategy of regulating synovial hyperplasia in RA.-Kim, E. Y., Sudini, K., Singh, A. K., Haque, M., Leaman, D., Khuder, S., Ahmed, S. Ursolic acid facilitates apoptosis in rheumatoid arthritis synovial fibroblasts by inducing SP1-mediated Noxa expression and proteasomal degradation of Mcl-1.

Tumors acquire inhibitor of apoptosis protein (IAP)-mediated apoptosis resistance through altered specificity of cytosolic proteolysis.

PubMed

Hong, Xu; Lei, Lu; Glas, Rickard

2003-06-16

Many tumors overexpress members of the inhibitor of apoptosis protein (IAP) family. IAPs contribute to tumor cell apoptosis resistance by the inhibition of caspases, and are degraded by the proteasome to allow further progression of apoptosis. Here we show that tumor cells can alter the specificity of cytosolic proteolysis in order to acquire apoptosis resistance, which promotes formation of rapidly growing tumors. Survival of tumor cells with low proteasomal activity can occur in the presence of high expression of Tri-peptidyl-peptidase II (TPP II), a large subtilisin-like peptidase that complements proteasomal activity. We find that this state leaves tumor cells unable of effectively degrading IAPs, and that cells in this state form rapidly growing tumors in vivo. We also find, in studies of apoptosis resistant cells derived from large in vivo tumors, that these have acquired an altered peptidase activity, with up-regulation of TPP II activity and decreased proteasomal activity. Importantly, we find that growth of subcutaneous tumors is limited by maintenance of the apoptosis resistant phenotype. The apoptosis resistant phenotype was reversed by increased expression of Smac/DIABLO, an antagonist of IAP molecules. Our data suggest a reversible mechanism in regulation of apoptosis resistance that drives tumor progression in vivo. These data are relevant in relation to the multitude of therapy-resistant clinical tumors that have increased levels of IAP molecules.

Quantitative Proteomics of the Root of Transgenic Wheat Expressing TaBWPR-1.2 Genes in Response to Waterlogging.

PubMed

Haque, Emdadul; Abe, Fumitaka; Mori, Masahiko; Nanjo, Yohei; Komatsu, Setsuko; Oyanagi, Atsushi; Kawaguchi, Kentaro

2014-11-04

Once candidate genes are available, the application of genetic transformation plays a major part to study their function in plants for adaptation to respective environmental stresses, including waterlogging (WL). The introduction of stress-inducible genes into wheat remains difficult because of low transformation and plant regeneration efficiencies and expression variability and instability. Earlier, we found two cDNAs encoding WL stress-responsive wheat pathogenesis-related proteins 1.2 ( TaBWPR-1.2 ), TaBWPR-1.2#2 and TaBWPR-1.2# 13. Using microprojectile bombardment, both cDNAs were introduced into "Bobwhite". Despite low transformation efficiency, four independent T₂ homozygous lines for each gene were isolated, where transgenes were ubiquitously and variously expressed. The highest transgene expression was obtained in Ubi: TaBWPR-1.2#2 L#11a and Ubi:TaBWPR-1.2#13 L#4a. Using quantitative proteomics, the root proteins of L#11a were analyzed to explore possible physiological pathways regulated by TaBWPR-1.2 under normal and waterlogged conditions. In L#11a, the abundance of proteasome subunit alpha type-3 decreased under normal conditions, whereas that of ferredoxin precursor and elongation factor-2 increased under waterlogged conditions in comparison with normal plants. Proteomic results suggest that L#11a is one of the engineered wheat plants where TaBWPR-1.2#2 is most probably involved in proteolysis, protein synthesis and alteration in the energy pathway in root tissues via the above proteins in order to gain metabolic adjustment to WL.

Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes

PubMed Central

Schreiner, Sabrina; Bürck, Carolin; Glass, Mandy; Groitl, Peter; Wimmer, Peter; Kinkley, Sarah; Mund, Andreas; Everett, Roger D.; Dobner, Thomas

2013-01-01

Death domain–associated protein (Daxx) cooperates with X-linked α-thalassaemia retardation syndrome protein (ATRX), a putative member of the sucrose non-fermentable 2 family of ATP-dependent chromatin-remodelling proteins, acting as the core ATPase subunit in this complex, whereas Daxx is the targeting factor, leading to histone deacetylase recruitment, H3.3 deposition and transcriptional repression of cellular promoters. Despite recent findings on the fundamental importance of chromatin modification in host-cell gene regulation, it remains unclear whether adenovirus type 5 (Ad5) transcription is regulated by cellular chromatin remodelling to allow efficient virus gene expression. Here, we focus on the repressive role of the Daxx/ATRX complex during Ad5 replication, which depends on intact protein–protein interaction, as negative regulation could be relieved with a Daxx mutant that is unable to interact with ATRX. To ensure efficient viral replication, Ad5 E1B-55K protein inhibits Daxx and targets ATRX for proteasomal degradation in cooperation with early region 4 open reading frame protein 6 and cellular components of a cullin-dependent E3-ubiquitin ligase. Our studies illustrate the importance and diversity of viral factors antagonizing Daxx/ATRX-mediated repression of viral gene expression and shed new light on the modulation of cellular chromatin remodelling factors by Ad5. We show for the first time that cellular Daxx/ATRX chromatin remodelling complexes play essential roles in Ad gene expression and illustrate the importance of early viral proteins to counteract cellular chromatin remodelling. PMID:23396441

Human Sex Determination at the Edge of Ambiguity: INHERITED XY SEX REVERSAL DUE TO ENHANCED UBIQUITINATION AND PROTEASOMAL DEGRADATION OF A MASTER TRANSCRIPTION FACTOR.

PubMed

Racca, Joseph D; Chen, Yen-Shan; Yang, Yanwu; Phillips, Nelson B; Weiss, Michael A

2016-10-14

A general problem is posed by analysis of transcriptional thresholds governing cell fate decisions in metazoan development. A model is provided by testis determination in therian mammals. Its key step, Sertoli cell differentiation in the embryonic gonadal ridge, is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in human SRY cause gonadal dysgenesis leading to XY female development (Swyer syndrome). Here, we have characterized an inherited mutation compatible with either male or female somatic phenotypes as observed in an XY father and XY daughter, respectively. The mutation (a crevice-forming substitution at a conserved back surface of the SRY high mobility group box) markedly destabilizes the domain but preserves specific DNA affinity and induced DNA bend angle. On transient transfection of diverse human and rodent cell lines, the variant SRY exhibited accelerated proteasomal degradation (relative to wild type) associated with increased ubiquitination; in vitro susceptibility to ubiquitin-independent ("default") cleavage by the 20S core proteasome was unchanged. The variant's gene regulatory activity (as assessed in a cellular model of the rat embryonic XY gonadal ridge) was reduced by 2-fold relative to wild-type SRY at similar levels of mRNA expression. Chemical proteasome inhibition restored native-like SRY expression and transcriptional activity in association with restored occupancy of a sex-specific enhancer element in principal downstream gene Sox9, demonstrating that the variant SRY exhibits essentially native activity on a per molecule basis. Our findings define a novel mechanism of impaired organogenesis, accelerated ubiquitin-directed proteasomal degradation of a master transcription factor leading to a developmental decision poised at the edge of ambiguity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Kanako; Department of Anesthesiology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501; Kanno, Takeshi

2008-03-07

The present study investigated proliferation of MKN28 and MKN45 human gastric cancer cells regulated by the N-methyl-D-aspartate (NMDA) receptor subunit. The NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (AP5) inhibited proliferation of MKN45 cells, but not MKN28 cells. Of the NMDA subunits such as NR1, NR2 (2A, 2B, 2C, and 2D), and NR3 (3A and 3B), all the NMDA subunit mRNAs except for the NR2B subunit mRNA were expressed in both MKN28 and MKN45 cells. MKN45 cells were characterized by higher expression of the NR2A subunit mRNA and lower expression of the NR1 subunit mRNA, but MKN28 otherwise by higher expression ofmore » the NR1 subunit mRNA and lower expression of the NR2A subunit mRNA. MKN45 cell proliferation was also inhibited by silencing the NR2A subunit-targeted gene. For MKN45 cells, AP5 or knocking-down the NR2A subunit increased the proportion of cells in the G{sub 1} phase of cell cycling and decreased the proportion in the S/G{sub 2} phase. The results of the present study, thus, suggest that blockage of NMDA receptors including the NR2A subunit suppresses MKN45 cell proliferation due to cell cycle arrest at the G{sub 1} phase; in other words, the NR2A subunit promotes MKN45 cell proliferation by accelerating cell cycling.« less

Association with β-COP Regulates the Trafficking of the Newly Synthesized Na,K-ATPase*

PubMed Central

Morton, Michael J.; Farr, Glen A.; Hull, Michael; Capendeguy, Oihana; Horisberger, Jean-Daniel; Caplan, Michael J.

2010-01-01

Plasma membrane expression of the Na,K-ATPase requires assembly of its α- and β-subunits. Using a novel labeling technique to identify Na,K-ATPase partner proteins, we detected an interaction between the Na,K-ATPase α-subunit and the coat protein, β-COP, a component of the COP-I complex. When expressed in the absence of the Na,K-ATPase β-subunit, the Na,K-ATPase α-subunit interacts with β-COP, is retained in the endoplasmic reticulum, and is targeted for degradation. In the presence of the Na,K-ATPase β-subunit, the α-subunit does not interact with β-COP and traffics to the plasma membrane. Pulse-chase experiments demonstrate that in cells expressing both the Na,K-ATPase α- and β-subunits, newly synthesized α-subunit associates with β-COP immediately after its synthesis but that this interaction does not constitute an obligate intermediate in the assembly of the α- and β-subunits to form the pump holoenzyme. The interaction with β-COP was reduced by mutating a dibasic motif at Lys54 in the Na,K-ATPase α-subunit. This mutant α-subunit is not retained in the endoplasmic reticulum and reaches the plasma membrane, even in the absence of Na,K-ATPase β-subunit expression. Although the Lys54 α-subunit reaches the cell surface without need for β-subunit assembly, it is only functional as an ion-transporting ATPase in the presence of the β-subunit. PMID:20801885

NSs protein of rift valley fever virus promotes posttranslational downregulation of the TFIIH subunit p62.

PubMed

Kalveram, Birte; Lihoradova, Olga; Ikegami, Tetsuro

2011-07-01

Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus.

Bortezomib as a new therapeutic approach for blastic plasmacytoid dendritic cell neoplasm

PubMed Central

Philippe, Laure; Ceroi, Adam; Bôle-Richard, Elodie; Jenvrin, Alizée; Biichle, Sabeha; Perrin, Sophie; Limat, Samuel; Bonnefoy, Francis; Deconinck, Eric; Saas, Philippe; Garnache-Ottou, Francine; Angelot-Delettre, Fanny

2017-01-01

Blastic plasmacytoid dendritic cell neoplasm is an aggressive hematologic malignancy with a poor prognosis. No consensus regarding optimal treatment modalities is currently available. Targeting the nuclear factor-kappa B pathway is considered a promising approach since blastic plasmacytoid dendritic cell neoplasm has been reported to exhibit constitutive activation of this pathway. Moreover, nuclear factor-kappa B inhibition in blastic plasmacytoid dendritic cell neoplasm cell lines, achieved using either an experimental specific inhibitor JSH23 or the clinical drug bortezomib, interferes in vitro with leukemic cell proliferation and survival. Here we extended these data by showing that primary blastic plasmacytoid dendritic cell neoplasm cells from seven patients were sensitive to bortezomib-induced cell death. We confirmed that bortezomib efficiently inhibits the phosphorylation of the RelA nuclear factor-kappa B subunit in blastic plasmacytoid dendritic cell neoplasm cell lines and primary cells from patients in vitro and in vivo in a mouse model. We then demonstrated that bortezomib can be associated with other drugs used in different chemotherapy regimens to improve its impact on leukemic cell death. Indeed, when primary blastic plasmacytoid dendritic cell neoplasm cells from a patient were grafted into mice, bortezomib treatment significantly increased the animals’ survival, and was associated with a significant decrease of circulating leukemic cells and RelA nuclear factor-kappa B subunit expression. Overall, our results provide a rationale for the use of bortezomib in combination with other chemotherapy for the treatment of patients with blastic plasmacytoid dendritic cell neoplasm. Based on our data, a prospective clinical trial combining proteasome inhibitor with classical drugs could be envisaged. PMID:28798071

Hinokitiol inhibits vasculogenic mimicry activity of breast cancer stem/progenitor cells through proteasome-mediated degradation of epidermal growth factor receptor

PubMed Central

TU, DOM-GENE; YU, YUN; LEE, CHE-HSIN; KUO, YU-LIANG; LU, YIN-CHE; TU, CHI-WEN; CHANG, WEN-WEI

2016-01-01

Hinokitiol, alternatively known as β-thujaplicin, is a tropolone-associated natural compound with antimicrobial, anti-inflammatory and antitumor activity. Breast cancer stem/progenitor cells (BCSCs) are a subpopulation of breast cancer cells associated with tumor initiation, chemoresistance and metastatic behavior, and may be enriched by mammosphere cultivation. Previous studies have demonstrated that BCSCs exhibit vasculogenic mimicry (VM) activity via the epidermal growth factor receptor (EGFR) signaling pathway. The present study investigated the anti-VM activity of hinokitiol in BCSCs. At a concentration below the half maximal inhibitory concentration, hinokitiol inhibited VM formation of mammosphere cells derived from two human breast cancer cell lines. Hinokitiol was additionally indicated to downregulate EGFR protein expression in mammosphere-forming BCSCs without affecting the expression of messenger RNA. The protein stability of EGFR in BCSCs was also decreased by hinokitiol. The EGFR protein expression and VM formation capability of hinokitiol-treated BCSCs were restored by co-treatment with MG132, a proteasome inhibitor. In conclusion, the present study indicated that hinokitiol may inhibit the VM activity of BCSCs through stimulating proteasome-mediated EGFR degradation. Hinokitiol may act as an anti-VM agent, and may be useful for the development of novel breast cancer therapeutic agents. PMID:27073579

Change in the Content of Immunoproteasomes and Macrophages in Rat Liver At the Induction of Donor-Specific Tolerance.

PubMed

Karpova, Ya D; Ustichenko, V D; Alabedal'karim, N M; Stepanova, A A; Lyupina, Yu V; Boguslavski, K I; Bozhok, G A; Sharova, N P

2017-01-01

Induction of donor specific tolerance (DST) by the introduction of donor cells into a recipient's portal vein is one of the approaches used to solve the problem of transplant engraftment. However, the mechanism of DST development remains unclear to this moment. In the present work, we first studied the change in the content of immunoproteasomes and macrophages of the liver at early stages of the development of allospecific portal tolerance in rats by Western blotting and flow cytofluorimetry. On the basis of the data obtained, we can conclude that the induction of DST is an active process characterized by two phases during which the level of the proteasome immune subunits LMP2 and LMP7 in liver mononuclear cells, including Kupffer cells, and the number of Kupffer cells change. The first phase lasts up to 5 days after the beginning of DST induction; the second phase - from 5 to 14 days. In both phases, the level of the subunits LMP2 and LMP7 in the total pool of mononuclear cells and Kupffer cells increases, with maximum values on days 1 and 7. In addition, the total number of Kupffer cells increases in both phases with a shift in several days. The most noticeable changes take place in the second phase. The third day is characterized by a lower content of mononuclear cells expressing immunoproteasomes compared to the control value in native animals. Presumably, at this time point a "window of opportunity" appears for subsequent filling of an empty niche with cells of different subpopulations and, depending on this fact, the development of tolerance or rejection. The results obtained raise the new tasks of finding ways to influence the cellular composition in the liver and the expression of immunoproteasomes on the third day after the beginning of DST induction to block the development of rejection.

Down regulated expression of the beta1 subunit of the big-conductance Ca2+ sensitive K+ channel in sphincter of Oddi cells from rabbits fed with a high cholesterol diet.

PubMed

Du, Pang; Cui, Guang-Bin; Wang, Ya-Rong; Zhang, Xiao-Yong; Ma, Ke-Jun; Wei, Jing-Guo

2006-12-01

Hypercholesterolemia, which is closely related to gallbladder bile stasis, can cause sphincter of Oddi dysfunction (SOD) by increasing the tension of sphincter of Oddi (SO). Intracellular calcium ion concentration ([Ca(2+)](i)) could influence the tension of SO. The beta1 subunit of the big-conductance Ca(2+) sensitive K(+) channel (BK(Ca)) can enhance the sensitivity of the BK(Ca) channel to [Ca(2+)](i). Absence and decline of the BKCa channel subunit beta1 could lead to many diseases. However, the relationship between hypercholesterolemia and the expression of beta1 subunit is not well understood. In this study, we successfully expressed and purified the rabbit BK(Ca) beta1 subunit protein and prepared its polyclonal antibody. The specificity of the prepared antibody was determined by Western blotting. A SOD rabbit model induced by a high cholesterol diet was established and the expression of the beta1 subunit of SO was determined by immunohistochemical staining and western blotting. Compared with the controls, our results demonstrated that hypercholesterolemia could decrease the expression of the beta1 subunit in the SO cells from rabbits. This indicates that lower expression of BKCa channel beta1 subunit might induce SOD.

Decreased surface expression of the δ subunit of the GABAA receptor contributes to reduced tonic inhibition in dentate granule cells in a mouse model of fragile X syndrome.

PubMed

Zhang, Nianhui; Peng, Zechun; Tong, Xiaoping; Lindemeyer, A Kerstin; Cetina, Yliana; Huang, Christine S; Olsen, Richard W; Otis, Thomas S; Houser, Carolyn R

2017-11-01

While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABA A receptors (GABA A Rs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABA A R, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABA A Rs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with immunolabeling at perisynaptic locations in Fmr1 KO mice. While α4 immunogold particles were also reduced at perisynaptic locations in the Fmr1 KO mice, the labeling was increased at synaptic sites. Together these findings suggest that, in the dentate gyrus, altered surface expression of the δ subunit, rather than a decrease in δ subunit expression alone, could be limiting δ subunit-mediated tonic inhibition in this model of FXS. Finding ways to increase surface expression of the δ subunit of the GABA A R could be a novel approach to treatment of hyperexcitability-related alterations in FXS. Copyright © 2017 Elsevier Inc. All rights reserved.

5-Hydroxy-3,6,7,8,3'4'-hexamethoxyflavone inhibits nitric oxide production in lipopolysaccharide-stimulated BV2 microglia via NF-κB suppression and Nrf-2-dependent heme oxygenase-1 induction.

PubMed

Kang, Chang-Hee; Kim, Min Jeong; Seo, Min Jeong; Choi, Yung Hyun; Jo, Wol Soon; Lee, Kyung-Tae; Jeong, Yong Kee; Kim, Gi-Young

2013-07-01

In this study, we found that 5-hydroxy-3,6,7,8,3'4'-hexamethoxyflavone (5HHMF) from Hizikia fusiforme considerably inhibits lipopolysaccharide (LPS)-stimulated NO production by suppressing the expression of inducible NO synthase (iNOS) in BV2 microglia. In addition, 5HHMF blocked LPS-induced phosphorylation of IκB, resulting in suppression of the nuclear translocation of nuclear factor-κB (NF-κB) subunits, namely p65 and p50, which are important molecules involved in the regulation of iNOS expression. Pyrrolidine dithiocarbamate (PDTC), a specific NF-κB inhibitor, along with 20S proteasome inhibitor (PSI) significantly inhibited LPS-induced iNOS expression, which indirectly suggested that 5HHMF downregulated iNOS expression by suppressing NF-κB activity. Thus, we found that 5HHMF enhances heme oxygenase-1 (HO-1) expression via nuclear factor-erythroid 2-related factor 2 (Nrf2) activation. In addition, cobalt protoporphyrin (CoPP), a specific HO-1 inducer, predominantly suppressed LPS-induced NO production. In contrast, zinc protoporphyrin (ZnPP), a specific HO-1 inhibitor, showed a partial suppressive effect of 5HHMF on LPS-induced NO production. Further, 5HHMF increased specific DNA-binding activity of Nrf2, and transient knockdown with Nrf2 siRNA subsequently reversed 5HHMF-induced NO inhibition, which was followed by suppression of HO-1 activity. Taken together, our findings indicate that 5HHMF suppresses NO production through modulation of iNOS, consequently suppressing NF-κB activity and induction of Nrf2-dependent HO-1 activity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Differential plasma membrane targeting of voltage-dependent calcium channel subunits expressed in a polarized epithelial cell line

PubMed Central

Brice, Nicola L; Dolphin, Annette C

1999-01-01

Voltage-dependent calcium channels (VDCCs) show a highly non-uniform distribution in many cell types, including neurons and other polarized secretory cells. We have examined whether this can be mimicked in a polarized epithelial cell line (Madin-Darby canine kidney), which has been used extensively to study the targeting of proteins. We expressed the VDCC α1A, α1B or α1C subunits either alone or in combination with accessory subunits α2-δ and the different β subunits, and examined their localization immunocytochemically. An α1 subunit was only targeted to the plasma membrane if co-expressed with the accessory subunits. The combination α1C/α2-δ and all β subunits was always localized predominantly to the basolateral membrane. It has been suggested that this is equivalent to somatodendritic targeting in neurons. In contrast, the α1B subunit was expressed at the apical membrane with all the accessory subunit combinations, by 24 h after microinjection. This membrane destination shows some parallels with axonal targeting in neurons. The α1A subunit was consistently observed at the apical membrane in the combinations α1A/α2-δ/β1b or β4. In contrast, when co-expressed with α2-δ/β2a, α1A was clearly targeted to the basolateral membrane. In conclusion, the VDCC α1 subunit appears to be the primary determinant for targeting the VDCC complex, but the β subunit can modify this destination, particularly for α1A. PMID:10066897

Targeted polypeptide degradation

DOEpatents

Church, George M [Brookline, MA; Janse, Daniel M [Brookline, MA

2008-05-13

This invention pertains to compositions, methods, cells and organisms useful for selectively localizing polypeptides to the proteasome for degradation. Therapeutic methods and pharmaceutical compositions for treating disorders associated with the expression and/or activity of a polypeptide by targeting these polypeptides for degradation, as well as methods for targeting therapeutic polypeptides for degradation and/or activating therapeutic polypeptides by degradation are provided. The invention provides methods for identifying compounds that mediate proteasome localization and/or polypeptide degradation. The invention also provides research tools for the study of protein function.

Release of skeletal muscle peptide fragments identifies individual proteins degraded during insulin deprivation in type 1 diabetic humans and mice.

PubMed

Robinson, Matthew M; Dasari, Surendra; Karakelides, Helen; Bergen, H Robert; Nair, K Sreekumaran

2016-09-01

Insulin regulates skeletal muscle protein degradation, but the types of proteins being degraded in vivo remain to be determined due to methodological limitations. We present a method to assess the types of skeletal muscle proteins that are degraded by extracting their degradation products as low-molecular weight (LMW) peptides from muscle samples. High-resolution mass spectrometry was used to identify the original intact proteins that generated the LMW peptides, which we validated in rodents and then applied to humans. We deprived insulin from insulin-treated streptozotocin (STZ) diabetic mice for 6 and 96 h and for 8 h in type 1 diabetic humans (T1D) for comparison with insulin-treated conditions. Protein degradation was measured using activation of autophagy and proteasome pathways, stable isotope tracers, and LMW approaches. In mice, insulin deprivation activated proteasome pathways and autophagy in muscle homogenates and isolated mitochondria. Reproducibility analysis of LMW extracts revealed that ∼80% of proteins were detected consistently. As expected, insulin deprivation increased whole body protein turnover in T1D. Individual protein degradation increased with insulin deprivation, including those involved in mitochondrial function, proteome homeostasis, nDNA support, and contractile/cytoskeleton. Individual mitochondrial proteins that generated more LMW fragment with insulin deprivation included ATP synthase subunit-γ (+0.5-fold, P = 0.007) and cytochrome c oxidase subunit 6 (+0.305-fold, P = 0.03). In conclusion, identifying LMW peptide fragments offers an approach to determine the degradation of individual proteins. Insulin deprivation increases degradation of select proteins and provides insight into the regulatory role of insulin in maintaining proteome homeostasis, especially of mitochondria. Copyright © 2016 the American Physiological Society.

Release of skeletal muscle peptide fragments identifies individual proteins degraded during insulin deprivation in type 1 diabetic humans and mice

PubMed Central

Robinson, Matthew M.; Dasari, Surendra; Karakelides, Helen; Bergen, H. Robert

2016-01-01

Insulin regulates skeletal muscle protein degradation, but the types of proteins being degraded in vivo remain to be determined due to methodological limitations. We present a method to assess the types of skeletal muscle proteins that are degraded by extracting their degradation products as low-molecular weight (LMW) peptides from muscle samples. High-resolution mass spectrometry was used to identify the original intact proteins that generated the LMW peptides, which we validated in rodents and then applied to humans. We deprived insulin from insulin-treated streptozotocin (STZ) diabetic mice for 6 and 96 h and for 8 h in type 1 diabetic humans (T1D) for comparison with insulin-treated conditions. Protein degradation was measured using activation of autophagy and proteasome pathways, stable isotope tracers, and LMW approaches. In mice, insulin deprivation activated proteasome pathways and autophagy in muscle homogenates and isolated mitochondria. Reproducibility analysis of LMW extracts revealed that ∼80% of proteins were detected consistently. As expected, insulin deprivation increased whole body protein turnover in T1D. Individual protein degradation increased with insulin deprivation, including those involved in mitochondrial function, proteome homeostasis, nDNA support, and contractile/cytoskeleton. Individual mitochondrial proteins that generated more LMW fragment with insulin deprivation included ATP synthase subunit-γ (+0.5-fold, P = 0.007) and cytochrome c oxidase subunit 6 (+0.305-fold, P = 0.03). In conclusion, identifying LMW peptide fragments offers an approach to determine the degradation of individual proteins. Insulin deprivation increases degradation of select proteins and provides insight into the regulatory role of insulin in maintaining proteome homeostasis, especially of mitochondria. PMID:27436610

Interaction of Ddc1 and RPA with single-stranded/double-stranded DNA junctions in yeast whole cell extracts: Proteolytic degradation of the large subunit of replication protein A in ddc1Δ strains.

PubMed

Sukhanova, Maria V; D'Herin, Claudine; Boiteux, Serge; Lavrik, Olga I

2014-10-01

To characterize proteins that interact with single-stranded/double-stranded (ss/ds) DNA junctions in whole cell free extracts of Saccharomyces cerevisiae, we used [(32)P]-labeled photoreactive partial DNA duplexes containing a 3'-ss/ds-junction (3'-junction) or a 5'-ss/ds-junction (5'-junction). Identification of labeled proteins was achieved by MALDI-TOF mass spectrometry peptide mass fingerprinting and genetic analysis. In wild-type extract, one of the components of the Ddc1-Rad17-Mec3 complex, Ddc1, was found to be preferentially photocrosslinked at a 3'-junction. On the other hand, RPAp70, the large subunit of the replication protein A (RPA), was the predominant crosslinking product at a 5'-junction. Interestingly, ddc1Δ extracts did not display photocrosslinking of RPAp70 at a 5'-junction. The results show that RPAp70 crosslinked to DNA with a 5'-junction is subject to limited proteolysis in ddc1Δ extracts, whereas it is stable in WT, rad17Δ, mec3Δ and mec1Δ extracts. The degradation of the RPAp70-DNA adduct in ddc1Δ extract is strongly reduced in the presence of the proteasome inhibitor MG 132. We also addressed the question of the stability of free RPA, using anti-RPA antibodies. The results show that RPAp70 is also subject to proteolysis without photocrosslinking to DNA upon incubation in ddc1Δ extract. The data point to a novel property of Ddc1, modulating the turnover of DNA binding proteins such as RPAp70 by the proteasome. Copyright © 2014 Elsevier B.V. All rights reserved.

Inhibin/activin-betaC and -betaE subunits in the Ishikawa human endometrial adenocarcinoma cell line.

PubMed

Kimmich, Tanja; Brüning, Ansgar; Käufl, Stephanie D; Makovitzky, Josef; Kuhn, Christina; Jeschke, Udo; Friese, Klaus; Mylonas, Ioannis

2010-08-01

Inhibins and activins are important regulators of the female reproductive system. Recently, two novel inhibin subunits, named betaC and betaE, have been identified and shown to be expressed in several human tissues. However, only limited data on the expression of these novel inhibin subunits in normal human endometrial tissue and endometrial adenocarcinoma cell lines exist. Samples of proliferative and secretory human endometrium were obtained from five premenopausal, non-pregnant patients undergoing gynecological surgery for benign diseases. Normal endometrial tissue and Ishikawa endometrial adenocarcinoma cell lines were analyzed by immunohistochemistry, immunofluorescence and RT-PCR. Expression of the inhibin betaC and betaE subunits could be demonstrated at the protein level by means of immunohistochemical evaluation and at the transcriptional level by establishing a betaC- and betaE-specific RT-PCR analysis in normal human endometrial tissue and the parental Ishikawa cell line. Interestingly, in a highly de-differentiated subclone of the Ishikawa cell line lacking estrogen receptor expression, the expression of the inhibin-betaC subunit appeared strongly reduced. Here, we show for the first time that the novel inhibin/activin-betaC and -betaE subunits are expressed in normal human endometrium and the estrogen receptor positive human endometrial carcinoma cell line Ishikawa using RT-PCR and immunohistochemical detection methods. Interestingly, the Ishikawa minus cell line (lacking estrogen receptor expression) demonstrated no to minimal expression of the betaC subunit as observed with immunofluorescence and RT-PCR, suggesting a possible hormone- dependency of this subunit in human endometrial cancer cells. Moreover, because the Ishikawa cell line minus is thought to be a more malignant endometrial cell line than its estrogen receptor positive counterpart, inhibin-betaC subunit might be substantially involved in the pathogenesis and malignant transformation in human endometrium.

Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis.

PubMed

Cheng, C; Prince, L S; Snyder, P M; Welsh, M J

1998-08-28

Three subunits, alpha, beta, and gamma, contribute to the formation of the epithelial Na+ channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the alpha subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual beta and gamma subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells and in vitro. When we co-expressed beta with alpha or with alpha plus gamma, the beta subunit assembled into a 25 S complex. Glycosylation of the alpha subunit was not required for assembly into a 25 S complex. We found that the alpha subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the alpha subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+ channel subunits form high order oligomeric complexes and that the alpha subunit contains the information that facilitates such formation. Interestingly, the ability of the alpha, but not the beta or gamma, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.

Properties of human brain sodium channel α-subunits expressed in HEK293 cells and their modulation by carbamazepine, phenytoin and lamotrigine

PubMed Central

Qiao, Xin; Sun, Guangchun; Clare, Jeffrey J; Werkman, Taco R; Wadman, Wytse J

2014-01-01

Background and purpose Voltage-activated Na+ channels contain one distinct α-subunit. In the brain NaV1.1, NaV1.2, NaV1.3 and NaV1.6 are the four most abundantly expressed α-subunits. The antiepileptic drugs (AEDs) carbamazepine, phenytoin and lamotrigine have voltage-gated Na+ channels as their primary therapeutic targets. This study provides a systematic comparison of the biophysical properties of these four α-subunits and characterizes their interaction with carbamazepine, phenytoin and lamotrigine. Experimental approach Na+ currents were recorded in voltage-clamp mode in HEK293 cells stably expressing one of the four α-subunits. Key results NaV1.2 and NaV1.3 subunits have a relatively slow recovery from inactivation, compared with the other subunits and NaV1.1 subunits generate the largest window current. Lamotrigine evokes a larger maximal shift of the steady-state inactivation relationship than carbamazepine or phenytoin. Carbamazepine shows the highest binding rate to the α-subunits. Lamotrigine binding to NaV1.1 subunits is faster than to the other α-subunits. Lamotrigine unbinding from the α-subunits is slower than that of carbamazepine and phenytoin. Conclusions and implications The four Na+ channel α-subunits show subtle differences in their biophysical properties, which, in combination with their (sub)cellular expression patterns in the brain, could contribute to differences in neuronal excitability. We also observed differences in the parameters that characterize AED binding to the Na+ channel subunits. Particularly, lamotrigine binding to the four α-subunits suggests a subunit-specific response. Such differences will have consequences for the clinical efficacy of AEDs. Knowledge of the biophysical and binding parameters could be employed to optimize therapeutic strategies and drug development. PMID:24283699

Short Chemical Ischemia Triggers Phosphorylation of eIF2α and Death of SH-SY5Y Cells but not Proteasome Stress and Heat Shock Protein Response in both SH-SY5Y and T98G Cells.

PubMed

Klacanova, Katarina; Pilchova, Ivana; Klikova, Katarina; Racay, Peter

2016-04-01

Both translation arrest and proteasome stress associated with accumulation of ubiquitin-conjugated protein aggregates were considered as a cause of delayed neuronal death after transient global brain ischemia; however, exact mechanisms as well as possible relationships are not fully understood. The aim of this study was to compare the effect of chemical ischemia and proteasome stress on cellular stress responses and viability of neuroblastoma SH-SY5Y and glioblastoma T98G cells. Chemical ischemia was induced by transient treatment of the cells with sodium azide in combination with 2-deoxyglucose. Proteasome stress was induced by treatment of the cells with bortezomib. Treatment of SH-SY5Y cells with sodium azide/2-deoxyglucose for 15 min was associated with cell death observed 24 h after treatment, while glioblastoma T98G cells were resistant to the same treatment. Treatment of both SH-SY5Y and T98G cells with bortezomib was associated with cell death, accumulation of ubiquitin-conjugated proteins, and increased expression of Hsp70. These typical cellular responses to proteasome stress, observed also after transient global brain ischemia, were not observed after chemical ischemia. Finally, chemical ischemia, but not proteasome stress, was in SH-SY5Y cells associated with increased phosphorylation of eIF2α, another typical cellular response triggered after transient global brain ischemia. Our results showed that short chemical ischemia of SH-SY5Y cells is not sufficient to induce both proteasome stress associated with accumulation of ubiquitin-conjugated proteins and stress response at the level of heat shock proteins despite induction of cell death and eIF2α phosphorylation.

Structure of a AAA+ unfoldase in the process of unfolding substrate

PubMed Central

Ripstein, Zev A; Huang, Rui; Augustyniak, Rafal; Kay, Lewis E; Rubinstein, John L

2017-01-01

AAA+ unfoldases are thought to unfold substrate through the central pore of their hexameric structures, but how this process occurs is not known. VAT, the Thermoplasma acidophilum homologue of eukaryotic CDC48/p97, works in conjunction with the proteasome to degrade misfolded or damaged proteins. We show that in the presence of ATP, VAT with its regulatory N-terminal domains removed unfolds other VAT complexes as substrate. We captured images of this transient process by electron cryomicroscopy (cryo-EM) to reveal the structure of the substrate-bound intermediate. Substrate binding breaks the six-fold symmetry of the complex, allowing five of the six VAT subunits to constrict into a tight helix that grips an ~80 Å stretch of unfolded protein. The structure suggests a processive hand-over-hand unfolding mechanism, where each VAT subunit releases the substrate in turn before re-engaging further along the target protein, thereby unfolding it. DOI: http://dx.doi.org/10.7554/eLife.25754.001 PMID:28390173

Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A.

PubMed

Wiley, J C; Wailes, L A; Idzerda, R L; McKnight, G S

1999-03-05

Regulation of protein kinase A by subcellular localization may be critical to target catalytic subunits to specific substrates. We employed epitope-tagged catalytic subunit to correlate subcellular localization and gene-inducing activity in the presence of regulatory subunit or protein kinase inhibitor (PKI). Transiently expressed catalytic subunit distributed throughout the cell and induced gene expression. Co-expression of regulatory subunit or PKI blocked gene induction and prevented nuclear accumulation. A mutant PKI lacking the nuclear export signal blocked gene induction but not nuclear accumulation, demonstrating that nuclear export is not essential to inhibit gene induction. When the catalytic subunit was targeted to the nucleus with a nuclear localization signal, it was not sequestered in the cytoplasm by regulatory subunit, although its activity was completely inhibited. PKI redistributed the nuclear catalytic subunit to the cytoplasm and blocked gene induction, demonstrating that the nuclear export signal of PKI can override a strong nuclear localization signal. With increasing PKI, the export process appeared to saturate, resulting in the return of catalytic subunit to the nucleus. These results demonstrate that both the regulatory subunit and PKI are able to completely inhibit the gene-inducing activity of the catalytic subunit even when the catalytic subunit is forced to concentrate in the nuclear compartment.

Vanillin-Ameliorated Development of Azoxymethane/Dextran Sodium Sulfate-Induced Murine Colorectal Cancer: The Involvement of Proteasome/Nuclear Factor-κB/Mitogen-Activated Protein Kinase Pathways.

PubMed

Li, Jung-Miao; Lee, Yu-Chen; Li, Chia-Cheng; Lo, Hsin-Yi; Chen, Feng-Yuan; Chen, Yi-Siou; Hsiang, Chien-Yun; Ho, Tin-Yun

2018-06-06

Vanillin is a natural dietary flavoring widely used in the food industry. Colorectal cancer (CRC) is one of the common malignancies in the world. Chronic intestinal inflammation is a risk factor for the development of CRC. We have previously found that vanillin improves and prevents colitis in mice. Here we evaluated the inhibitory activities of vanillin on a mouse model of colitis-induced CRC. Mice were challenged intraperitoneally with azoxymethane (AOM) and orally with dextran sodium sulfate (DSS). Various dosages of vanillin were orally administered for 13 consecutive weeks. Vanillin alleviated the development of tumors in AOM/DSS-induced mice. The total number of tumors in 100 mg/kg vanillin group was significantly reduced by 57.14 ± 7.67%, compared with sham group. Gene expression analysis showed that vanillin downregulated the expression levels of proteasome genes in colon tissues. Moreover, vanillin at 10 mM significantly suppressed proteasome activities in HCT-116 cells by 41.27 ± 0.41%. Furthermore, vanillin diminished the phosphorylation of mitogen-activated protein kinases (MAPKs) and reduced the number of p65-positive cells, proliferating cells, and granulocytes in colon tissues with statistical significance. In conclusion, our data suggested that vanillin was a bioactive compound that ameliorated the development of AOM/DSS-induced colon cancer in mice. Moreover, the amelioration of vanillin might be associated with the downregulation of proteasome, nuclear factor-κB, and MAPK pathways.

A Double-Edged Sword Role for Ubiquitin-Proteasome System in Brain Stem Cardiovascular Regulation During Experimental Brain Death

PubMed Central

Wu, Carol H. Y.; Chan, Julie Y. H.; Chan, Samuel H. H.; Chang, Alice Y. W.

2011-01-01

Background Brain stem cardiovascular regulatory dysfunction during brain death is underpinned by an upregulation of nitric oxide synthase II (NOS II) in rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from blood pressure of comatose patients that disappears before brain death ensues. Furthermore, the ubiquitin-proteasome system (UPS) may be involved in the synthesis and degradation of NOS II. We assessed the hypothesis that the UPS participates in brain stem cardiovascular regulation during brain death by engaging in both synthesis and degradation of NOS II in RVLM. Methodology/Principal Findings In a clinically relevant experimental model of brain death using Sprague-Dawley rats, pretreatment by microinjection into the bilateral RVLM of proteasome inhibitors (lactacystin or proteasome inhibitor II) antagonized the hypotension and reduction in the life-and-death signal elicited by intravenous administration of Escherichia coli lipopolysaccharide (LPS). On the other hand, pretreatment with an inhibitor of ubiquitin-recycling (ubiquitin aldehyde) or ubiquitin C-terminal hydrolase isozyme L1 (UCH-L1) potentiated the elicited hypotension and blunted the prevalence of the life-and-death signal. Real-time polymerase chain reaction, Western blot, electrophoresis mobility shift assay, chromatin immunoprecipitation and co-immunoprecipitation experiments further showed that the proteasome inhibitors antagonized the augmented nuclear presence of NF-κB or binding between NF-κB and nos II promoter and blunted the reduced cytosolic presence of phosphorylated IκB. The already impeded NOS II protein expression by proteasome inhibitor II was further reduced after gene-knockdown of NF-κB in RVLM. In animals pretreated with UCH-L1 inhibitor and died before significant increase in nos II mRNA occurred, NOS II protein expression in RVLM was considerably elevated. Conclusions/Significance We conclude that UPS participates in the defunct and maintained brain stem cardiovascular regulation during experimental brain death by engaging in both synthesis and degradation of NOS II at RVLM. Our results provide information on new therapeutic initiatives against this fatal eventuality. PMID:22110641

Trafficking regulates the subcellular distribution of voltage-gated sodium channels in primary sensory neurons.

PubMed

Bao, Lan

2015-09-30

Voltage-gated sodium channels (Navs) comprise at least nine pore-forming α subunits. Of these, Nav1.6, Nav1.7, Nav1.8 and Nav1.9 are the most frequently studied in primary sensory neurons located in the dorsal root ganglion and are mainly localized to the cytoplasm. A large pool of intracellular Navs raises the possibility that changes in Nav trafficking could alter channel function. The molecular mediators of Nav trafficking mainly consist of signals within the Navs themselves, interacting proteins and extracellular factors. The surface expression of Navs is achieved by escape from the endoplasmic reticulum and proteasome degradation, forward trafficking and plasma membrane anchoring, and it is also regulated by channel phosphorylation and ubiquitination in primary sensory neurons. Axonal transport and localization of Navs in afferent fibers involves the motor protein KIF5B and scaffold proteins, including contactin and PDZ domain containing 2. Localization of Nav1.6 to the nodes of Ranvier in myelinated fibers of primary sensory neurons requires node formation and the submembrane cytoskeletal protein complex. These findings inform our understanding of the molecular and cellular mechanisms underlying Nav trafficking in primary sensory neurons.

Hypoxia and hypoxia-inducible factors in neuroblastoma.

PubMed

Påhlman, Sven; Mohlin, Sofie

2018-05-01

Hypoxia (i.e., low oxygen levels) is a known feature of aggressive tumors. Cells, including tumor cells, respond to conditions of insufficient oxygen by activating a transcriptional program mainly driven by hypoxia-inducible factors (HIF)-1 and HIF-2. Both HIF-1α and HIF-2α expression levels have been shown to correlate to patient outcome in various tumor forms and in neuroblastoma, a solid childhood tumor of the sympathetic nervous system, in particular, HIF-2α marks a subpopulation of immature neural crest-like perivascularly located cells and associates with aggressive disease and distant metastasis. It has for long been recognized that the HIF-α subunits are oxygen-dependently regulated at the post-translational level, via ubiquitination and proteasomal degradation. Evidence of oxygen-independent mechanisms of regulation, transcriptional control of EPAS1/HIF2A and possible cytoplasmic activities of HIF-2α has also emerged during recent years. In this review, we discuss these non-conventional actions of HIF-2α, its putative role as a therapeutic target and the constraints it carries, as well as the importance of HIF-2 activity in a vascularized setting, the so-called pseudo-hypoxic state.

Scythe regulates apoptosis through modulating ubiquitin-mediated proteolysis of the Xenopus elongation factor XEF1AO

PubMed Central

Minami, Ryosuke; Shimada, Masumi; Yokosawa, Hideyoshi; Kawahara, Hiroyuki

2007-01-01

Scythe was originally identified as a novel Reaper-binding anti-apoptotic protein, although the mechanisms of its functions remain largely obscure. Our previous analysis revealed that Scythe can bind to a proteasomal subunit via N-terminal domains and that the domains are required for appropriate development of Xenopus embryos. In the present study, we show evidence that the N-terminus of Scythe interacts with XEF1AO, a maternal form of Xenopus laevis EF1A that was suggested to be a potential inducer of apoptosis in vertebrates, and that the binding enhances the poly-ubiquitin modification and subsequent degradation of XEF1AO. Scythe is required for degradation of XEF1AO, since immunodepletion of Scythe from embryonic extracts stabilized XEF1AO significantly. Furthermore, we show that apoptosis induced by accumulation of XEF1AO can be suppressed by co-expression of the full-length form of Scythe. These observations indicate that the proteolytic regulation of XEF1AO, mediated through Scythe, is essential to prevent inappropriate accumulation of XEF1AO and resulting apoptotic events during the course of Xenopus development. PMID:17428197

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jack Preiss

Conversion of the Potato tuber ADP-glucose Pyrophopshorylase Regulatory Subunit into a Catalytic Subunit. ADP-glucose synthesis, a rate-limiting reaction in starch synthesis, is catalyzed by ADP-glucose pyrophosphorylase (ADPGlc PPase). The enzyme in plants is allosterically activated by 3-phosphoglycerate (3PGA) and inhibited by inorganic phosphate (Pi) and is composed of two subunits as a heterotetramer, a2b2. Subunit a is the catalytic subunit and subunit b is designated as the regulatory subunit.The b subunit increases the affinty of the activator for the catalytic subunit. Recent results have shown that the subunits are derived from the same ancestor subunit as the regulatory subunit canmore » be converted to a catalytically subunit via mutation of just two amino acids. Lys44 and Thr54 in the large subunit from potato tuber were converted to the homologous catalytic subunit residues, Arg33 and Lys43. The activity of the large subunit mutants cannot be readily tested with a co-expressed wild-type small (catalytic) subunit because of the intrinsic activity of the latter. We co-expressed the regulatory-subunit mutants with SmallD145N, an inactive S subunit in which the catalytic Asp145 was mutated. The activity of the small (catalytic) subunit was reduced more than three orders of magnitude. Coexpression of the L subunit double mutant LargeK44R/T54K with SmallD145N generated an enzyme with considerable activity, 10% and 18% of the wildtype enzyme, in the ADP-glucose synthetic and pyrophosphorolytic direction, respectively. Replacement of those two residues in the small subunit by the homologous amino acids in the L subunits (mutations R33K and K43T) decreased the activity one and two orders of magnitude. The wild-type enzyme and SmallD145NLargeK44R/T54K had very similar kinetic properties indicating that the substrate site has been conserved. The fact that only two mutations in the L subunit restored enzyme activity is very strong evidence that the large subunit is derived from the catalytic ancestor. Previous results showed that Asp145 in the small subunit of the wild-type is essential for catalysis, whereas the homologous Asp160 in the Large WT subunit is not. However, in this study, mutation D160N or D160E in the LK44R/T54K subunit abolished the activity, which shows the ancestral essential role of this residue and confirms that the catalysis of SmallD145NLarge K44R/T54K occurs in the L(b) subunit. A phylogenetic tree of the ADP-Glc PPases present in photosynthetic eukaryotes also sheds information about the origin of the subunits. The tree showed that plant Small and Large subunits can be divided into two and four distinct groups, respectively. The two main groups of S subunits are from dicot and monocot plants, whereas Large subunit groups correlate better with their documented tissue expression. The first Large-subunit group is generally expressed in photosynthetic tissues and comprises Large subunits from dicots and monocots. Group II displays a broader expression pattern, whereas groups III and IV are expressed in storage organs (roots, stems, tubers, seeds). Subunits from group III are only from dicot plants, whereas group IV are seed-specific subunits from monocots. These last two groups stem from the same branch of the phylogenetic tree and split before monocot and dicot separation. Thus few as two mutations turned the L subunit from Solanum tuberosum catalytic, showing that L and S subunits share a common catalytic ancestor, rather than a non-catalytic one. The L subunit evolved to have a regulatory role, lost catalytic residues more than 130 million years ago before monocots and dicots diverged, and preserved, possibly as a byproduct, the active site domain.« less

GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

NASA Technical Reports Server (NTRS)

Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

1995-01-01

The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of schizophrenics, the previously reported upregulation of muscimol binding sites and downregulation of benzodiazepine binding sites in the prefrontal and adjacent cingulate cortex of schizophrenics are possibly due to posttranscriptional modifications of mRNAs and their translated polypeptides.

Roles of F-box proteins in human digestive system tumors (Review).

PubMed

Gong, Jian; Lv, Liang; Huo, Jirong

2014-12-01

F-box proteins (FBPs), the substrate-recognition subunit of E3 ubiquitin (Ub) ligase, are the important components of Ub proteasome system (UPS). FBPs are involved in multiple cellular processes through ubiquitylation and subsequent degradation of their target proteins. Many studies have described the roles of FBPs in human cancers. Digestive system tumors account for a large proportion of all the tumors, and their mortality is very high. This review summarizes for the first time the roles of FBPs in digestive system tumorige-nesis and tumor progression, aiming at finding new routes for the rational design of targeted anticancer therapies in digestive system tumors.

pVHL's kryptonite: E2-EPF UCP.

PubMed

Ohh, Michael

2006-08-01

E2-EPF ubiquitin carrier protein (UCP) is a member of an E2 family of enzymes that catalyzes the ligation of ubiquitin to proteins targeted for destruction by the proteasome. UCP is overexpressed in common human cancers, suggesting its involvement in oncogenesis, but a physiologic target of UCP has not been identified. In a recent report published in Nature Medicine, Jung et al. identified von Hippel-Lindau (VHL) tumor suppressor protein, which targets the alpha subunit of hypoxia-inducible factor (HIF) for ubiquitin-mediated destruction, as a bona fide substrate of UCP and demonstrated a potential pVHL-HIF pathway-dependent role for UCP in cancer development.

NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR

PubMed Central

Kainulainen, Markus; Lau, Simone; Samuel, Charles E.; Hornung, Veit

2016-01-01

ABSTRACT Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. IMPORTANCE Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs requires E3 ubiquitin ligase complexes of the SCF (Skp1, Cul1, F-box protein) type to destroy PKR. SCF-type complexes can engage variant ubiquitination substrate recognition subunits, and we found the F-box proteins FBXW11 and β-TRCP1 to be relevant for the action of NSs against PKR. Thus, we identified the host cell factors that are critically needed by Rift Valley fever virus to uphold its replication against the potent antiviral kinase PKR. PMID:27122577

Memory in aged mice is rescued by enhanced expression of the GluN2B subunit of the NMDA receptor

PubMed Central

Brim, B. L.; Haskell, R.; Awedikian, R.; Ellinwood, N.M.; Jin, L.; Kumar, A.; Foster, T.C.; Magnusson, K.

2012-01-01

The GluN2B subunit of the N-methyl-D-aspartate (NMDA) receptor shows age-related declines in expression across the frontal cortex and hippocampus. This decline is strongly correlated to age-related memory declines. This study was designed to determine if increasing GluN2B subunit expression in the frontal lobe or hippocampus would improve memory in aged mice. Mice were injected bilaterally with either the GluN2B vector, containing cDNA specific for the GluN2B subunit and enhanced Green Fluorescent Protein (eGFP); a control vector or vehicle. Spatial memory, cognitive flexibility, and associative memory were assessed using the Morris water maze. Aged mice, with increased GluN2B subunit expression, exhibited improved long-term spatial memory, comparable to young mice. However, memory was rescued on different days in the Morris water maze; early for hippocampal GluN2B subunit enrichment and later for the frontal lobe. A higher concentration of the GluN2B antagonist, Ro 25-6981, was required to impair long-term spatial memory in aged mice with enhanced GluN2B expression, as compared to aged controls, suggesting there was an increase in the number of GluN2B-containing NMDA receptors. In addition, hippocampal slices from aged mice with increased GluN2B subunit expression exhibited enhanced NMDA receptor-mediated excitatory post-synaptic potentials (EPSP). Treatment with Ro 25-6981 showed that a greater proportion of the NMDA receptor-mediated EPSP was due to the GluN2B subunit in these animals, as compared to aged controls. These results suggest that increasing the production of the GluN2B subunit in aged animals enhances memory and synaptic transmission. Therapies that enhance GluN2B subunit expression within the aged brain may be useful for ameliorating age-related memory declines. PMID:23103326

The cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells.

PubMed

Cho, Eun-Ah; Juhnn, Yong-Sung

2012-06-01

Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on γ-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (GαsQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of GαsQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after γ-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2'-O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2'-O-Me-cAMP and restored XRCC1 protein level following γ-ray irradiation. From these results, we conclude that the cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting the ubiquitin-proteasome dependent degradation of XRCC1 in an Epac-dependent pathway in lung cancer cells. Copyright © 2012 Elsevier Inc. All rights reserved.

Sphingosine 1-phosphate lyase ablation disrupts presynaptic architecture and function via an ubiquitin- proteasome mediated mechanism.

PubMed

Mitroi, Daniel N; Deutschmann, André U; Raucamp, Maren; Karunakaran, Indulekha; Glebov, Konstantine; Hans, Michael; Walter, Jochen; Saba, Julie; Gräler, Markus; Ehninger, Dan; Sopova, Elena; Shupliakov, Oleg; Swandulla, Dieter; van Echten-Deckert, Gerhild

2016-11-24

The bioactive lipid sphingosine 1-phosphate (S1P) is a degradation product of sphingolipids that are particularly abundant in neurons. We have shown previously that neuronal S1P accumulation is toxic leading to ER-stress and an increase in intracellular calcium. To clarify the neuronal function of S1P, we generated brain-specific knockout mouse models in which S1P-lyase (SPL), the enzyme responsible for irreversible S1P cleavage was inactivated. Constitutive ablation of SPL in the brain (SPL fl/fl/Nes ) but not postnatal neuronal forebrain-restricted SPL deletion (SPL fl/fl/CaMK ) caused marked accumulation of S1P. Hence, altered presynaptic architecture including a significant decrease in number and density of synaptic vesicles, decreased expression of several presynaptic proteins, and impaired synaptic short term plasticity were observed in hippocampal neurons from SPL fl/fl/Nes mice. Accordingly, these mice displayed cognitive deficits. At the molecular level, an activation of the ubiquitin-proteasome system (UPS) was detected which resulted in a decreased expression of the deubiquitinating enzyme USP14 and several presynaptic proteins. Upon inhibition of proteasomal activity, USP14 levels, expression of presynaptic proteins and synaptic function were restored. These findings identify S1P metabolism as a novel player in modulating synaptic architecture and plasticity.

Ubiquitin specific protease 2 acts as a key modulator for the regulation of cell cycle by adiponectin and leptin in cancer cells.

PubMed

Nepal, Saroj; Shrestha, Anup; Park, Pil-Hoon

2015-09-05

Adiponectin and leptin, both produced from adipose tissue, cause cell cycle arrest and progression, respectively in cancer cells. Ubiquitin specific protease-2 (USP-2), a deubiquitinating enzyme, is known to impair proteasome-induced degradation of cyclin D1, a critical cell cycle regulator. Herein, we investigated the effects of these adipokines on USP-2 expression and its potential role in the modulation of cell cycle. Treatment with globular adiponectin (gAcrp) decreased, whereas leptin increased USP-2 expression both in human hepatoma and breast cancer cells. In addition, overexpression or gene silencing of USP-2 affected cyclin D1 expression and cell cycle progression/arrest by adipokines. Adiponectin and leptin also modulated in vitro proteasomal activity, which was partially dependent on USP-2 expression. Taken together, our results reveal that modulation of USP-2 expression plays a crucial role in cell cycle regulation by adipokines. Thus, USP-2 would be a promising therapeutic target for the modulation of cancer cell growth by adipokines. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit p62▿

PubMed Central

Kalveram, Birte; Lihoradova, Olga; Ikegami, Tetsuro

2011-01-01

Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) is an important emerging pathogen of humans and ruminants. Its NSs protein has previously been identified as a major virulence factor that suppresses host defense through three distinct mechanisms: it directly inhibits beta interferon (IFN-β) promoter activity, it promotes the degradation of double-stranded RNA-dependent protein kinase (PKR), and it suppresses host transcription by disrupting the assembly of the basal transcription factor TFIIH through sequestration of its p44 subunit. Here, we report that in addition to PKR, NSs also promotes the degradation of the TFIIH subunit p62. Infection of cells with the RVFV MP-12 vaccine strain reduced p62 protein levels to below the detection limit early in the course of infection. This NSs-mediated downregulation of p62 was posttranslational, as it was unaffected by pharmacological inhibition of transcription or translation and MP-12 infection had no effect on p62 mRNA levels. Treatment of cells with proteasome inhibitors but not inhibition of lysosomal acidification or nuclear export resulted in a stabilization of p62 in the presence of NSs. Furthermore, p62 could be coprecipitated with NSs from lysates of infected cells. These data suggest that the RVFV NSs protein is able to interact with the TFIIH subunit p62 inside infected cells and promotes its degradation, which can occur directly in the nucleus. PMID:21543505

Beta3 subunits promote expression and nicotine-induced up-regulation of human nicotinic alpha6* nicotinic acetylcholine receptors expressed in transfected cell lines.

PubMed

Tumkosit, Prem; Kuryatov, Alexander; Luo, Jie; Lindstrom, Jon

2006-10-01

Nicotinic acetylcholine receptors (AChRs) containing alpha6 subunits are typically found at aminergic nerve endings where they play important roles in nicotine addiction and Parkinson's disease. alpha6* AChRs usually contain beta3 subunits. beta3 subunits are presumed to assemble only in the accessory subunit position within AChRs where they do not participate in forming acetylcholine binding sites. Assembly of subunits in the accessory position may be a critical final step in assembly of mature AChRs. Human alpha6 AChRs subtypes were permanently transfected into human tsA201 human embryonic kidney (HEK) cell lines. alpha6beta2beta3 and alpha6beta4beta3 cell lines were found to express much larger amounts of AChRs and were more sensitive to nicotine-induced increase in the amount of AChRs than were alpha6beta2 or alpha6beta4 cell lines. The increased sensitivity to nicotine-induced up-regulation was due not to a beta3-induced increase in affinity for nicotine but probably to a direct effect on assembly of AChR subunits. HEK cells express only a small amount of mature alpha6beta2 AChRs, but many of these subunits are on the cell surface. This contrasts with Xenopus laevis oocytes, which express a large amount of incorrectly assembled alpha6beta2 subunits that bind cholinergic ligands but form large amorphous intracellular aggregates. Monoclonal antibodies (mAbs) were made to the alpha6 and beta3 subunits to aid in the characterization of these AChRs. The alpha6 mAbs bind to epitopes C-terminal of the extracellular domain. These data demonstrate that both cell type and the accessory subunit beta3 can play important roles in alpha6* AChR expression, stability, and up-regulation by nicotine.

Multiple alpha subunits of integrin are involved in cell-mediated responses of the Manduca immune system.

PubMed

Zhuang, Shufei; Kelo, Lisha; Nardi, James B; Kanost, Michael R

2008-01-01

The cell-mediated responses of the insect innate immune system-phagocytosis, nodulation, encapsulation-involve multiple cell adhesion molecules of hemocyte surfaces. A hemocyte-specific (HS) integrin and a member of the immunoglobulin (Ig) superfamily (neuroglian) are involved in the encapsulation response of hemocytes in Manduca sexta. In addition, two new integrin alpha (alpha) subunits have been found on these hemocytes. The alpha2 subunit is mainly expressed in epidermis and Malphigian tubules, whereas the alpha3 subunit is primarily expressed on hemocytes and fat body cells. Of the three known alpha subunits, the alpha1 subunit found in HS integrin is the predominant subunit of hemocytes. Cell adhesion assays indicate that alpha2 belongs to the integrin family with RGD-binding motifs, confirming the phylogenetic analysis of alpha subunits based on the amino-acid sequence alignment of different alpha subunits. Double-stranded RNAs (dsRNAs) targeting each of these three integrin alpha subunits not only specifically decreased transcript expression of each alpha subunit in hemocytes, but also abolished the cell-mediated encapsulation response of hemocytes to foreign surfaces. The individual alpha subunits of M. sexta integrins, like their integrin counterparts in mammalian immune systems, have critical, individual roles in cell-substrate and cell-cell interactions during immune responses.

PSMB4 promotes multiple myeloma cell growth by activating NF-κB-miR-21 signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Peihao; Guo, Honggang; Li, Guangchao

2015-03-06

Proteasomal subunit PSMB4, was recently identified as potential cancer driver genes in several tumors. However, the regulatory mechanism of PSMB4 on carcinogenesis process remains unclear. In this study, we investigated the expression and roles of PSMB4 in multiple myeloma (MM). We found a significant up-regulation of PSMB4 in MM plasma and cell lines. Ectopic overexpression of PSMB4 promoted cell growth and colony forming ability of MM cells, whereas inhibition of PSMB4 led to a decrease of such events. Furthermore, our results demonstrated the up-regulation of miR-21 and a positive correlation between the levels of miR-21 and PSMB4 in MM. Re-expressionmore » of miR-21 markedly rescued PSMB4 knockdown-mediated suppression of cell proliferation and clone-formation. Additionally, while enforced expression of PSMB4 profoundly increased NF-κB activity and the level of miR-21, PSMB4 knockdown or NF-κB inhibition suppressed miR-21 expression in MM cells. Taken together, our results demonstrated that PSMB4 regulated MM cell growth in part by activating NF-κB-miR-21 signaling, which may represent promising targets for novel specific therapies. - Highlights: • First reported upregulation of PSMB4 in MM plasma and cell lines. • PSMB4 promoted MM cell growth and colony forming ability. • Further found miR-21 was up-regulated by PSMB4 in MM plasma and cell lines. • PSMB4-induced miR-21 expression was modulated by NF-κB. • PSMB4-NF-κB-miR-21 axis may be potential therapeutic targets of MM.« less

Comparative proteome analysis of drought-sensitive and drought-tolerant rapeseed roots and their hybrid F1 line under drought stress.

PubMed

Mohammadi, Payam Pour; Moieni, Ahmad; Komatsu, Setsuko

2012-11-01

Rapeseed (Brassica napus L.), which is the third leading source of vegetable oil, is sensitive to drought stress during the early vegetative growth stage. To investigate the initial response of rapeseed to drought stress, changes in the protein expression profiles of drought-sensitive (RGS-003) and drought-tolerant lines (SLM-003), and their F1 hybrid, were analyzed using a proteomics approach. Seven-day-old rapeseed seedlings were treated with drought stress by restricting water for 7 days, and proteins were extracted from roots and separated by two-dimensional polyacrylamide gel electrophoresis. In the sensitive rapeseed line, 35 protein spots were differentially expressed under drought stress, and proteins related to metabolism, energy, disease/defense, and transport were decreased. In the tolerant line, 32 protein spots were differentially expressed under drought stress, and proteins involved in metabolism, disease/defense, and transport were increased, while energy-related proteins were decreased. Six protein spots in F1 hybrid were common among expressed proteins in the drought-sensitive and -tolerant lines. Notably, tubulin beta-2 and heat shock protein 70 were decreased in the drought-sensitive line and hybrid F1 plants, while jasmonate-inducible protein and 20S proteasome subunit PAF1 were increased in the F1 hybrids and drought-tolerant line. These results indicate that (1) V-type H(+) ATPase, plasma-membrane associated cation-binding protein, HSP 90, and elongation factor EF-2 have a role in the drought tolerance of rapeseed; (2) The decreased levels of heat shock protein 70 and tubulin beta-2 in the drought-sensitive and hybrid F1 lines might explain the reduced growth of these lines in drought conditions.

INTRINSIC REGULATION OF HEMOGLOBIN EXPRESSION BY VARIABLE SUBUNIT INTERFACE STRENGTHS

PubMed Central

Manning, James M.; Popowicz, Anthony M.; Padovan, Julio C.; Chait, Brian T.; Manning, Lois R.

2012-01-01

SUMMARY The expression of the six types of human hemoglobin subunits over time is currently considered to be regulated mainly by transcription factors that bind to upstream control regions of the gene (the “extrinsic” component of regulation). Here we describe how subunit pairing and further assembly to tetramers in the liganded state is influenced by the affinity of subunits for one another (the “intrinsic” component of regulation). The adult hemoglobin dimers have the strongest subunit interfaces and the embryonic hemoglobins are the weakest with fetal hemoglobins of intermediate strength, corresponding to the temporal order of their expression. These variable subunit binding strengths and the attenuating effects of acetylation contribute to the differences with which these hemoglobin types form functional O2-binding tetramers consistent with gene switching. PMID:22129306

Heat shock proteins and proteasomal degradation in normal and tumor cells.

PubMed

Karademir, Betul; Bozaykut, Perinur; Kartal Ozer, Nesrin

2014-10-01

Proteasomal degradation of oxidized proteins is a crucial mechanism to prevent the accumulation of cellular damage. The removal of the damage is generally a required process for healthy organisms to keep the integrity while in cancer cells the situation may be different. In normal conditions, cancer cells have higher proteasome activity compared to normal cells. During cancer treatment, cellular damage by chemotherapy is an expected process to be able to kill the tumor cells. And the accumulation of this damage accompanied by the decrease in protein repair and removal systems may increase the efficacy of the cancer therapy. Heat shock proteins (Hsp) as molecular chaperones are involved in the folding, activation and assembly of a variety of proteins. Among these Hsp40, Hsp70 and Hsp90 are believed to act as a chaperone system to regulate the proteasomal degradation. In this study, we tested the role of heat stress response on the proteasomal degradation of oxidized proteins. We used two different cell lines to observe the difference in normal and tumor cells. First the effect of heat stress (42°C, 1h) were tested in terms of protein oxidation tested by protein carbonyl formation and proteasomal degradation. The results were extremely different in normal fibroblast cells and hippocampal tumor cells. In the same direction, the expressions of Hsp40, Hsp70 and Hsp90 were affected in a different manner in two cell lines, will be discussed in detail. Supported by TUBITAK COST-CM1001-110S281. Copyright © 2014. Published by Elsevier Inc.

Continued 26S proteasome dysfunction in mouse brain cortical neurons impairs autophagy and the Keap1-Nrf2 oxidative defence pathway.

PubMed

Ugun-Klusek, Aslihan; Tatham, Michael H; Elkharaz, Jamal; Constantin-Teodosiu, Dumitru; Lawler, Karen; Mohamed, Hala; Paine, Simon M L; Anderson, Glen; John Mayer, R; Lowe, James; Ellen Billett, E; Bedford, Lynn

2017-01-05

The ubiquitin-proteasome system (UPS) and macroautophagy (autophagy) are central to normal proteostasis and interdependent in that autophagy is known to compensate for the UPS to alleviate ensuing proteotoxic stress that impairs cell function. UPS and autophagy dysfunctions are believed to have a major role in the pathomechanisms of neurodegenerative disease. Here we show that continued 26S proteasome dysfunction in mouse brain cortical neurons causes paranuclear accumulation of fragmented dysfunctional mitochondria, associated with earlier recruitment of Parkin and lysine 48-linked ubiquitination of mitochondrial outer membrane (MOM) proteins, including Mitofusin-2. Early events also include phosphorylation of p62/SQSTM1 (p62) and increased optineurin, as well as autophagosomal LC3B and removal of some mitochondria, supporting the induction of selective autophagy. Inhibition of the degradation of ubiquitinated MOM proteins with continued 26S proteasome dysfunction at later stages may impede efficient mitophagy. However, continued 26S proteasome dysfunction also decreases the levels of essential autophagy proteins ATG9 and LC3B, which is characterised by decreases in their gene expression, ultimately leading to impaired autophagy. Intriguingly, serine 351 phosphorylation of p62 did not enhance its binding to Keap1 or stabilise the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor in this neuronal context. Nrf2 protein levels were markedly decreased despite transcriptional activation of the Nrf2 gene. Our study reveals novel insights into the interplay between the UPS and autophagy in neurons and is imperative to understanding neurodegenerative disease where long-term proteasome inhibition has been implicated.

A Comprehensive Proteomics Analysis Reveals a Secretory Path- and Status-Dependent Signature of Exosomes Released from Tumor-Associated Macrophages.

PubMed

Zhu, Yinghui; Chen, Xianwei; Pan, Qingfei; Wang, Yang; Su, Siyuan; Jiang, Cuicui; Li, Yang; Xu, Ningzhi; Wu, Lin; Lou, Xiaomin; Liu, Siqi

2015-10-02

Exosomes are 30-120 nm-sized membrane vesicles of endocytic origin that are released into the extracellular environment and play roles in cell-cell communication. Tumor-associated macrophages (TAMs) are important constituents of the tumor microenvironment; thus, it is critical to study the features and complex biological functions of TAM-derived exosomes. Here, we constructed a TAM cell model from a mouse macrophage cell line, Ana-1, and performed comparative proteomics on exosomes, exosome-free media, and cells between TAMs and Ana-1. Proteomic analysis between exosome and exosome-free fractions indicated that the functions of exosome dominant proteins were mainly enriched in RNA processing and proteolysis. TAM status dramatically affected the abundances of 20S proteasome subunits and ribosomal proteins in their exosomes. The 20S proteasome activity assay strongly indicated that TAM exosomes possessed higher proteolytic activity. In addition, Ana-1- and TAM-derived exosomes have different RNA profiles, which may result from differential RNA processing proteins. Taken together, our comprehensive proteomics study provides novel views for understanding the complicated roles of macrophage-derived exosomes in the tumor microenvironment.

CCAAT/enhancer binding protein beta (C/EBPβ) isoform balance as a regulator of epithelial-mesenchymal transition in mouse mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miura, Yuka; Hagiwara, Natsumi; Radisky, Derek C.

2014-09-10

Activation of the epithelial-mesenchymal transition (EMT) program promotes cell invasion and metastasis, and is reversed through mesenchymal-epithelial transition (MET) after formation of distant metastases. Here, we show that an imbalance of gene products encoded by the transcriptional factor C/EBPβ, LAP (liver-enriched activating protein) and LIP (liver-enriched inhibitory protein), can regulate both EMT- and MET-like phenotypic changes in mouse mammary epithelial cells. By using tetracycline repressive LIP expression constructs, we found that SCp2 cells, a clonal epithelial line of COMMA1-D cells, expressed EMT markers, lost the ability to undergo alveolar-like morphogenesis in 3D Matrigel, and acquired properties of benign adenoma cells.more » Conversely, we found that inducible expression of LAP in SCg6 cells, a clonal fibroblastic line of COMMA1-D cells, began to express epithelial keratins with suppression of proliferation. The overexpression of the C/EBPβ gene products in these COMMA1-D derivatives was suppressed by long-term cultivation on tissue culture plastic, but gene expression was maintained in cells grown on Matrigel or exposed to proteasome inhibitors. Thus, imbalances of C/EBPβ gene products in mouse mammary epithelial cells, which are affected by contact with basement membrane, are defined as a potential regulator of metastatic potential. - Highlights: • We created a temporal imbalance of C/EBPβ gene products in the mammary model cells. • The temporal up-regulation of LIP protein induced EMT-like cell behaviors. • The temporal up-regulation of LAP protein induced MET-like cell behaviors. • Excess amount of C/EBPβ gene products were eliminated by proteasomal-degradation. • Basement membrane components attenuated proteasome-triggered protein elimination.« less

MCPIP1 contributes to the toxicity of proteasome inhibitor MG-132 in HeLa cells by the inhibition of NF-κB.

PubMed

Skalniak, Lukasz; Dziendziel, Monika; Jura, Jolanta

2014-10-01

Recently, we have shown that the treatment of cells with proteasome inhibitor MG-132 results in the induction of expression of monocyte chemotactic protein-1 induced protein 1 (MCPIP1). MCPIP1 is a ribonuclease, responsible for the degradation of transcripts encoding certain pro-inflammatory cytokines. The protein is also known as an inhibitor of NF-κB transcription factor. Thanks to its molecular properties, MCPIP1 is considered as a regulator of inflammation, differentiation, and survival. Using siRNA technology, we show here that MCPIP1 expression contributes to the toxic properties of MG-132 in HeLa cells. The inhibition of proteasome by MG-132 and epoxomicin markedly increased MCPIP1 expression. While MG-132 induces HeLa cell death, down-regulation of MCPIP1 expression by siRNA partially protects HeLa cells from MG-132 toxicity and restores Nuclear factor-κB (NF-κB) activity, inhibited by MG-132 treatment. Inversely, overexpression of MCPIP1 decreased constitutive activity of NF-κB and limited the survival of HeLa cells, as we have shown in the previous study. Interestingly, although MG-132 decreased the expression of IκBα and increased p65 phosphorylation, the inhibition of constitutive NF-κB activity was observed in MG-132-treated cells. Since the elevated constitutive activity of NF-κB is one of the mechanisms providing increased survival of cancer cells, including HeLa cells, we propose that death-promoting properties of MCPIP1 in MG-132-treated HeLa cells may, at least partially, derive from the negative effect on the constitutive NF-κB activity.

Bortezomib as a new therapeutic approach for blastic plasmacytoid dendritic cell neoplasm.

PubMed

Philippe, Laure; Ceroi, Adam; Bôle-Richard, Elodie; Jenvrin, Alizée; Biichle, Sabeha; Perrin, Sophie; Limat, Samuel; Bonnefoy, Francis; Deconinck, Eric; Saas, Philippe; Garnache-Ottou, Francine; Angelot-Delettre, Fanny

2017-11-01

Blastic plasmacytoid dendritic cell neoplasm is an aggressive hematologic malignancy with a poor prognosis. No consensus regarding optimal treatment modalities is currently available. Targeting the nuclear factor-kappa B pathway is considered a promising approach since blastic plasmacytoid dendritic cell neoplasm has been reported to exhibit constitutive activation of this pathway. Moreover, nuclear factor-kappa B inhibition in blastic plasmacytoid dendritic cell neoplasm cell lines, achieved using either an experimental specific inhibitor JSH23 or the clinical drug bortezomib, interferes in vitro with leukemic cell proliferation and survival. Here we extended these data by showing that primary blastic plasmacytoid dendritic cell neoplasm cells from seven patients were sensitive to bortezomib-induced cell death. We confirmed that bortezomib efficiently inhibits the phosphorylation of the RelA nuclear factor-kappa B subunit in blastic plasmacytoid dendritic cell neoplasm cell lines and primary cells from patients in vitro and in vivo in a mouse model. We then demonstrated that bortezomib can be associated with other drugs used in different chemotherapy regimens to improve its impact on leukemic cell death. Indeed, when primary blastic plasmacytoid dendritic cell neoplasm cells from a patient were grafted into mice, bortezomib treatment significantly increased the animals' survival, and was associated with a significant decrease of circulating leukemic cells and RelA nuclear factor-kappa B subunit expression. Overall, our results provide a rationale for the use of bortezomib in combination with other chemotherapy for the treatment of patients with blastic plasmacytoid dendritic cell neoplasm. Based on our data, a prospective clinical trial combining proteasome inhibitor with classical drugs could be envisaged. Copyright© Ferrata Storti Foundation.

Roles for N-terminal Extracellular Domains of Nicotinic Acetylcholine Receptor (nAChR) β3 Subunits in Enhanced Functional Expression of Mouse α6β2β3- and α6β4β3-nAChRs*

PubMed Central

Dash, Bhagirathi; Li, Ming D.; Lukas, Ronald J.

2014-01-01

Functional heterologous expression of naturally expressed mouse α6*-nicotinic acetylcholine receptors (mα6*-nAChRs; where “*” indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mα6mβ4hβ3- (∼5–8-fold) or WT mα6mβ4mβ3-nAChRs (∼2-fold) yielded higher function than mα6mβ4-nAChRs. Function was not detected when mα6 and mβ2 subunits were expressed together or in the additional presence of hβ3 or mβ3 subunits. However, function emerged upon expression of mα6mβ2mβ3V9′S-nAChRs containing β3 subunits having gain-of-function V9′S (valine to serine at the 9′-position) mutations in transmembrane domain II and was further elevated 9-fold when hβ3V9′S subunits were substituted for mβ3V9′S subunits. Studies involving WT or gain-of-function chimeric mouse/human β3 subunits narrowed the search for domains that influence functional expression of mα6*-nAChRs. Using hβ3 subunits as templates for site-directed mutagenesis studies, substitution with mβ3 subunit residues in extracellular N-terminal domain loops “C” (Glu221 and Phe223), “E” (Ser144 and Ser148), and “β2-β3” (Gln94 and Glu101) increased function of mα6mβ2*- (∼2–3-fold) or mα6mβ4* (∼2–4-fold)-nAChRs. EC50 values for nicotine acting at mα6mβ4*-nAChR were unaffected by β3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops β2-β3 and E) interfaces of β3 subunits are some of the molecular impediments for functional expression of mα6mβ2β3- or mα6mβ4β3-nAChRs. PMID:25028511

Inactivation properties of voltage-gated K+ channels altered by presence of beta-subunit.

PubMed

Rettig, J; Heinemann, S H; Wunder, F; Lorra, C; Parcej, D N; Dolly, J O; Pongs, O

1994-05-26

Structural and functional diversity of voltage-gated Kv1-type potassium channels in rat brain is enhanced by the association of two different types of subunits, the membrane-bound, poreforming alpha-subunits and a peripheral beta-subunit. We have cloned a beta-subunit (Kv beta 1) that is specifically expressed in the rat nervous system. Association of Kv beta 1 with alpha-subunits confers rapid A-type inactivation on non-inactivating Kv1 channels (delayed rectifiers) in expression systems in vitro. This effect is mediated by an inactivating ball domain in the Kv beta 1 amino terminus.

Characterization of Puma-Dependent and Puma-Independent Neuronal Cell Death Pathways following Prolonged Proteasomal Inhibition▿

PubMed Central

Tuffy, Liam P.; Concannon, Caoimhín G.; D'Orsi, Beatrice; King, Matthew A.; Woods, Ina; Huber, Heinrich J.; Ward, Manus W.; Prehn, Jochen H. M.

2010-01-01

Proteasomal stress and the accumulation of polyubiquitinated proteins are key features of numerous neurodegenerative disorders. Previously we demonstrated that stabilization of p53 and activation of its target gene, puma (p53-upregulated mediator of apoptosis), mediated proteasome inhibitor-induced apoptosis in cancer cells. Here we demonstrated that Puma also contributed to proteasome inhibitor-induced apoptosis in mouse neocortical neurons. Although protection afforded by puma gene deletion was incomplete, we found little evidence indicating contributions from other proapoptotic BH3-only proteins. Attenuation of bax expression did not further reduce Puma-independent apoptosis, suggesting that pathways other than the mitochondrial apoptosis pathway were activated. Real-time imaging experiments in wild-type and puma-deficient neurons using a fluorescence resonance energy transfer (FRET)-based caspase sensor confirmed the involvement of a second cell death pathway characterized by caspase activation prior to mitochondrial permeabilization and, more prominently, a third, caspase-independent and Puma-independent pathway characterized by rapid cell shrinkage and nuclear condensation. This pathway involved lysosomal permeabilization in the absence of autophagy activation and was sensitive to cathepsin but not autophagy inhibition. Our data demonstrate that proteasomal stress activates distinct cell death pathways in neurons, leading to both caspase-dependent and caspase-independent apoptosis, and demonstrate independent roles for Puma and lysosomal permeabilization in this model. PMID:20921277

AMPA, NMDA and kainate glutamate receptor subunits are expressed in human peripheral blood mononuclear cells (PBMCs) where the expression of GluK4 is altered by pregnancy and GluN2D by depression in pregnant women.

PubMed

Bhandage, Amol K; Jin, Zhe; Hellgren, Charlotte; Korol, Sergiy V; Nowak, Krzysztof; Williamsson, Louise; Sundström-Poromaa, Inger; Birnir, Bryndis

2017-04-15

The amino acid glutamate opens cation permeable ion channels, the iGlu receptors. These ion channels are abundantly expressed in the mammalian brain where glutamate is the main excitatory neurotransmitter. The neurotransmitters and their receptors are being increasingly detected in the cells of immune system. Here we examined the expression of the 18 known subunits of the iGlu receptors families; α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate, N-methyl-d-aspartate (NMDA) and delta in human peripheral blood mononuclear cells (PBMCs). We compared the expression of the subunits between four groups: men, non-pregnant women, healthy pregnant women and depressed pregnant women. Out of 18 subunits of the iGlu receptors, mRNAs for 11 subunits were detected in PBMCs from men and non-pregnant women; AMPA: GluA3, GluA4, kainate: GluK2, GluK4, GluK5, NMDA: GluN1, GluN2C, GluN2D, GluN3A, GluN3B, and delta: GluD1. In the healthy and the depressed pregnant women, in addition, the delta GluD2 subunit was identified. The mRNAs for GluK4, GluK5, GluN2C and GluN2D were expressed at a higher level than other subunits. Gender, pregnancy or depression during pregnancy altered the expression of GluA3, GluK4, GluN2D, GluN3B and GluD1 iGlu subunit mRNAs. The greatest changes recorded were the lower GluA3 and GluK4 mRNA levels in pregnant women and the higher GluN2D mRNA level in healthy but not in depressed pregnant women as compared to non-pregnant individuals. Using subunit specific antibodies, the GluK4, GluK5, GluN1, GluN2C and GluN2D subunit proteins were identified in the PBMCs. The results show expression of specific iGlu receptor subunit in the PBMCs and support the idea of physiology-driven changes of iGlu receptors subtypes in the immune cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of sulfate concentrations on the expression of a soybean seed storage protein gene and its reversibility in transgenic Arabidopsis thaliana.

PubMed

Hirai, M Y; Fujiwara, T; Chino, M; Naito, S

1995-10-01

Transgenic expression of genes encoding the alpha' and beta subunits of beta-conglycinin, one of the major seed storage proteins of soybean (Glycine max [L.] Merr.), was analyzed in Arabidopsis thaliana (L.) Heynh. under conditions of sulfate deficiency. Temporal patterns of expression of both the intact beta subunit gene and the beta subunit gene promoter fused to the beta-glucuronidase (GUS) gene are similar in soil-less cultures using rockwool, suggesting that the response to sulfate deficiency is regulated mainly at the level of transcription. In hydroponic cultures with various concentrations of sulfate, expression of both the intact beta subunit gene and the beta subunit gene promoter-GUS fusion gene were negatively correlated to increased sulfate concentrations in the culture medium. Transfer of transgenic A. thaliana plants carrying the beta subunit gene promoter-GUS fusion from sulfate-deficient to sulfate-sufficient control medium caused GUS activity in developing siliques to be repressed within two days. A reverse shift, where the plants were transferred from the control to sulfate-deficient medium, caused GUS activity to become higher than that in seeds of the control plants within two days. These results indicate that the expression of the beta subunit gene promoter responds rapidly to changes of sulfate availability.

Ubiquitin-proteasomal degradation of COX-2 in TGF-β stimulated human endometrial cells is mediated through endoplasmic reticulum mannosidase I.

PubMed

Singh, Mohan; Chaudhry, Parvesh; Parent, Sophie; Asselin, Eric

2012-01-01

Cyclooxygenase (COX)-2 is a key regulatory enzyme in the production of prostaglandins (PG) during various physiological processes. Mechanisms of COX-2 regulation in human endometrial stromal cells (human endometrial stromal cells) are not fully understood. In this study, we investigate the role of TGF-β in the regulation of COX-2 in human uterine stromal cells. Each TGF-β isoform decreases COX-2 protein level in human uterine stromal cells in Smad2/3-dependent manner. The decrease in COX-2 is accompanied by a decrease in PG synthesis. Knockdown of Smad4 using specific small interfering RNA prevents the decrease in COX-2 protein, confirming that Smad pathway is implicated in the regulation of COX-2 expression in human endometrial stromal cells. Pretreatment with 26S proteasome inhibitor, MG132, significantly restores COX-2 protein and PG synthesis, indicating that COX-2 undergoes proteasomal degradation in the presence of TGF-β. In addition, each TGF-β isoform up-regulates endoplasmic reticulum (ER)-mannosidase I (ERManI) implying that COX-2 degradation is mediated through ER-associated degradation pathway in these cells. Furthermore, inhibition of ERManI activity using the mannosidase inhibitor (kifunensine), or small interfering RNA-mediated knockdown of ERManI, prevents TGF-β-induced COX-2 degradation. Taken together, these studies suggest that TGF-β promotes COX-2 degradation in a Smad-dependent manner by up-regulating the expression of ERManI and thereby enhancing ER-associated degradation and proteasomal degradation pathways.

Human Sex Determination at the Edge of Ambiguity

PubMed Central

Racca, Joseph D.; Chen, Yen-Shan; Yang, Yanwu; Phillips, Nelson B.; Weiss, Michael A.

2016-01-01

A general problem is posed by analysis of transcriptional thresholds governing cell fate decisions in metazoan development. A model is provided by testis determination in therian mammals. Its key step, Sertoli cell differentiation in the embryonic gonadal ridge, is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in human SRY cause gonadal dysgenesis leading to XY female development (Swyer syndrome). Here, we have characterized an inherited mutation compatible with either male or female somatic phenotypes as observed in an XY father and XY daughter, respectively. The mutation (a crevice-forming substitution at a conserved back surface of the SRY high mobility group box) markedly destabilizes the domain but preserves specific DNA affinity and induced DNA bend angle. On transient transfection of diverse human and rodent cell lines, the variant SRY exhibited accelerated proteasomal degradation (relative to wild type) associated with increased ubiquitination; in vitro susceptibility to ubiquitin-independent (“default”) cleavage by the 20S core proteasome was unchanged. The variant's gene regulatory activity (as assessed in a cellular model of the rat embryonic XY gonadal ridge) was reduced by 2-fold relative to wild-type SRY at similar levels of mRNA expression. Chemical proteasome inhibition restored native-like SRY expression and transcriptional activity in association with restored occupancy of a sex-specific enhancer element in principal downstream gene Sox9, demonstrating that the variant SRY exhibits essentially native activity on a per molecule basis. Our findings define a novel mechanism of impaired organogenesis, accelerated ubiquitin-directed proteasomal degradation of a master transcription factor leading to a developmental decision poised at the edge of ambiguity. PMID:27576690

Tripeptidyl peptidase II. An oligomeric protease complex from Arabidopsis.

PubMed

Book, Adam J; Yang, Peizhen; Scalf, Mark; Smith, Lloyd M; Vierstra, Richard D

2005-06-01

The breakdown of most nuclear and cytoplasmic proteins involves their partial cleavage by the 26S proteasome followed by further disassembly to free amino acids by the combined action of endo- and exopeptidases. In animals, one important intermediate exopeptidase is tripeptidyl peptidase (TPP)II, which digests peptide products of the 26S proteasome and other endopeptidases into tripeptides. Here, we describe the purification and characterization of TPPII from Arabidopsis (Arabidopsis thaliana). Like its animal counterparts, Arabidopsis TPPII exists as a soluble, approximately 5- to 9-MD complex. Two related species of 153 and 142 kD are present in the purified preparations that are derived from a single TPP2 gene. Sequencing by Edman degradation of the intact polypeptides and mass spectrometry of proteolytic fragments demonstrated that the 142-kD form mainly differs from the 153-kD form by a truncation at the C-terminal end. This serine protease is a member of the subtilisin superfamily and is sensitive to the inhibitors alanine-alanine-phenylalanine-chloromethylketone and butabindide, which are diagnostic for the TPPII subfamily. The Arabidopsis TPP2 gene is widely expressed in many tissue types with related genes evident in other plant genomes. Whereas the 26S proteasome is essential, TPPII appears not as important for plant physiology. An Arabidopsis T-DNA mutant defective in TPP2 expression displays no phenotypic abnormalities and is not hypersensitive to either amino acid analogs or the 26S proteasome inhibitor MG132. As a consequence, plants likely contain other intermediate exopeptidases that assist in amino acid recycling.

Tripeptidyl Peptidase II. An Oligomeric Protease Complex from Arabidopsis1

PubMed Central

Book, Adam J.; Yang, Peizhen; Scalf, Mark; Smith, Lloyd M.; Vierstra, Richard D.

2005-01-01

The breakdown of most nuclear and cytoplasmic proteins involves their partial cleavage by the 26S proteasome followed by further disassembly to free amino acids by the combined action of endo- and exopeptidases. In animals, one important intermediate exopeptidase is tripeptidyl peptidase (TPP)II, which digests peptide products of the 26S proteasome and other endopeptidases into tripeptides. Here, we describe the purification and characterization of TPPII from Arabidopsis (Arabidopsis thaliana). Like its animal counterparts, Arabidopsis TPPII exists as a soluble, approximately 5- to 9-MD complex. Two related species of 153 and 142 kD are present in the purified preparations that are derived from a single TPP2 gene. Sequencing by Edman degradation of the intact polypeptides and mass spectrometry of proteolytic fragments demonstrated that the 142-kD form mainly differs from the 153-kD form by a truncation at the C-terminal end. This serine protease is a member of the subtilisin superfamily and is sensitive to the inhibitors alanine-alanine-phenylalanine-chloromethylketone and butabindide, which are diagnostic for the TPPII subfamily. The Arabidopsis TPP2 gene is widely expressed in many tissue types with related genes evident in other plant genomes. Whereas the 26S proteasome is essential, TPPII appears not as important for plant physiology. An Arabidopsis T-DNA mutant defective in TPP2 expression displays no phenotypic abnormalities and is not hypersensitive to either amino acid analogs or the 26S proteasome inhibitor MG132. As a consequence, plants likely contain other intermediate exopeptidases that assist in amino acid recycling. PMID:15908606

γ-Aminobutyric Acid Type A α4, β2, and δ Subunits Assemble to Produce More Than One Functionally Distinct Receptor Type

PubMed Central

Eaton, Megan M.; Bracamontes, John; Shu, Hong-Jin; Li, Ping; Mennerick, Steven; Steinbach, Joe Henry

2014-01-01

Native γ-aminobutyric acid (GABA)A receptors consisting of α4, β1–3, and δ subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed α4βδ receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of α4β2δ receptors employing free subunits and concatemeric constructs, expressed in Xenopus oocytes, HEK 293 cells, and cultured hippocampal neurons. The expression system had a strong effect on the properties of receptors containing free subunits. The midpoint of GABA activation curve was 10 nM for receptors in oocytes versus 2300 nM in HEK cells. Receptors activated by the steroid alfaxalone had an estimated maximal open probability of 0.6 in oocytes and 0.01 in HEK cells. Irrespective of the expression system, receptors resulting from combining the tandem construct β2-δ and a free α4 subunit exhibited large steroid responses. We propose that free α4, β2, and δ subunits assemble in different configurations with distinct properties in oocytes and HEK cells, and that subunit linkage can overcome the expression system-dependent preferential assembly of free subunits. Hippocampal neurons transfected with α4 and the picrotoxin-resistant δ(T269Y) subunit showed large responses to alfaxalone in the presence of picrotoxin, suggesting that α4βδ receptors may assemble in a similar configuration in neurons and oocytes. PMID:25238745

cIAPs promote the proteasomal degradation of mutant SOD1 linked to familial amyotrophic lateral sclerosis.

PubMed

Choi, Jin Sun; Kim, Kidae; Lee, Do Hee; Cho, Sayeon; Ha, Jae Du; Park, Byoung Chul; Kim, Sunhong; Park, Sung Goo; Kim, Jeong-Hoon

2016-11-18

Although the ubiquitin-proteasome system is believed to play an important role in the pathogenesis of familial amyotrophic lateral sclerosis (FALS), caused by mutations in Cu/Zn-superoxide dismutase 1 (SOD1), the mechanism of how mutant SOD1 protein is regulated in cells is still poorly understood. Here we have demonstrated that cellular inhibitor of apoptosis proteins (cIAPs) are specifically associated with FALS-linked mutant SOD1 (mSOD1) and that this interaction promotes the ubiquitin-dependent proteasomal degradation of mutant SOD1. By utilizing cumate inducible SOD1 cells, we also showed that knock-down or pharmacologic depletion of cIAPs leads to H 2 O 2 induced cytotoxicity in mSOD1 expressing cells. Altogether, our results reveal a novel role of cIAPs in FALS-associated mutant SOD1 regulation. Copyright © 2016 Elsevier Inc. All rights reserved.

Ubiquitin and Proteasomes in Transcription

PubMed Central

Geng, Fuqiang; Wenzel, Sabine; Tansey, William P.

2013-01-01

Regulation of gene transcription is vitally important for the maintenance of normal cellular homeostasis. Failure to correctly regulate gene expression, or to deal with problems that arise during the transcription process, can lead to cellular catastrophe and disease. One of the ways cells cope with the challenges of transcription is by making extensive use of the proteolytic and nonproteolytic activities of the ubiquitin-proteasome system (UPS). Here, we review recent evidence showing deep mechanistic connections between the transcription and ubiquitin-proteasome systems. Our goal is to leave the reader with a sense that just about every step in transcription—from transcription initiation through to export of mRNA from the nucleus—is influenced by the UPS and that all major arms of the system—from the first step in ubiquitin (Ub) conjugation through to the proteasome—are recruited into transcriptional processes to provide regulation, directionality, and deconstructive power. PMID:22404630

Post-Translational Regulation of Polycystin-2 Protein Expression as a Novel Mechanism of Cholangiocyte Reaction and Repair from Biliary Damage

PubMed Central

Spirli, Carlo; Villani, Ambra; Mariotti, Valeria; Fabris, Luca; Fiorotto, Romina; Strazzabosco, Mario

2015-01-01

Polycystin-2 (PC2 /TRPP2), a member of the transient receptor potential channels (TRP) family, is a non-selective calcium channel. Mutations in PC2/TRPP2 are associated with Polycystic Liver Diseases. PC2-defective cholangiocytes shows increased production of cAMP, PKA-dependent activation of the ERK1/2 pathway, HIF1α-mediated VEGF production, and stimulation of cyst growth and progression. Activation of the ERK/HIF1α/VEGF pathway in cholangiocytes plays a key role during repair from biliary damage. We hypothesized that PC2 levels are modulated during biliary damage/repair, resulting in activation of the ERK/HIF1α/VEGF pathway. Results PC2 protein expression, but not its gene expression, was significantly reduced in mouse livers with biliary damage (Mdr2−/−-KO, bile duct ligation, DDC-treatment). Treatment of colangiocytes with pro-inflammatory cytokines, nitric oxide (NO) donors and ER stressors), increased ERK1/2 phosphorylation, HIF1α transcriptional activity, secretion of VEGF, VEGFR2 phosphorylation and downregulated PC2 protein expression without affecting PC2 gene expression. Expression of Herp and NEK, ubiquitin-like proteins that promote proteosomal PC2 degradation was increased. Pre-treatment with the proteasome inhibitor MG-132 restored the expression of PC2 in cells treated with cytokines but not in cells treated with NO donors or with ER stressors. In these conditions, PC2 degradation was instead inhibited by interfering with the autophagy pathway. Treatment of DDC-mice and of Mdr2−/−-mice with the proteasome inhibitor bortezomib, restored PC2 expression and significantly reduced the ductular reaction, fibrosis and p-ERK1/2. In conclusion, in response to biliary damage, PC2 expression is modulated post-translationally by the proteasome or the autophagy pathways. PC2-dowregulation is associated with activation of ERK1/2 and increase of HIF1α-mediated VEGF secretion. Treatments able to restore PC2 expression and to reduce ductular reaction and fibrosis may represent a new therapeutic approach in biliary diseases. PMID:26313562

A binuclear complex constituted by diethyldithiocarbamate and copper(I) functions as a proteasome activity inhibitor in pancreatic cancer cultures and xenografts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han, Jinbin, E-mail: hanjinbin@gmail.com; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032; Shanghai Clinical Center, Chinese Academy of Sciences/Xuhui Central Hospital, Shanghai 200031

2013-12-15

It is a therapeutic strategy for cancers including pancreatic to inhibit proteasome activity. Disulfiram (DSF) may bind copper (Cu) to form a DSF–Cu complex. DSF–Cu is capable of inducing apoptosis in cancer cells by inhibiting proteasome activity. DSF is rapidly converted to diethyldithiocarbamate (DDTC) within bodies. Copper(II) absorbed by bodies is reduced to copper(I) when it enters cells. We found that DDTC and copper(I) could form a binuclear complex which might be entitled DDTC–Cu(I), and it had been synthesized by us in the laboratory. This study is to investigate the anticancer potential of this complex on pancreatic cancer and themore » possible mechanism. Pancreatic cancer cell lines, SW1990, PANC-1 and BXPC-3 were used for in vitro assays. Female athymic nude mice grown SW1990 xenografts were used as animal models. Cell counting kit-8 (cck-8) assay and flow cytometry were used for analyzing apoptosis in cells. A 20S proteasome assay kit was used in proteasome activity analysis. Western blot (WB) and immunohistochemistry (IHC) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used in tumor sample analysis. The results suggest that DDTC–Cu(I) inhibit pancreatic cancer cell proliferation and proteasome activity in vitro and in vivo. Accumulation of ubiquitinated proteins, and increased p27 as well as decreased NF-κB expression were detected in tumor tissues of DDTC–Cu(I)-treated group. Our data indicates that DDTC–Cu(I) is an effective proteasome activity inhibitor with the potential to be explored as a drug for pancreatic cancer. - Highlights: • A new structure of DDTC–Cu(I) was reported for the first time. • DDTC–Cu(I) dissolved directly in water was for in vitro and in vivo uses. • DDTC–Cu(I) demonstrated significant anticancer effect in vitro and in vivo. • DDTC–Cu(I) is capable of inhibiting proteasome activity in vitro and in vivo.« less

The Na, K-ATPase β-Subunit Isoforms Expression in Glioblastoma Multiforme: Moonlighting Roles

PubMed Central

Rotoli, Deborah; Cejas, Mariana-Mayela; Maeso, María-del-Carmen; Pérez-Rodríguez, Natalia-Dolores; Morales, Manuel; Ávila, Julio

2017-01-01

Glioblastoma multiforme (GBM) is the most common form of malignant glioma. Recent studies point out that gliomas exploit ion channels and transporters, including Na, K-ATPase, to sustain their singular growth and invasion as they invade the brain parenchyma. Moreover, the different isoforms of the β-subunit of Na, K-ATPase have been implicated in regulating cellular dynamics, particularly during cancer progression. The aim of this study was to determine the Na, K-ATPase β subunit isoform subcellular expression patterns in all cell types responsible for microenvironment heterogeneity of GBM using immunohistochemical analysis. All three isoforms, β1, β2/AMOG (Adhesion Molecule On Glia) and β3, were found to be expressed in GBM samples. Generally, β1 isoform was not expressed by astrocytes, in both primary and secondary GBM, although other cell types (endothelial cells, pericytes, telocytes, macrophages) did express this isoform. β2/AMOG and β3 positive expression was observed in the cytoplasm, membrane and nuclear envelope of astrocytes and GFAP (Glial Fibrillary Acidic Protein) negative cells. Interestingly, differences in isoforms expression have been observed between primary and secondary GBM: in secondary GBM, β2 isoform expression in astrocytes was lower than that observed in primary GBM, while the expression of the β3 subunit was more intense. These changes in β subunit isoforms expression in GBM could be related to a different ionic handling, to a different relationship between astrocyte and neuron (β2/AMOG) and to changes in the moonlighting roles of Na, K-ATPase β subunits as adaptor proteins and transcription factors. PMID:29117147

Immunohistochemical analyses of alpha1 and alpha3 Na+/K+-ATPase subunit expression in medulloblastomas.

PubMed

Suñol, Mariona; Cusi, Victoria; Cruz, Ofelia; Kiss, Robert; Lefranc, Florence

2011-03-01

The levels of expression of the α1 and α3 subunits of the Na(+)/K(+)-ATPase (the NaK sodium pump) in medulloblastomas are unclear. This study investigated the expression of the NaK subunits using immunohistochemical methods in 29 medulloblastomas including 23 classic, three large-cell/anaplastic and three nodular/desmoplastic medulloblastomas, as well as in three atypical teratoid/rhabdoid tumors (AT/RTs). There was overexpression of the α1 or α3 NaK subunits in more than half of the medulloblastomas and atypical AT/RTs, with about one-third of these tumours displaying overexpression of both subunits. These preliminary data suggest that targeting these subunits in AT/RTs and medulloblastomas that overexpress these proteins may lead to therapeutic benefit. These findings warrant confirmation in larger numbers of patients than those used in this study. Moreover, it should be determined whether inhibition of the α1/α3 NaK subunits can be integrated into the risk stratification schemes already in use for medulloblastoma patients.

The importance of immunohistochemical analyses in evaluating the phenotype of Kv channel knockout mice.

PubMed

Menegola, Milena; Clark, Eliana; Trimmer, James S

2012-06-01

To gain insights into the phenotype of voltage-gated potassium (Kv)1.1 and Kv4.2 knockout mice, we used immunohistochemistry to analyze the expression of component principal or α subunits and auxiliary subunits of neuronal Kv channels in knockout mouse brains. Genetic ablation of the Kv1.1 α subunit did not result in compensatory changes in the expression levels or subcellular distribution of related ion channel subunits in hippocampal medial perforant path and mossy fiber nerve terminals, where high levels of Kv1.1 are normally expressed. Genetic ablation of the Kv4.2 α subunit did not result in altered neuronal cytoarchitecture of the hippocampus. Although Kv4.2 knockout mice did not exhibit compensatory changes in the expression levels or subcellular distribution of the related Kv4.3 α subunit, we found dramatic decreases in the cellular and subcellular expression of specific Kv channel interacting proteins (KChIPs) that reflected their degree of association and colocalization with Kv4.2 in wild-type mouse and rat brains. These studies highlight the insights that can be gained by performing detailed immunohistochemical analyses of Kv channel knockout mouse brains. Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.

Essential function of VCP/p97 in infection cycle of the nucleopolyhedrovirus AcMNPV in Spodoptera frugiperda Sf9 cells.

PubMed

Lyupina, Yulia V; Erokhov, Pavel A; Kravchuk, Oksana I; Finoshin, Alexander D; Abaturova, Svetlana B; Orlova, Olga V; Beljelarskaya, Svetlana N; Kostyuchenko, Margarita V; Mikhailov, Victor S

2018-06-08

The protein VCP/p97 (also named CDC48 and TER94) belongs to a type II subfamily of the AAA+ATPases and controls cellular proteostasis by acting upstream of proteasomes in the ubiquitin-proteasome protein degradation pathway. The function of VCP/p97 in the baculovirus infection cycle in insect cells remains unknown. Here, we identified VCP/p97 in the fall armyworm Spodoptera frugiperda (Sf9) cells and analyzed the replication of the Autographa californica multiple nucleopolyhedrovirus, AcMNPV, in Sf9 cells in which the VCP/p97 function was inhibited. The specific allosteric inhibitor of the VCP/p97 ATPase activity, NMS-873, did not deplete VCP/p97 in infected cells but caused a dose-dependent inhibition of viral DNA synthesis and efficiently suppressed expression of viral proteins and production of budded virions. NMS-873 caused accumulation of ubiquitinated proteins in a manner similar to the inhibitor of proteasome activity, Bortezomib. This suggests the essential function of VCP/p97 in the baculovirus infection cycle might be associated, at least in part, with the ubiquitin-proteasome system. Copyright © 2018 Elsevier B.V. All rights reserved.

Pterostilbene is equally potent as resveratrol in inhibiting 12-O-tetradecanoylphorbol-13-acetate activated NFkappaB, AP-1, COX-2, and iNOS in mouse epidermis.

PubMed

Cichocki, Michal; Paluszczak, Jaroslaw; Szaefer, Hanna; Piechowiak, Adriana; Rimando, Agnes M; Baer-Dubowska, Wanda

2008-06-01

Resveratrol, a phytoalexin present in grapes, has been reported to inhibit multistage mouse skin carcinogenesis. Recent studies showed that topically applied resveratrol significantly inhibited cyclooxygenase-2 (COX-2) expression and activation of nuclear factor-kappaB (NF-kappaB) induced by tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis. The aim of the present study was to further explore the effect of resveratrol on TPA-induced signaling pathways in mouse epidermis and to compare with its dimethylether, pterostilbene. Resveratrol and pterostilbene significantly reduced activator protein 1 (AP-1) and NF-kappaB activation. In the case of AP-1, the binding of c-Jun subunit was particularly affected, while only slight effect on c-Fos binding to TPA-responsive element (AP-1 binding consensus sequence) (TRE) site was observed. Both stilbenes inhibited the activation of NF-kappaB by blocking the translocation of p65 to the nucleus and increasing the retention of IkappaBa in the cytosol. The latter might be related to decreased activity of IkappaB kinase and/or proteasome 20S. Reduced activation of transcription factors decreased the expression and activity of COX-2 and inducible nitric oxide synthase (iNOS). In most assays, pterostilbene was either equally or significantly more potent than resveratrol. Pterostilbene might show higher biological activity due to its possible better bioavailability, since substitution of hydroxy with methoxy group increases lipophilicity.

Quantitative proteomic analysis of Parkin substrates in Drosophila neurons.

PubMed

Martinez, Aitor; Lectez, Benoit; Ramirez, Juanma; Popp, Oliver; Sutherland, James D; Urbé, Sylvie; Dittmar, Gunnar; Clague, Michael J; Mayor, Ugo

2017-04-11

Parkin (PARK2) is an E3 ubiquitin ligase that is commonly mutated in Familial Parkinson's Disease (PD). In cell culture models, Parkin is recruited to acutely depolarised mitochondria by PINK1. PINK1 activates Parkin activity leading to ubiquitination of multiple proteins, which in turn promotes clearance of mitochondria by mitophagy. Many substrates have been identified using cell culture models in combination with depolarising drugs or proteasome inhibitors, but not in more physiological settings. Here we utilized the recently introduced BioUb strategy to isolate ubiquitinated proteins in flies. Following Parkin Wild-Type (WT) and Parkin Ligase dead (LD) expression we analysed by mass spectrometry and stringent bioinformatics analysis those proteins differentially ubiquitinated to provide the first survey of steady state Parkin substrates using an in vivo model. We further used an in vivo ubiquitination assay to validate one of those substrates in SH-SY5Y cells. We identified 35 proteins that are more prominently ubiquitinated following Parkin over-expression. These include several mitochondrial proteins and a number of endosomal trafficking regulators such as v-ATPase sub-units, Syx5/STX5, ALiX/PDCD6IP and Vps4. We also identified the retromer component, Vps35, another PD-associated gene that has recently been shown to interact genetically with parkin. Importantly, we validated Parkin-dependent ubiquitination of VPS35 in human neuroblastoma cells. Collectively our results provide new leads to the possible physiological functions of Parkin activity that are not overtly biased by acute mitochondrial depolarisation.

Identification of prostate cancer biomarkers in urinary exosomes

PubMed Central

Øverbye, Anders; Skotland, Tore; Koehler, Christian J.; Thiede, Bernd; Seierstad, Therese; Berge, Viktor; Sandvig, Kirsten; Llorente, Alicia

2015-01-01

Exosomes have recently appeared as a novel source of non-invasive cancer biomarkers since tumour-specific molecules can be found in exosomes isolated from biological fluids. We have here investigated the proteome of urinary exosomes by using mass spectrometry to identify proteins differentially expressed in prostate cancer patients compared to healthy male controls. In total, 15 control and 16 prostate cancer samples of urinary exosomes were analyzed. Importantly, 246 proteins were differentially expressed in the two groups. The majority of these proteins (221) were up-regulated in exosomes from prostate cancer patients. These proteins were analyzed according to specific criteria to create a focus list that contained 37 proteins. At 100% specificity, 17 of these proteins displayed individual sensitivities above 60%. Even though several of these proteins showed high sensitivity and specificity for prostate cancer as individual biomarkers, combining them in a multi-panel test has the potential for full differentiation of prostate cancer from non-disease controls. The highest sensitivity, 94%, was observed for transmembrane protein 256 (TM256; chromosome 17 open reading frame 61). LAMTOR proteins were also distinctly enriched with very high specificity for patient samples. TM256 and LAMTOR1 could be used to augment the sensitivity to 100%. Other prominent proteins were V-type proton ATPase 16 kDa proteolipid subunit (VATL), adipogenesis regulatory factor (ADIRF), and several Rab-class members and proteasomal proteins. In conclusion, this study clearly shows the potential of using urinary exosomes in the diagnosis and clinical management of prostate cancer. PMID:26196085

Identification of prostate cancer biomarkers in urinary exosomes.

PubMed

Øverbye, Anders; Skotland, Tore; Koehler, Christian J; Thiede, Bernd; Seierstad, Therese; Berge, Viktor; Sandvig, Kirsten; Llorente, Alicia

2015-10-06

Exosomes have recently appeared as a novel source of non-invasive cancer biomarkers since tumour-specific molecules can be found in exosomes isolated from biological fluids. We have here investigated the proteome of urinary exosomes by using mass spectrometry to identify proteins differentially expressed in prostate cancer patients compared to healthy male controls. In total, 15 control and 16 prostate cancer samples of urinary exosomes were analyzed. Importantly, 246 proteins were differentially expressed in the two groups. The majority of these proteins (221) were up-regulated in exosomes from prostate cancer patients. These proteins were analyzed according to specific criteria to create a focus list that contained 37 proteins. At 100% specificity, 17 of these proteins displayed individual sensitivities above 60%. Even though several of these proteins showed high sensitivity and specificity for prostate cancer as individual biomarkers, combining them in a multi-panel test has the potential for full differentiation of prostate cancer from non-disease controls. The highest sensitivity, 94%, was observed for transmembrane protein 256 (TM256; chromosome 17 open reading frame 61). LAMTOR proteins were also distinctly enriched with very high specificity for patient samples. TM256 and LAMTOR1 could be used to augment the sensitivity to 100%. Other prominent proteins were V-type proton ATPase 16 kDa proteolipid subunit (VATL), adipogenesis regulatory factor (ADIRF), and several Rab-class members and proteasomal proteins. In conclusion, this study clearly shows the potential of using urinary exosomes in the diagnosis and clinical management of prostate cancer.

Sulforaphane enhances proteasomal and autophagic activities in mice and is a potential therapeutic reagent for Huntington's disease.

PubMed

Liu, Yanying; Hettinger, Casey L; Zhang, Dong; Rezvani, Khosrow; Wang, Xuejun; Wang, Hongmin

2014-05-01

The ubiquitin proteasome system (UPS) is impaired in Huntington's disease, a devastating neurodegenerative disorder. Sulforaphane, a naturally occurring compound, has been shown to stimulate UPS activity in cell cultures. To test whether sulforaphane enhances UPS function in vivo, we treated UPS function reporter mice ubiquitously expressing the green fluorescence protein (GFP) fused to a constitutive degradation signal that promotes its rapid degradation in the conditions of a healthy UPS. The modified GFP is termed GFP UPS reporter (GFPu). We found that both GFPu and ubiquitinated protein levels were significantly reduced and the three peptidase activities of the proteasome were increased in the brain and peripheral tissues of the mice. Interestingly, sulforaphane treatment also enhanced autophagy activity in the brain and the liver. To further examine whether sulforaphane promotes mutant huntingtin (mHtt) degradation, we treated Huntington's disease cells with sulforaphane and found that sulforaphane not only enhanced mHtt degradation but also reduced mHtt cytotoxicity. Sulforaphane-mediated mHtt degradation was mainly through the UPS pathway as the presence of a proteasome inhibitor abolished this effect. Taken together, these data indicate that sulforaphane activates protein degradation machineries in both the brain and peripheral tissues and may be a therapeutic reagent for Huntington's disease and other intractable disorders. Accumulation of mutant huntingtin (mHtt) protein causes Huntington's disease (HD). Sulforaphane (SFN), a naturally occurring compound, increased proteasome and autophagy activities in vivo and enhanced mHtt turnover and cell survival in HD cell models. SFN-mediated mHtt degradation is mainly through the proteasome pathway. These data suggest that SFN can be a therapeutic reagent for treating HD and other intractable disorders. © 2014 International Society for Neurochemistry.

Ubiquitin-proteasome system impairment caused by a missense cardiac myosin-binding protein C mutation and associated with cardiac dysfunction in hypertrophic cardiomyopathy.

PubMed

Bahrudin, Udin; Morisaki, Hiroko; Morisaki, Takayuki; Ninomiya, Haruaki; Higaki, Katsumi; Nanba, Eiji; Igawa, Osamu; Takashima, Seiji; Mizuta, Einosuke; Miake, Junichiro; Yamamoto, Yasutaka; Shirayoshi, Yasuaki; Kitakaze, Masafumi; Carrier, Lucie; Hisatome, Ichiro

2008-12-26

The ubiquitin-proteasome system is responsible for the disappearance of truncated cardiac myosin-binding protein C, and the suppression of its activity contributes to cardiac dysfunction. This study investigated whether missense cardiac myosin-binding protein C gene (MYBPC3) mutation in hypertrophic cardiomyopathy (HCM) leads to destabilization of its protein, causes UPS impairment, and is associated with cardiac dysfunction. Mutations were identified in Japanese HCM patients using denaturing HPLC and sequencing. Heterologous expression was investigated in COS-7 cells as well as neonatal rat cardiac myocytes to examine protein stability and proteasome activity. The cardiac function was measured using echocardiography. Five novel MYBPC3 mutations -- E344K, DeltaK814, Delta2864-2865GC, Q998E, and T1046M -- were identified in this study. Compared with the wild type and other mutations, the E334K protein level was significantly lower, it was degraded faster, it had a higher level of polyubiquination, and increased in cells pretreated with the proteasome inhibitor MG132 (50 microM, 6 h). The electrical charge of its amino acid at position 334 influenced its stability, but E334K did not affect its phosphorylation. The E334K protein reduced cellular 20 S proteasome activity, increased the proapoptotic/antiapoptotic protein ratio, and enhanced apoptosis in transfected Cos-7 cells and neonatal rat cardiac myocytes. Patients carrying the E334K mutation presented significant left ventricular dysfunction and dilation. The conclusion is the missense MYBPC3 mutation E334K destabilizes its protein through UPS and may contribute to cardiac dysfunction in HCM through impairment of the ubiquitin-proteasome system.

PML clastosomes prevent nuclear accumulation of mutant ataxin-7 and other polyglutamine proteins

PubMed Central

Janer, Alexandre; Martin, Elodie; Muriel, Marie-Paule; Latouche, Morwena; Fujigasaki, Hiroto; Ruberg, Merle; Brice, Alexis; Trottier, Yvon; Sittler, Annie

2006-01-01

The pathogenesis of spinocerebellar ataxia type 7 and other neurodegenerative polyglutamine (polyQ) disorders correlates with the aberrant accumulation of toxic polyQ-expanded proteins in the nucleus. Promyelocytic leukemia protein (PML) nuclear bodies are often present in polyQ aggregates, but their relation to pathogenesis is unclear. We show that expression of PML isoform IV leads to the formation of distinct nuclear bodies enriched in components of the ubiquitin-proteasome system. These bodies recruit soluble mutant ataxin-7 and promote its degradation by proteasome-dependent proteolysis, thus preventing the aggregate formation. Inversely, disruption of the endogenous nuclear bodies with cadmium increases the nuclear accumulation and aggregation of mutant ataxin-7, demonstrating their role in ataxin-7 turnover. Interestingly, β-interferon treatment, which induces the expression of endogenous PML IV, prevents the accumulation of transiently expressed mutant ataxin-7 without affecting the level of the endogenous wild-type protein. Therefore, clastosomes represent a potential therapeutic target for preventing polyQ disorders. PMID:16818720

The carboxypeptidase angiotensin converting enzyme (ACE) shapes the MHC class I peptide repertoire

PubMed Central

Shen, Xiao Z.; Billet, Sandrine; Lin, Chentao; Okwan-Duodu, Derick; Chen, Xu; Lukacher, Aron E.; Bernstein, Kenneth E.

2011-01-01

The surface presentation of peptides by major histocompatibility complex (MHC) class I molecules is critical to CD8+ T cell mediated adaptive immune responses. Aminopeptidases are implicated in the editing of peptides for MHC class I loading, but C-terminal editing is thought due to proteasome cleavage. By comparing genetically deficient, wild-type and over-expressing mice, we now identify the dipeptidase angiotensin-converting enzyme (ACE) as playing a physiologic role in peptide processing for MHC class I. ACE edits the C-termini of proteasome-produced class I peptides. The lack of ACE exposes novel antigens but also abrogates some self-antigens. ACE has major effects on surface MHC class I expression in a haplotype-dependent manner. We propose a revised model of MHC class I peptide processing by introducing carboxypeptidase activity. PMID:21964607

Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells

PubMed Central

Li, Hewang; Armando, Ines; Yu, Peiying; Escano, Crisanto; Mueller, Susette C.; Asico, Laureano; Pascua, Annabelle; Lu, Quansheng; Wang, Xiaoyan; Villar, Van Anthony M.; Jones, John E.; Wang, Zheng; Periasamy, Ammasi; Lau, Yuen-Sum; Soares-da-Silva, Patricio; Creswell, Karen; Guillemette, Gaétan; Sibley, David R.; Eisner, Gilbert; Felder, Robin A.; Jose, Pedro A.

2008-01-01

Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R. PMID:18464932

Proteasome inhibitor bortezomib enhances the effect of standard chemotherapy in small cell lung cancer

PubMed Central

Taromi, Sanaz; Lewens, Florentine; Arsenic, Ruza; Sedding, Dagmar; Sänger, Jörg; Kunze, Almut; Möbs, Markus; Benecke, Joana; Freitag, Helma; Christen, Friederike; Kaemmerer, Daniel; Lupp, Amelie; Heilmann, Mareike; Lammert, Hedwig; Schneider, Claus-Peter; Richter, Karen; Hummel, Michael; Siegmund, Britta; Burger, Meike; Briest, Franziska; Grabowski, Patricia

2017-01-01

Small cell lung cancer (SCLC) is an aggressive cancer showing a very poor prognosis because of metastasis formation at an early stage and acquisition of chemoresistance. One key driver of chemoresistance is the transcription factor Forkhead box protein M1 (FOXM1) that regulates cell cycle proliferation, maintenance of genomic stability, DNA damage response, and cell differentiation in numerous tumor entities. In this study we investigated the role of FOXM1 in SCLC progression and analyzed the effect of FOXM1 inhibition using two proteasome inhibitors, bortezomib and siomycin A. FOXM1 was strongly expressed in patient-derived SCLC samples (n=123) and its nuclear localization was associated with the proliferation marker Ki-67. Both proteasome inhibitors successfully inhibited FOXM1 expression leading to a significantly reduced proliferation and a decreased mitotic rate along with cell cycle arrest and apoptosis induction. These effects were further enhanced by addition of bortezomib to standard chemotherapy. Treatment of mice bearing chemoresistant SCLC xenografts with bortezomib reduced the mean bioluminescence signal of tumors by 54%. Similarly, treatment with cisplatin as a standard chemotherapy reduced the mean bioluminescence signal of tumors by 58%. However, in combination with standard chemotherapy bortezomib further reduced the mean bioluminescence signal by 93% (p=0.0258). In conclusion, we demonstrate the effect of bortezomib in inhibiting FOXM1 expression and thus in sensitizing resistant SCLC cells to standard chemotherapy. Thus, addition of bortezomib to standard chemotherapy might potently improve SCLC therapy, particularly in an extensive cancer stage. PMID:29228593

Proteasome inhibitor bortezomib enhances the effect of standard chemotherapy in small cell lung cancer.

PubMed

Taromi, Sanaz; Lewens, Florentine; Arsenic, Ruza; Sedding, Dagmar; Sänger, Jörg; Kunze, Almut; Möbs, Markus; Benecke, Joana; Freitag, Helma; Christen, Friederike; Kaemmerer, Daniel; Lupp, Amelie; Heilmann, Mareike; Lammert, Hedwig; Schneider, Claus-Peter; Richter, Karen; Hummel, Michael; Siegmund, Britta; Burger, Meike; Briest, Franziska; Grabowski, Patricia

2017-11-14

Small cell lung cancer (SCLC) is an aggressive cancer showing a very poor prognosis because of metastasis formation at an early stage and acquisition of chemoresistance. One key driver of chemoresistance is the transcription factor Forkhead box protein M1 (FOXM1) that regulates cell cycle proliferation, maintenance of genomic stability, DNA damage response, and cell differentiation in numerous tumor entities. In this study we investigated the role of FOXM1 in SCLC progression and analyzed the effect of FOXM1 inhibition using two proteasome inhibitors, bortezomib and siomycin A. FOXM1 was strongly expressed in patient-derived SCLC samples (n=123) and its nuclear localization was associated with the proliferation marker Ki-67. Both proteasome inhibitors successfully inhibited FOXM1 expression leading to a significantly reduced proliferation and a decreased mitotic rate along with cell cycle arrest and apoptosis induction. These effects were further enhanced by addition of bortezomib to standard chemotherapy. Treatment of mice bearing chemoresistant SCLC xenografts with bortezomib reduced the mean bioluminescence signal of tumors by 54%. Similarly, treatment with cisplatin as a standard chemotherapy reduced the mean bioluminescence signal of tumors by 58%. However, in combination with standard chemotherapy bortezomib further reduced the mean bioluminescence signal by 93% (p=0.0258). In conclusion, we demonstrate the effect of bortezomib in inhibiting FOXM1 expression and thus in sensitizing resistant SCLC cells to standard chemotherapy. Thus, addition of bortezomib to standard chemotherapy might potently improve SCLC therapy, particularly in an extensive cancer stage.

Effects of a Proteasome Inhibitor on Cardiomyocytes in a Pressure-Overload Hypertrophy Rat Model: An Animal Study.

PubMed

Kim, In-Sub; Jo, Won-Min

2017-06-01

The ubiquitin-proteasome system (UPS) is an important pathway of proteolysis in pathologic hypertrophic cardiomyocytes. We hypothesize that MG132, a proteasome inhibitor, might prevent hypertrophic cardiomyopathy (CMP) by blocking the UPS. Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and androgen receptor (AR) have been reported to be mediators of CMP and heart failure. This study drew upon pathophysiologic studies and the analysis of NF-κB and AR to assess the cardioprotective effects of MG132 in a left ventricular hypertrophy (LVH) rat model. We constructed a transverse aortic constriction (TAC)-induced LVH rat model with 3 groups: sham (TAC-sham, n=10), control (TAC-cont, n=10), and MG132 administration (TAC-MG132, n=10). MG-132 (0.1 mg/kg) was injected for 4 weeks in the TAC-MG132 group. Pathophysiologic evaluations were performed and the expression of AR and NF-κB was measured in the left ventricle. Fibrosis was prevalent in the pathologic examination of the TAC-cont model, and it was reduced in the TAC-MG132 group, although not significantly. Less expression of AR, but not NF-κB, was found in the TAC-MG132 group than in the TAC-cont group (p<0.05). MG-132 was found to suppress AR in the TAC-CMP model by blocking the UPS, which reduced fibrosis. However, NF-κB expression levels were not related to UPS function.

Advances in understanding the pathogenesis of primary familial and congenital polycythaemia.

PubMed

Huang, Lily J; Shen, Yu-Min; Bulut, Gamze B

2010-03-01

Primary familial and congenital polycythemia (PFCP) is an autosomal-dominant proliferative disorder characterized by erythrocytosis and hypersensitivity of erythroid progenitors to erythropoietin (Epo). Several lines of evidence suggest a causal role of truncated erythropoietin receptor (EpoR) in this disease. In this review, we discuss PFCP in the context of erythrocytosis and EpoR signalling. We focus on recent studies describing mechanisms underlying Epo-dependent EpoR down-regulation. One mechanism depends on internalization mediated through the p85 regulatory subunit of the Phosphoinositide 3-Kinase, and the other utilizes ubiquitin-based proteasomal degradation. Truncated PFCP EpoRs are not properly down-regulated upon stimulation, underscoring the importance of these mechanisms in the pathogenesis of PFCP.

The effect of α2-δ and other accessory subunits on expression and properties of the calcium channel α1G

PubMed Central

Dolphin, A C; Wyatt, C N; Richards, J; Beattie, R E; Craig, P; Lee, J-H; Cribbs, L L; Volsen, S G; Perez-Reyes, E

1999-01-01

The effect has been examined of the accessory α2-δ and β subunits on the properties of α1G currents expressed in monkey COS-7 cells and Xenopus oocytes. In immunocytochemical experiments, the co-expression of α2-δ increased plasma membrane localization of expressed α1G and conversely, the heterologous expression of α1G increased immunostaining for endogenous α2-δ, suggesting an interaction between the two subunits. Heterologous expression of α2-δ together with α1G in COS-7 cells increased the amplitude of expressed α1G currents by about 2-fold. This finding was confirmed in the Xenopus oocyte expression system. The truncated δ construct did not increase α1G current amplitude, or increase its plasma membrane expression. This indicates that it is the exofacial α2 domain that is involved in the enhancement by α2-δ. β1b also produced an increase of functional expression of α1G, either in the absence or the presence of heterologously expressed α2-δ, whereas the other β subunits had much smaller effects. None of the accessory subunits had any marked influence on the voltage dependence or kinetics of the expressed α1G currents. These results therefore suggest that α2-δ and β1b interact with α1G to increase trafficking of, or stabilize, functional α1G channels expressed at the plasma membrane. PMID:10432337

Genetic expansion of chaperonin-containing TCP-1 (CCT/TRiC) complex subunits yields testis-specific isoforms required for spermatogenesis in planarian flatworms.

PubMed

Counts, Jenna T; Hester, Tasha M; Rouhana, Labib

2017-12-01

Chaperonin-containing Tail-less complex polypeptide 1 (CCT) is a highly conserved, hetero-oligomeric complex that ensures proper folding of actin, tubulin, and regulators of mitosis. Eight subunits (CCT1-8) make up this complex, and every subunit has a homolog expressed in the testes and somatic tissue of the planarian flatworm Schmidtea mediterranea. Gene duplications of four subunits in the genomes of S. mediterranea and other planarian flatworms created paralogs to CCT1, CCT3, CCT4, and CCT8 that are expressed exclusively in the testes. Functional analyses revealed that each CCT subunit expressed in the S. mediterranea soma is essential for homeostatic integrity and survival, whereas sperm elongation defects were observed upon knockdown of each individual testis-specific paralog (Smed-cct1B; Smed-cct3B; Smed-cct4A; and Smed-cct8B), regardless of potential redundancy with paralogs expressed in both testes and soma (Smed-cct1A; Smed-cct3A; Smed-cct4B; and Smed-cct8A). Yet, no detriment was observed in the number of adult somatic stem cells (neoblasts) that maintain differentiated tissue in planarians. Thus, expression of all eight CCT subunits is required to execute the essential functions of the CCT complex. Furthermore, expression of the somatic paralogs in planarian testes is not sufficient to complete spermatogenesis when testis-specific paralogs are knocked down, suggesting that the evolution of chaperonin subunits may drive changes in the development of sperm structure and that correct CCT subunit stoichiometry is crucial for spermiogenesis. © 2017 Wiley Periodicals, Inc.

The sodium pump α1 sub-unit: a disease progression–related target for metastatic melanoma treatment

PubMed Central

Mathieu, Véronique; Pirker, Christine; Martin de Lassalle, Elisabeth; Vernier, Mathieu; Mijatovic, Tatjana; DeNeve, Nancy; Gaussin, Jean-François; Dehoux, Mischael; Lefranc, Florence; Berger, Walter; Kiss, Robert

2009-01-01

Melanomas remain associated with dismal prognosis because they are naturally resistant to apoptosis and they markedly metastasize. Up-regulated expression of sodium pump α sub-units has previously been demonstrated when comparing metastatic to non-metastatic melanomas. Our previous data revealed that impairing sodium pump α1 activity by means of selective ligands, that are cardiotonic steroids, markedly impairs cell migration and kills apoptosis-resistant cancer cells. The objective of this study was to determine the expression levels of sodium pump α sub-units in melanoma clinical samples and cell lines and also to characterize the role of α1 sub-units in melanoma cell biology. Quantitative RT-PCR, Western blotting and immunohistochemistry were used to determine the expression levels of sodium pump α sub-units. In vitro cytotoxicity of various cardenolides and of an anti-α1 siRNA was evaluated by means of MTT assay, quantitative videomicroscopy and through apoptosis assays. The in vivo activity of a novel cardenolide UNBS1450 was evaluated in a melanoma brain metastasis model. Our data show that all investigated human melanoma cell lines expressed high levels of the α1 sub-unit, and 33% of human melanomas displayed significant α1 sub-unit expression in correlation with the Breslow index. Furthermore, cardenolides (notably UNBS1450; currently in Phase I clinical trials) displayed marked anti-tumour effects against melanomas in vitro. This activity was closely paralleled by decreases in cMyc expression and by increases in apoptotic features. UNBS1450 also displayed marked anti-tumour activity in the aggressive human metastatic brain melanoma model in vivo. The α1 sodium pump sub-unit could represent a potential novel target for combating melanoma. PMID:19243476

α2-containing GABAA receptors expressed in hippocampal region CA3 control fast network oscillations

PubMed Central

Heistek, Tim S; Ruiperez-Alonso, Marta; Timmerman, A Jaap; Brussaard, Arjen B; Mansvelder, Huibert D

2013-01-01

GABAA receptors are critically involved in hippocampal oscillations. GABAA receptor α1 and α2 subunits are differentially expressed throughout the hippocampal circuitry and thereby may have distinct contributions to oscillations. It is unknown which GABAA receptor α subunit controls hippocampal oscillations and where these receptors are expressed. To address these questions we used transgenic mice expressing GABAA receptor α1 and/or α2 subunits with point mutations (H101R) that render these receptors insensitive to allosteric modulation at the benzodiazepine binding site, and tested how increased or decreased function of α subunits affects hippocampal oscillations. Positive allosteric modulation by zolpidem prolonged decay kinetics of hippocampal GABAergic synaptic transmission and reduced the frequency of cholinergically induced oscillations. Allosteric modulation of GABAergic receptors in CA3 altered oscillation frequency in CA1, while modulation of GABA receptors in CA1 did not affect oscillations. In mice having a point mutation (H101R) at the GABAA receptor α2 subunit, zolpidem effects on cholinergically induced oscillations were strongly reduced compared to wild-type animals, while zolpidem modulation was still present in mice with the H101R mutation at the α1 subunit. Furthermore, genetic knockout of α2 subunits strongly reduced oscillations, whereas knockout of α1 subunits had no effect. Allosteric modulation of GABAergic receptors was strongly reduced in unitary connections between fast spiking interneurons and pyramidal neurons in CA3 of α2H101R mice, but not of α1H101R mice, suggesting that fast spiking interneuron to pyramidal neuron synapses in CA3 contain α2 subunits. These findings suggest that α2-containing GABAA receptors expressed in the CA3 region provide the inhibition that controls hippocampal rhythm during cholinergically induced oscillations. PMID:23109109

Enhanced p62 expression through impaired proteasomal degradation is involved in caspase-1 activation in monosodium urate crystal-induced interleukin-1b expression.

PubMed

Choe, Jung-Yoon; Jung, Hyun-Young; Park, Ki-Yeun; Kim, Seong-Kyu

2014-06-01

Evidence for the role of autophagy in the regulation of inflammation, especially IL-1b expression in response to monosodium urate (MSU) crystals, is presented. This study investigated the role of p62, a selective autophagy receptor in autophagy, in IL-1b production in MSU crystal-induced inflammation. IL-1b, TNF-a and IL-6 mRNA expression was measured by quantitative real-time PCR (qRTPCR). Autophagy-related molecules such as p62, Cullin-3, microtubule-associated protein 1 light-chain 3 (LC3) I/II, ubiquitin, caspase-1 and mitogen-activated protein kinase (MAPK)-related proteins were measured by immunoblotting. Small interfering RNAs (siRNAs) for Atg16L1, IL-1b and p62 were used to silence each target gene. MSU crystals accelerate the process of autophagosome formation and also induce impairment of proteasomal degradation, resulting in p62 accumulation in autophagy. Enhanced p62 accumulation by MSU crystals leads to IL-1b expression through activation of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), but not p38, of the MAPK pathway and is also involved in activation of caspase-1 in inflammasomes. Impaired autophagosome formation by Atg16L1 siRNA significantly amplified p62 levels, thereby producing enhanced inflammatory responses, including overexpression of IL-1b under stimulation of MSU crystals. IL-1b also induces p62 protein, and blocking IL-1b under stimulation of MSU crystals greatly reduced p62 levels. This study demonstrates that enhanced p62 expression through impaired proteasomal degradation by MSU crystals plays a crucial role in caspase-1 activation in MSU crystal-induced IL-1b production. p62 is required for activation of inflammasomes during acute inflammation in gout.

Astrocytes express specific variants of CaM KII delta and gamma, but not alpha and beta, that determine their cellular localizations.

PubMed

Vallano, M L; Beaman-Hall, C M; Mathur, A; Chen, Q

2000-04-01

Multiple isoforms of type II Ca(2+)-calmodulin-dependent kinase (CaM KII) are composed of two major neuron-specific subunits, designated alpha and beta, and two less well-characterized subunits that are also expressed in non-neuronal tissues, designated delta and gamma. Regulated expression of these 4 gene products, and several variants produced by alternative splicing, shows temporal and regional specificity and influences intracellular targeting. We used immunoblotting and RT-PCR to analyze subunit and variant expression and distribution in cultured cerebellar astrocytes and neurons, and whole cerebellar cortex from rodent brain. The data indicate that: (i) astrocytes express a single splice variant of delta, namely delta(2); (ii) like neurons, astrocytes express two forms of CaM KII gamma; gamma(B) and gamma(A); (iii) these CaM KII variants are enriched in the supernate fraction in astrocytes, and the particulate fraction in neurons; (iv) unlike neurons, astrocytes do not express detectable levels of alpha or beta subunits or their respective splice variants. The results indicate that neurons and astrocytes express distinct CaM KII subunits and variants that localize to distinct subcellular compartments and, by inference, exert distinct cellular functions. Copyright 2000 Wiley-Liss, Inc.

Expression profiles of the Gα subunits during Xenopus tropicalis embryonic development.

PubMed

Fuentealba, Jaime; Toro-Tapia, Gabriela; Rodriguez, Marion; Arriagada, Cecilia; Maureira, Alejandro; Beyer, Andrea; Villaseca, Soraya; Leal, Juan I; Hinrichs, Maria V; Olate, Juan; Caprile, Teresa; Torrejón, Marcela

2016-09-01

Heterotrimeric G protein signaling plays major roles during different cellular events. However, there is a limited understanding of the molecular mechanisms underlying G protein control during embryogenesis. G proteins are highly conserved and can be grouped into four subfamilies according to sequence homology and function. To further studies on G protein function during embryogenesis, the present analysis identified four Gα subunits representative of the different subfamilies and determined their spatiotemporal expression patterns during Xenopus tropicalis embryogenesis. Each of the Gα subunit transcripts was maternally and zygotically expressed, and, as development progressed, dynamic expression patterns were observed. In the early developmental stages, the Gα subunits were expressed in the animal hemisphere and dorsal marginal zone. While expression was observed at the somite boundaries, in vascular structures, in the eye, and in the otic vesicle during the later stages, expression was mainly found in neural tissues, such as the neural tube and, especially, in the cephalic vesicles, neural crest region, and neural crest-derived structures. Together, these results support the pleiotropism and complexity of G protein subfamily functions in different cellular events. The present study constitutes the most comprehensive description to date of the spatiotemporal expression patterns of Gα subunits during vertebrate development. Copyright © 2016 Elsevier B.V. All rights reserved.

The genes for the {alpha}-type HC3 (PMSA2) and {beta}-type HC5 (PMSB1) subunits of human proteasomes map to chromosomes 6q27 and 7p12-p13 by fluorescence in situ hybridization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okumura, Katsuzumi; Nogami, Masahiro; Taguchi, Hiroshi

1995-05-20

The authors have determined the locations of the genes for the two subunits, HC3 and HC5, by fluorescence in situ hybridization (FISH). Chromosome spreads were obtained from phytohemagglutinin-stimulated blood lymphocytes of a healthy donor after thymidine synchronization and bromodeoxyuridine incorporation by the method of Takahashi et al. Genomic DNA fragments of HC3 (4.3 kb, including exons 3, 4, and 5) and HC5 (7.5 kb including exons 1 and 2) (11) were labeled with biotin-16-dUTP by nick-translation. In situ hybridization was performed according to Lichter et al. in the presence of COT-1 DNA as a competitor. Hybridized probe was detected withmore » FITC-conjugated avidin without further signal amplification. Comparison of the fluorescence signals and the banding patterns of the chromosomes indicated that the HC3 and HC5 genes were located on chromosome band 6q27 and 7p12-p13, respectively.« less

FTY720 and (S)-FTY720 vinylphosphonate inhibit sphingosine kinase 1 and promote its proteasomal degradation in human pulmonary artery smooth muscle, breast cancer and androgen-independent prostate cancer cells

PubMed Central

Tonelli, Francesca; Lim, Keng Gat; Loveridge, Carolyn; Long, Jaclyn; Pitson, Stuart M.; Tigyi, Gabor; Bittman, Robert; Pyne, Susan; Pyne, Nigel J.

2010-01-01

Sphingosine kinase 1 (SK1) is an enzyme that catalyses the phosphorylation of sphingosine to produce the bioactive lipid sphingosine 1-phosphate (S1P). We demonstrate here that FTY720 (Fingolimod™) and (S)-FTY720 vinylphosphonate are novel inhibitors of SK1 catalytic activity and induce the proteasomal degradation of this enzyme in human pulmonary artery smooth muscle cells, MCF-7 breast cancer cells and androgen-independent LNCaP-AI prostate cancer cells. Proteasomal degradation of SK1 in response to FTY720 and (S)-FTY720 vinylphosphonate is associated with the down-regulation of the androgen receptor in LNCaP-AI cells. (S)-FTY720 vinylphosphonate also induces the apoptosis of these cells. These findings indicate that SK1 is involved in protecting LNCaP-AI from apoptosis. This protection might be mediated by so-called ‘inside-out’ signalling by S1P, as LNCaP-AI cells exhibit increased expression of S1P2/3 receptors and reduced lipid phosphate phosphatase expression (compared with androgen-sensitive LNCaP cells) thereby potentially increasing the bioavailability of S1P at S1P2/3 receptors. PMID:20570726

The ECS(SPSB) E3 ubiquitin ligase is the master regulator of the lifetime of inducible nitric-oxide synthase.

PubMed

Matsumoto, Kazuma; Nishiya, Tadashi; Maekawa, Satoshi; Horinouchi, Takahiro; Ogasawara, Kouetsu; Uehara, Takashi; Miwa, Soichi

2011-05-27

The ubiquitin-proteasome pathway is an important regulatory system for the lifetime of inducible nitric-oxide synthase (iNOS), a high-output isoform compared to neuronal NOS (nNOS) and endothelial NOS (eNOS), to prevent overproduction of NO that could trigger detrimental effects such as cytotoxicity. Two E3 ubiquitin ligases, Elongin B/C-Cullin-5-SPRY domain- and SOCS box-containing protein [ECS(SPSB)] and the C-terminus of Hsp70-interacting protein (CHIP), recently have been reported to target iNOS for proteasomal degradation. However, the significance of each E3 ubiquitin ligase for the proteasomal degradation of iNOS remains to be determined. Here, we show that ECS(SPSB) specifically interacted with iNOS, but not nNOS and eNOS, and induced the subcellular redistribution of iNOS from dense regions to diffused expression as well as the ubiquitination and proteasomal degradation of iNOS, whereas CHIP neither interacted with iNOS nor had any effects on the subcellular localization, ubiquitination, and proteasomal degradation of iNOS. These results differ from previous reports. Furthermore, the lifetime of the iNOS(N27A) mutant, a form of iNOS that does not bind to ECS(SPSB), was substantially extended in macrophages. These results demonstrate that ECS(SPSB), but not CHIP, is the master regulator of the iNOS lifetime. Copyright © 2011 Elsevier Inc. All rights reserved.

Inhibition of the ubiquitin-proteasome pathway does not protect against ventilator-induced accelerated proteolysis or atrophy in the diaphragm.

PubMed

Smuder, Ashley J; Nelson, W Bradley; Hudson, Matthew B; Kavazis, Andreas N; Powers, Scott K

2014-07-01

Mechanical ventilation (MV) is a life-saving intervention in patients with acute respiratory failure. However, prolonged MV results in ventilator-induced diaphragm dysfunction (VIDD), a condition characterized by both diaphragm fiber atrophy and contractile dysfunction. Previous work has shown that calpain, caspase-3, and the ubiquitin-proteasome pathway (UPP) are all activated in the diaphragm during prolonged MV. However, although it is established that both calpain and caspase-3 are important contributors to VIDD, the role that the UPP plays in the development of VIDD remains unknown. These experiments tested the hypothesis that inhibition of the UPP will protect the diaphragm against VIDD. The authors tested this prediction in an established animal model of MV using a highly specific UPP inhibitor, epoxomicin, to prevent MV-induced activation of the proteasome in the diaphragm (n = 8 per group). The results of this study reveal that inhibition of the UPP did not prevent ventilator-induced diaphragm muscle fiber atrophy and contractile dysfunction during 12 h of MV. Also, inhibition of the UPP does not affect MV-induced increases in calpain and caspase-3 activity in the diaphragm. Finally, administration of the proteasome inhibitor did not protect against the MV-induced increases in the expression of the E3 ligases, muscle ring finger-1 (MuRF1), and atrogin-1/MaFbx. Collectively, these results indicate that proteasome activation does not play a required role in VIDD development during the first 12 h of MV.

Ectopic Expression of α6 and δ GABAA Receptor Subunits in Hilar Somatostatin Neurons Increases Tonic Inhibition and Alters Network Activity in the Dentate Gyrus

PubMed Central

Tong, Xiaoping; Peng, Zechun; Zhang, Nianhui; Cetina, Yliana; Huang, Christine S.; Wallner, Martin; Otis, Thomas S.

2015-01-01

The role of GABAA receptor (GABAAR)-mediated tonic inhibition in interneurons remains unclear and may vary among subgroups. Somatostatin (SOM) interneurons in the hilus of the dentate gyrus show negligible expression of nonsynaptic GABAAR subunits and very low tonic inhibition. To determine the effects of ectopic expression of tonic GABAAR subtypes in these neurons, Cre-dependent viral vectors were used to express GFP-tagged GABAAR subunits (α6 and δ) selectively in hilar SOM neurons in SOM-Cre mice. In single-transfected animals, immunohistochemistry demonstrated strong expression of either the α6 or δ subunit; in cotransfected animals, both subunits were consistently expressed in the same neurons. Electrophysiology revealed a robust increase of tonic current, with progressively larger increases following transfection of δ, α6, and α6/δ subunits, respectively, indicating formation of functional receptors in all conditions and likely coassembly of the subunits in the same receptor following cotransfection. An in vitro model of repetitive bursting was used to determine the effects of increased tonic inhibition in hilar SOM interneurons on circuit activity in the dentate gyrus. Upon cotransfection, the frequency of GABAAR-mediated bursting in granule cells was reduced, consistent with a reduction in synchronous firing among hilar SOM interneurons. Moreover, in vivo studies of Fos expression demonstrated reduced activation of α6/δ-cotransfected neurons following acute seizure induction by pentylenetetrazole. The findings demonstrate that increasing tonic inhibition in hilar SOM interneurons can alter dentate gyrus circuit activity during strong stimulation and suggest that tonic inhibition of interneurons could play a role in regulating excessive synchrony within the network. SIGNIFICANCE STATEMENT In contrast to many hippocampal interneurons, somatostatin (SOM) neurons in the hilus of the dentate gyrus have very low levels of nonsynaptic GABAARs and exhibit very little tonic inhibition. In an effort to increase tonic inhibition selectively in these interneurons, we used Cre-dependent viral vectors in SOM-Cre mice to achieve interneuron-specific expression of the nonsynaptic GABAAR subunits (α6 and δ) in vivo. We show, for the first time, that such recombinant GFP-tagged GABAAR subunits are expressed robustly, assemble to form functional receptors, substantially increase tonic inhibition in SOM interneurons, and alter circuit activity within the dentate gyrus. PMID:26658866

Signal transduction and downregulation of C-MET in HGF stimulated low and highly metastatic human osteosarcoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Husmann, Knut, E-mail: khusmann@research.balgrist.ch; Ducommun, Pascal; Division of Plastic Surgery and Hand Surgery, Department of Surgery, University Hospital Zurich, Zurich

2015-09-04

The poor outcome of osteosarcoma (OS), particularly in patients with metastatic disease and a five-year survival rate of only 20%, asks for more effective therapeutic strategies targeting malignancy-promoting mechanisms. Dysregulation of C-MET, its ligand hepatocyte growth factor (HGF) and the fusion oncogene product TPR-MET, first identified in human MNNG-HOS OS cells, have been described as cancer-causing factors in human cancers. Here, the expression of these molecules at the mRNA and the protein level and of HGF-stimulated signaling and downregulation of C-MET was compared in the parental low metastatic HOS and MG63 cell lines and the respective highly metastatic MNNG-HOS andmore » 143B and the MG63-M6 and MG63-M8 sublines. Interestingly, expression of TPR-MET was only observed in MNNG-HOS cells. HGF stimulated the phosphorylation of Akt and Erk1/2 in all cell lines investigated, but phospho-Stat3 remained at basal levels. Downregulation of HGF-stimulated Akt and Erk1/2 phosphorylation was much faster in the HGF expressing MG63-M8 cells than in HOS cells. Degradation of HGF-activated C-MET occurred predominantly through the proteasomal and to a lesser extent the lysosomal pathway in the cell lines investigated. Thus, HGF-stimulated Akt and Erk1/2 signaling as well as proteasomal degradation of HGF activated C-MET are potential therapeutic targets in OS. - Highlights: • Expression of TPR-MET was only observed in MNNG-HOS cells. • HGF stimulated the phosphorylation of Akt and Erk1/2 but not of Stat3 in osteosarcoma cell lines. • Degradation of HGF-activated C-MET occurred predominantly through the proteasomal pathway.« less

Efficient TGF-β/SMAD signaling in human melanoma cells associated with high c-SKI/SnoN expression

PubMed Central

2011-01-01

Background SKI and SnoN proteins have been shown to inhibit TGF-β signaling, acting both as transcriptional co-repressors in the cell nucleus, and as sequestrators of SMAD proteins in the cytoplasm. TGF-β, on the other hand, induces rapid, proteasome-mediated, degradation of both proteins. How elevated SKI and SnoN protein levels co-exist with active autocrine TGF-β signaling in cancer cells is yet to be understood. Results In this study, we found elevated SKI and SnoN protein levels in a panel of melanoma cell lines, as compared to normal melanocytes. There was no correlation between SKI protein content and the capacity of melanoma cells to invade Matrigel™, to form subcutaneous tumors, or to metastasize to bone after intracardiac inoculation into nude mice. Nor did we find a correlation between SKI expression and histopathological staging of human melanoma. TGF-β induced a rapid and dose-dependent degradation of SKI protein, associated with SMAD3/4 specific transcriptional response and induction of pro-metastatic target genes, partially prevented by pharmacologic blockade of proteasome activity. SKI knockdown in 1205Lu melanoma cells did not alter their invasive capacity or transcriptional responses to TGF-β, and did not allow p21 expression in response to TGF-β or reveal any growth inhibitory activity of TGF-β. Conclusions Despite high expression in melanoma cells, the role of SKI in melanoma remains elusive: SKI does not efficiently interfere with the pro-oncogenic activities of TGF-β, unless stabilized by proteasome blockade. Its highly labile nature makes it an unlikely target for therapeutic intervention. PMID:21211030

Mechanisms of action of acetaldehyde in the up-regulation of the human α2(I) collagen gene in hepatic stellate cells: key roles of Ski, SMAD3, SMAD4, and SMAD7.

PubMed

Reyes-Gordillo, Karina; Shah, Ruchi; Arellanes-Robledo, Jaime; Hernández-Nazara, Zamira; Rincón-Sánchez, Ana Rosa; Inagaki, Yutaka; Rojkind, Marcos; Lakshman, M Raj

2014-05-01

Alcohol-induced liver fibrosis and eventually cirrhosis is a leading cause of death. Acetaldehyde, the first metabolite of ethanol, up-regulates expression of the human α2(I) collagen gene (COL1A2). Early acetaldehyde-mediated effects involve phosphorylation and nuclear translocation of SMAD3/4-containing complexes that bind to COL1A2 promoter to induce fibrogenesis. We used human and mouse hepatic stellate cells to elucidate the mechanisms whereby acetaldehyde up-regulates COL1A2 by modulating the role of Ski and the expression of SMADs 3, 4, and 7. Acetaldehyde induced up-regulation of COL1A2 by 3.5-fold, with concomitant increases in the mRNA (threefold) and protein (4.2- and 3.5-fold) levels of SMAD3 and SMAD4, respectively. It also caused a 60% decrease in SMAD7 expression. Ski, a member of the Ski/Sno oncogene family, is colocalized in the nucleus with SMAD4. Acetaldehyde induces translocation of Ski and SMAD4 to the cytoplasm, where Ski undergoes proteasomal degradation, as confirmed by the ability of the proteasomal inhibitor lactacystin to blunt up-regulation of acetaldehyde-dependent COL1A2, but not of the nonspecific fibronectin gene (FN1). We conclude that acetaldehyde up-regulates COL1A2 by enhancing expression of the transactivators SMAD3 and SMAD4 while inhibiting the repressor SMAD7, along with promoting Ski translocation from the nucleus to cytoplasm. We speculate that drugs that prevent proteasomal degradation of repressors targeting COL1A2 may have antifibrogenic properties. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Mechanisms of Action of Acetaldehyde in the Up-Regulation of the Human α2(I) Collagen Gene in Hepatic Stellate Cells

PubMed Central

Reyes-Gordillo, Karina; Shah, Ruchi; Arellanes-Robledo, Jaime; Hernández-Nazara, Zamira; Rincón-Sánchez, Ana Rosa; Inagaki, Yutaka; Rojkind, Marcos; Lakshman, M. Raj

2015-01-01

Alcohol-induced liver fibrosis and eventually cirrhosis is a leading cause of death. Acetaldehyde, the first metabolite of ethanol, up-regulates expression of the human α2(I) collagen gene (COL1A2). Early acetaldehyde-mediated effects involve phosphorylation and nuclear translocation of SMAD3/4–containing complexes that bind to COL1A2 promoter to induce fibrogenesis. We used human and mouse hepatic stellate cells to elucidate the mechanisms whereby acetaldehyde up-regulates COL1A2 by modulating the role of Ski and the expression of SMADs 3, 4, and 7. Acetaldehyde induced up-regulation of COL1A2 by 3.5-fold, with concomitant increases in the mRNA (threefold) and protein (4.2- and 3.5-fold) levels of SMAD3 and SMAD4, respectively. It also caused a 60% decrease in SMAD7 expression. Ski, a member of the Ski/Sno oncogene family, is colocalized in the nucleus with SMAD4. Acetaldehyde induces translocation of Ski and SMAD4 to the cytoplasm, where Ski undergoes proteasomal degradation, as confirmed by the ability of the proteasomal inhibitor lactacystin to blunt up-regulation of acetaldehyde-dependent COL1A2, but not of the nonspecific fibronectin gene (FN1). We conclude that acetaldehyde up-regulates COL1A2 by enhancing expression of the transactivators SMAD3 and SMAD4 while inhibiting the repressor SMAD7, along with promoting Ski translocation from the nucleus to cytoplasm. We speculate that drugs that prevent proteasomal degradation of repressors targeting COL1A2 may have antifibrogenic properties. PMID:24641900

Involvement of Parkin in the ubiquitin proteasome system-mediated degradation of N-type voltage-gated Ca2+ channels.

PubMed

Grimaldo, Lizbeth; Sandoval, Alejandro; Garza-López, Edgar; Felix, Ricardo

2017-01-01

N-type calcium (CaV2.2) channels are widely expressed in the brain and the peripheral nervous system, where they play important roles in the regulation of transmitter release. Although CaV2.2 channel expression levels are precisely regulated, presently little is known regarding the molecules that mediate its synthesis and degradation. Previously, by using a combination of biochemical and functional analyses, we showed that the complex formed by the light chain 1 of the microtubule-associated protein 1B (LC1-MAP1B) and the ubiquitin-proteasome system (UPS) E2 enzyme UBE2L3, may interact with the CaV2.2 channels promoting ubiquitin-mediated degradation. The present report aims to gain further insights into the possible mechanism of degradation of the neuronal CaV2.2 channel by the UPS. First, we identified the enzymes UBE3A and Parkin, members of the UPS E3 ubiquitin ligase family, as novel CaV2.2 channel binding partners, although evidence to support a direct protein-protein interaction is not yet available. Immunoprecipitation assays confirmed the interaction between UBE3A and Parkin with CaV2.2 channels heterologously expressed in HEK-293 cells and in neural tissues. Parkin, but not UBE3A, overexpression led to a reduced CaV2.2 protein level and decreased current density. Electrophysiological recordings performed in the presence of MG132 prevented the actions of Parkin suggesting enhanced channel proteasomal degradation. Together these results unveil a novel functional coupling between Parkin and the CaV2.2 channels and provide a novel insight into the basic mechanisms of CaV channels protein quality control and functional expression.

HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway (Biochemical and Biophysical Research Communications, v. 444 issue 3)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xiaozhen; Institute of Systems Biology, Peking University, Beijing 100191; Lu, Guang

2014-02-14

Highlights: • The 2000–2634 aa region of HUWE1 mediates the interaction with BRCA1 degron. • HUWE1 promotes the degradation of BRCA1 through the ubiquitin–proteasome pathway. • HUWE1 expression is inversely correlated with BRCA1 in breast cancer cells. • RNAi inhibition of HUWE1 confers increased resistance of MCF-10F cells to IR and MMC. - Abstract: The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the controlmore » of BRCA1 protein level. HUWE1binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.« less

HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway (Biochemical and Biophysical Research Communications, v. 444, isse 4)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xiaozhen; Institute of Systems Biology, Peking University, Beijing 100191; Lu, Guang

2014-02-21

Highlights: • The 2000–2634aa region of HUWE1 mediates the interaction with BRCA1 degron. • HUWE1 promotes the degradation of BRCA1 through the ubiquitin–proteasome pathway. • HUWE1 expression is inversely correlated with BRCA1 in breast cancer cells. • RNAi inhibition of HUWE1 confers increased resistance of MCF-10F cells to IR and MMC. - Abstract: The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control ofmore » BRCA1 protein level. HUWE1 binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.« less

Endothelin-1 (ET-1) induces resistance to bortezomib in human multiple myeloma cells via a pathway involving the ETB receptor and upregulation of proteasomal activity.

PubMed

Vaiou, Maria; Pangou, Evanthia; Liakos, Panagiotis; Sakellaridis, Nikos; Vassilopoulos, George; Dimas, Konstantinos; Papandreou, Christos

2016-10-01

Bortezomib (BTZ) is used for the treatment of multiple myeloma (MM). However, a significant proportion of patients may be refractory to the drug. This study aimed to investigate whether the endothelin (ET-1) axis may act as an escape mechanism to treatment with bortezomib in MM cells. NCI-H929 and RPMI-8226 (human MM cell lines) were cultured with or without ET-1, BTZ, and inhibitors of the endothelin receptors. ET-1 levels were determined by ELISA, while the protein levels of its receptors and of the PI3K and MAPK pathways' components by western blot. Effects of ET-1 on cell proliferation were studied by MTT and on the ubiquitin proteasome pathway by assessing the chymotryptic activity of the 20S proteasome in cell lysates. Endothelin receptors A and B (ETAR and ETBR, respectively) were found to be expressed in both cell lines, with the RPMI-8226 cells that are considered resistant to BTZ, expressing higher levels of ETBR and in addition secreting ET-1. Treatment of the NCI-H929 cells with ET-1 increased proliferation, while co-incubation of these cells with ET-1 and BTZ decreased BTZ efficacy with concomitant upregulation of 20S proteasomal activity. Si-RNA silencing or chemical blockade of ETBR abrogated the protective effects of ET-1. Finally, data suggest that the predominant signaling pathway involved in ET-1/ETBR-induced BTZ resistance in MM cells may be the MAPK pathway. Our data suggest a possible role of the ET-1/ETBR axis in regulating the sensitivity of MM cells to BTZ. Thus, combining bortezomib with strategies to target the ET-1 axis could prove to be a novel promising therapeutic approach in MM.

NMDA receptor expression and C terminus structure in the rotifer Brachionus plicatilis and long-term potentiation across the Metazoa.

PubMed

Kenny, Nathan J; Dearden, Peter K

2013-12-01

The C termini of N-methyl-D-aspartate (NMDA) receptor NR2 subunits are thought to play a major role in the molecular establishment of memory across the Bilateria, via the phenomenon known as long-term potentiation (LTP). Despite their long history of use as models in the study of memory, the expression and structure of the NR2 subunit in the Lophotrochozoa has remained uncategorized. Here, we report the phylogenic relationships of NR subunits across the Bilateria, and the cloning and in situ analysis of expression of NMDA NR1 and NR2 subunits in the monogont rotifer Brachionus plicatilis. RNA in situ hybridization suggests expression of NMDA receptor subunits in B. plicatilis is neural, consistent with expression observed in other species, and ours is the first report confirming NR2 expression in the lophotrochozoan clade. However, the single NR2 subunit identified in B. plicatilis was found to lack the long C terminal domain found in vertebrates, which is believed to modulate LTP. Further investigation revealed that mollusc and annelid NR2 subunits possess long intracellular C terminal domains. As data from molluscs (and particularly Aplysia californica) are the basis for much of our understanding of LTP, understanding how these diverse lophotrochozoan C termini function in vivo will have many implications for how we consider the evolution of the molecular control of learning and memory across the Metazoa as a whole and interpret the results of experiments into this vital component of cognition.

Role of Plant-Specific N-Terminal Domain of Maize CK2β1 Subunit in CK2β Functions and Holoenzyme Regulation

PubMed Central

Vélez-Bermúdez, Isabel C.; Carretero-Paulet, Lorenzo; Lumbreras, Victoria; Pagès, Montserrat

2011-01-01

Protein kinase CK2 is a highly pleiotropic Ser/Thr kinase ubiquituous in eukaryotic organisms. CK2 is organized as a heterotetrameric enzyme composed of two types of subunits: the catalytic (CK2α) and the regulatory (CK2β). The CK2β subunits enhance the stability, activity and specificity of the holoenzyme, but they can also perform functions independently of the CK2 tetramer. CK2β regulatory subunits in plants differ from their animal or yeast counterparts, since they present an additional specific N-terminal extension of about 90 aminoacids that shares no homology with any previously characterized functional domain. Sequence analysis of the N-terminal domain of land plant CK2β subunit sequences reveals its arrangement through short, conserved motifs, some of them including CK2 autophosphorylation sites. By using maize CK2β1 and a deleted version (ΔNCK2β1) lacking the N-terminal domain, we have demonstrated that CK2β1 is autophosphorylated within the N-terminal domain. Moreover, the holoenzyme composed with CK2α1/ΔNCK2β1 is able to phosphorylate different substrates more efficiently than CK2α1/CK2β1 or CK2α alone. Transient overexpression of CK2β1 and ΔNCK2β1 fused to GFP in different plant systems show that the presence of N-terminal domain enhances aggregation in nuclear speckles and stabilizes the protein against proteasome degradation. Finally, bimolecular fluorescence complementation (BiFC) assays show the nuclear and cytoplasmic location of the plant CK2 holoenzyme, in contrast to the individual CK2α/β subunits mainly observed in the nucleus. All together, our results support the hypothesis that the plant-specific N-terminal domain of CK2β subunits is involved in the down-regulation of the CK2 holoenzyme activity and in the stabilization of CK2β1 protein. In summary, the whole amount of data shown in this work suggests that this domain was acquired by plants for regulatory purposes. PMID:21789193

Integrin distributions in renal cell carcinomas of various grades of malignancy.

PubMed Central

Korhonen, M.; Laitinen, L.; Ylänne, J.; Koukoulis, G. K.; Quaranta, V.; Juusela, H.; Gould, V. E.; Virtanen, I.

1992-01-01

We studied 41 renal cell carcinomas, classified according to histologic grades G1 through G3, by indirect immunofluorescence microscopy using a panel of monoclonal antibodies (MAb) against various integrin subunits, and the basement membrane (BM) components laminin and collagen type IV. Selected cases also were immunostained using the avidin-biotin-complex method. The alpha 3 and beta 1 integrin subunits were detected in tumor cells of all the carcinomas. All G1 carcinomas, like normal tubular epithelial cells, expressed the alpha 6 subunit, whereas it was lacking in 20% and 40% of G2 and G3 carcinomas, respectively. Furthermore, when alpha 6 was expressed, a lack of basally polarized organization of the subunit, coupled with disorganization of the BM components, correlated with histologic grade. Another feature that appeared to characterize the more anaplastic tumors was their high level (80%) of the alpha v subunit expression as compared with its absence in the G1 carcinomas. Stromal myofibroblasts, identified by double-labeling with anti-myosin, were often characterized by the expression of the alpha 1, alpha 3, alpha 5 and beta 1 subunits. These results indicate that changes in integrin expression in renal cell carcinomas may be correlated with their degree of histologic malignancy. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:1443050

Specific roles for the Ccr4-Not complex subunits in expression of the genome

PubMed Central

Azzouz, Nowel; Panasenko, Olesya O.; Deluen, Cécile; Hsieh, Julien; Theiler, Grégory; Collart, Martine A.

2009-01-01

In this work we used micro-array experiments to determine the role of each nonessential subunit of the conserved Ccr4-Not complex in the control of gene expression in the yeast Saccharomyces cerevisiae. The study was performed with cells growing exponentially in high glucose and with cells grown to glucose depletion. Specific patterns of gene deregulation were observed upon deletion of any given subunit, revealing the specificity of each subunit's function. Consistently, the purification of the Ccr4-Not complex through Caf40p by tandem affinity purification from wild-type cells or cells lacking individual subunits of the Ccr4-Not complex revealed that each subunit had a particular impact on complex integrity. Furthermore, the micro-arrays revealed that the role of each subunit was specific to the growth conditions. From the study of only two different growth conditions, revealing an impact of the Ccr4-Not complex on more than 85% of all studied genes, we can infer that the Ccr4-Not complex is important for expression of most of the yeast genome. PMID:19155328

Identification of Susceptibility Loci and Genes for Colorectal Cancer Risk.

PubMed

Zeng, Chenjie; Matsuda, Koichi; Jia, Wei-Hua; Chang, Jiang; Kweon, Sun-Seog; Xiang, Yong-Bing; Shin, Aesun; Jee, Sun Ha; Kim, Dong-Hyun; Zhang, Ben; Cai, Qiuyin; Guo, Xingyi; Long, Jirong; Wang, Nan; Courtney, Regina; Pan, Zhi-Zhong; Wu, Chen; Takahashi, Atsushi; Shin, Min-Ho; Matsuo, Keitaro; Matsuda, Fumihiko; Gao, Yu-Tang; Oh, Jae Hwan; Kim, Soriul; Jung, Keum Ji; Ahn, Yoon-Ok; Ren, Zefang; Li, Hong-Lan; Wu, Jie; Shi, Jiajun; Wen, Wanqing; Yang, Gong; Li, Bingshan; Ji, Bu-Tian; Brenner, Hermann; Schoen, Robert E; Küry, Sébastien; Gruber, Stephen B; Schumacher, Fredrick R; Stenzel, Stephanie L; Casey, Graham; Hopper, John L; Jenkins, Mark A; Kim, Hyeong-Rok; Jeong, Jin-Young; Park, Ji Won; Tajima, Kazuo; Cho, Sang-Hee; Kubo, Michiaki; Shu, Xiao-Ou; Lin, Dongxin; Zeng, Yi-Xin; Zheng, Wei

2016-06-01

Known genetic factors explain only a small fraction of genetic variation in colorectal cancer (CRC). We conducted a genome-wide association study to identify risk loci for CRC. This discovery stage included 8027 cases and 22,577 controls of East-Asian ancestry. Promising variants were evaluated in studies including as many as 11,044 cases and 12,047 controls. Tumor-adjacent normal tissues from 188 patients were analyzed to evaluate correlations of risk variants with expression levels of nearby genes. Potential functionality of risk variants were evaluated using public genomic and epigenomic databases. We identified 4 loci associated with CRC risk; P values for the most significant variant in each locus ranged from 3.92 × 10(-8) to 1.24 × 10(-12): 6p21.1 (rs4711689), 8q23.3 (rs2450115, rs6469656), 10q24.3 (rs4919687), and 12p13.3 (rs11064437). We also identified 2 risk variants at loci previously associated with CRC: 10q25.2 (rs10506868) and 20q13.3 (rs6061231). These risk variants, conferring an approximate 10%-18% increase in risk per allele, are located either inside or near protein-coding genes that include transcription factor EB (lysosome biogenesis and autophagy), eukaryotic translation initiation factor 3, subunit H (initiation of translation), cytochrome P450, family 17, subfamily A, polypeptide 1 (steroidogenesis), splA/ryanodine receptor domain and SOCS box containing 2 (proteasome degradation), and ribosomal protein S2 (ribosome biogenesis). Gene expression analyses showed a significant association (P < .05) for rs4711689 with transcription factor EB, rs6469656 with eukaryotic translation initiation factor 3, subunit H, rs11064437 with splA/ryanodine receptor domain and SOCS box containing 2, and rs6061231 with ribosomal protein S2. We identified susceptibility loci and genes associated with CRC risk, linking CRC predisposition to steroid hormone, protein synthesis and degradation, and autophagy pathways and providing added insight into the mechanism of CRC pathogenesis. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

The functional interplay between the HIF pathway and the ubiquitin system - more than a one-way road.

PubMed

Günter, Julia; Ruiz-Serrano, Amalia; Pickel, Christina; Wenger, Roland H; Scholz, Carsten C

2017-07-15

The hypoxia inducible factor (HIF) pathway and the ubiquitin system represent major cellular processes that are involved in the regulation of a plethora of cellular signaling pathways and tissue functions. The ubiquitin system controls the ubiquitination of proteins, which is the covalent linkage of one or several ubiquitin molecules to specific targets. This ubiquitination is catalyzed by approximately 1000 different E3 ubiquitin ligases and can lead to different effects, depending on the type of internal ubiquitin chain linkage. The best-studied function is the targeting of proteins for proteasomal degradation. The activity of E3 ligases is antagonized by proteins called deubiquitinases (or deubiquitinating enzymes), which negatively regulate ubiquitin chains. This is performed in most cases by the catalytic removal of these chains from the targeted protein. The HIF pathway is regulated in an oxygen-dependent manner by oxygen-sensing hydroxylases. Covalent modification of HIFα subunits leads to the recruitment of an E3 ligase complex via the von Hippel-Lindau (VHL) protein and the subsequent polyubiquitination and proteasomal degradation of HIFα subunits, demonstrating the regulation of the HIF pathway by the ubiquitin system. This unidirectional effect of an E3 ligase on the HIF pathway is the best-studied example for the interplay between these two important cellular processes. However, additional regulatory mechanisms of the HIF pathway through the ubiquitin system are emerging and, more recently, also the reciprocal regulation of the ubiquitin system through components of the HIF pathway. Understanding these mechanisms and their relevance for the activity of each other is of major importance for the comprehensive elucidation of the oxygen-dependent regulation of cellular processes. This review describes the current knowledge of the functional bidirectional interplay between the HIF pathway and the ubiquitin system on the protein level. Copyright © 2017 Elsevier Inc. All rights reserved.

NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR.

PubMed

Kainulainen, Markus; Lau, Simone; Samuel, Charles E; Hornung, Veit; Weber, Friedemann

2016-07-01

Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells. Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs requires E3 ubiquitin ligase complexes of the SCF (Skp1, Cul1, F-box protein) type to destroy PKR. SCF-type complexes can engage variant ubiquitination substrate recognition subunits, and we found the F-box proteins FBXW11 and β-TRCP1 to be relevant for the action of NSs against PKR. Thus, we identified the host cell factors that are critically needed by Rift Valley fever virus to uphold its replication against the potent antiviral kinase PKR. Copyright © 2016 Kainulainen et al.

Both Ubiquitin Ligases FBXW8 and PARK2 Are Sequestrated into Insolubility by ATXN2 PolyQ Expansions, but Only FBXW8 Expression Is Dysregulated

PubMed Central

Halbach, Melanie Vanessa; Stehning, Tanja; Damrath, Ewa; Jendrach, Marina; Şen, Nesli Ece; Başak, A. Nazlı; Auburger, Georg

2015-01-01

The involvement of the ubiquitin-proteasome system (UPS) in the course of various age-associated neurodegenerative diseases is well established. The single RING finger type E3 ubiquitin-protein ligase PARK2 is mutated in a Parkinson’s disease (PD) variant and was found to interact with ATXN2, a protein where polyglutamine expansions cause Spinocerebellar ataxia type 2 (SCA2) or increase the risk for Levodopa-responsive PD and for the motor neuron disease Amyotrophic lateral sclerosis (ALS). We previously reported evidence for a transcriptional induction of the multi-subunit RING finger Skp1/Cul/F-box (SCF) type E3 ubiquitin-protein ligase complex component FBXW8 in global microarray profiling of ATXN2-expansion mouse cerebellum and demonstrated its role for ATXN2 degradation in vitro. Now, we documented co-localization in vitro and co-immunoprecipitations both in vitro and in vivo, which indicate associations of FBXW8 with ATXN2 and PARK2. Both FBXW8 and PARK2 proteins are driven into insolubility by expanded ATXN2. Whereas the FBXW8 transcript upregulation by ATXN2- expansion was confirmed also in qPCR of skin fibroblasts and blood samples of SCA2 patients, a FBXW8 expression dysregulation was not observed in ATXN2-deficient mice, nor was a PARK2 transcript dysregulation observed in any samples. Jointly, all available data suggest that the degradation of wildtype and mutant ATXN2 is dependent on FBXW8, and that ATXN2 accumulation selectively modulates FBXW8 levels, while PARK2 might act indirectly through FBXW8. The effects of ATXN2-expansions on FBXW8 expression in peripheral tissues like blood may become useful for clinical diagnostics. PMID:25790475

HIV-1 Vpr Induces the Degradation of ZIP and sZIP, Adaptors of the NuRD Chromatin Remodeling Complex, by Hijacking DCAF1/VprBP

PubMed Central

Maudet, Claire; Sourisce, Adèle; Dragin, Loïc; Lahouassa, Hichem; Rain, Jean-Christophe; Bouaziz, Serge; Ramirez, Bertha Cécilia; Margottin-Goguet, Florence

2013-01-01

The Vpr protein from type 1 and type 2 Human Immunodeficiency Viruses (HIV-1 and HIV-2) is thought to inactivate several host proteins through the hijacking of the DCAF1 adaptor of the Cul4A ubiquitin ligase. Here, we identified two transcriptional regulators, ZIP and sZIP, as Vpr-binding proteins degraded in the presence of Vpr. ZIP and sZIP have been shown to act through the recruitment of the NuRD chromatin remodeling complex. Strikingly, chromatin is the only cellular fraction where Vpr is present together with Cul4A ubiquitin ligase subunits. Components of the NuRD complex and exogenous ZIP and sZIP were also associated with this fraction. Several lines of evidence indicate that Vpr induces ZIP and sZIP degradation by hijacking DCAF1: (i) Vpr induced a drastic decrease of exogenously expressed ZIP and sZIP in a dose-dependent manner, (ii) this decrease relied on the proteasome activity, (iii) ZIP or sZIP degradation was impaired in the presence of a DCAF1-binding deficient Vpr mutant or when DCAF1 expression was silenced. Vpr-mediated ZIP and sZIP degradation did not correlate with the growth-related Vpr activities, namely G2 arrest and G2 arrest-independent cytotoxicity. Nonetheless, infection with HIV-1 viruses expressing Vpr led to the degradation of the two proteins. Altogether our results highlight the existence of two host transcription factors inactivated by Vpr. The role of Vpr-mediated ZIP and sZIP degradation in the HIV-1 replication cycle remains to be deciphered. PMID:24116224

Activated Rac1 requires gp130 for Stat3 activation, cell proliferation and migration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arulanandam, Rozanne; Geletu, Mulu; Feracci, Helene

2010-03-10

Rac1 (Rac) is a member of the Rho family of small GTPases which controls cell migration by regulating the organization of actin filaments. Previous results suggested that mutationally activated forms of the Rho GTPases can activate the Signal Transducer and Activator of Transcription-3 (Stat3), but the exact mechanism is a matter of controversy. We recently demonstrated that Stat3 activity of cultured cells increases dramatically following E-cadherin engagement. To better understand this pathway, we now compared Stat3 activity levels in mouse HC11 cells before and after expression of the mutationally activated Rac1 (Rac{sup V12}), at different cell densities. The results revealedmore » for the first time a dramatic increase in protein levels and activity of both the endogenous Rac and Rac{sup V12} with cell density, which was due to inhibition of proteasomal degradation. In addition, Rac{sup V12}-expressing cells had higher Stat3, tyrosine-705 phosphorylation and activity levels at all densities, indicating that Rac{sup V12} is able to activate Stat3. Further examination of the mechanism of Stat3 activation showed that Rac{sup V12} expression caused a surge in mRNA of Interleukin-6 (IL6) family cytokines, known potent Stat3 activators. Knockdown of gp130, the common subunit of this family reduced Stat3 activity, indicating that these cytokines may be responsible for the Stat3 activation by Rac{sup V12}. The upregulation of IL6 family cytokines was required for cell migration and proliferation induced by Rac{sup V12}, as shown by gp130 knockdown experiments, thus demonstrating that the gp130/Stat3 axis represents an essential effector of activated Rac for the regulation of key cellular functions.« less

FABP4/aP2 Regulates Macrophage Redox Signaling and Inflammasome Activation via Control of UCP2.

PubMed

Steen, Kaylee A; Xu, Hongliang; Bernlohr, David A

2017-01-15

Obesity-linked metabolic disease is mechanistically associated with the accumulation of proinflammatory macrophages in adipose tissue, leading to increased reactive oxygen species (ROS) production and chronic low-grade inflammation. Previous work has demonstrated that deletion of the adipocyte fatty acid-binding protein (FABP4/aP2) uncouples obesity from inflammation via upregulation of the uncoupling protein 2 (UCP2). Here, we demonstrate that ablation of FABP4/aP2 regulates systemic redox capacity and reduces cellular protein sulfhydryl oxidation and, in particular, oxidation of mitochondrial protein cysteine residues. Coincident with the loss of FABP4/aP2 is the upregulation of the antioxidants superoxide dismutase (SOD2), catalase, methionine sulfoxide reductase A, and the 20S proteasome subunits PSMB5 and αβ. Reduced mitochondrial protein oxidation in FABP4/aP2 -/- macrophages attenuates the mitochondrial unfolded-protein response (mtUPR) as measured by expression of heat shock protein 60, Clp protease, and Lon peptidase 1. Consistent with a diminished mtUPR, FABP4/aP2 -/- macrophages exhibit reduced expression of cleaved caspase-1 and NLRP3. Secretion of interleukin 1β (IL-1β), in response to inflammasome activation, is ablated in FABP4/aP2 -/- macrophages, as well as in FABP4/aP2 inhibitor-treated cells, but partially rescued in FABP4/aP2-null macrophages when UCP2 is silenced. Collectively, these data offer a novel pathway whereby FABP4/aP2 regulates macrophage redox signaling and inflammasome activation via control of UCP2 expression. Copyright © 2017 American Society for Microbiology.

BCAS2 interacts with HSF4 and negatively regulates its protein stability via ubiquitination.

PubMed

Liao, Shengjie; Du, Rong; Wang, Lei; Qu, Zhen; Cui, Xiukun; Li, Chang; Liu, Fei; Huang, Mi; Wang, Jiuxiang; Chen, Jiaxiang; Gao, Meng; Yu, Shanshan; Tang, Zhaohui; Li, David Wan-Cheng; Jiang, Tao; Liu, Mugen

2015-11-01

Heat shock factor 4 (HSF4) is an important transcriptional factor that plays a vital role in lens development and differentiation, but the mechanism underlying the regulation of HSF4 is ambiguous. BCAS2 was reported to be an essential subunit of pre-mRNA splicing complex. Here, we identified BCAS2 as a novel HSF4 interacting partner. High expression of BCAS2 in the lens epithelium cells and the bow region of mouse lens was detected by immunohistochemistry. In human lens epithelial cells, BCAS2 negatively regulates HSF4 protein level and transcriptional activity, whereas in BCAS2 knockdown cells, HSF4 protein stability was increased significantly. We further demonstrated that the prolonged protein half-time of HSF4 in BCAS2 knockdown cells was due to reduced ubiquitination. Moreover, we have identified the lysine 206 of HSF4 as the key residue for ubiquitination. The HSF4-K206R mutant blocked the impact of BCAS2 on HSF4 protein stability. Taken together, we identified a pathway for HSF4 degradation through the ubiquitin-proteasome system, and a novel function for BCAS2 that may act as a negative regulatory factor for modulating HSF4 protein homeostasis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Cep55 regulates embryonic growth and development by promoting Akt stability in zebrafish.

PubMed

Jeffery, Jessie; Neyt, Christine; Moore, Wade; Paterson, Scott; Bower, Neil I; Chenevix-Trench, Georgia; Verkade, Heather; Hogan, Benjamin M; Khanna, Kum Kum

2015-05-01

CEP55 was initially described as a centrosome- and midbody-associated protein and a key mediator of cytokinesis. More recently, it has been implicated in PI3K/AKT pathway activation via an interaction with the catalytic subunit of PI3K. However, its role in embryonic development is unknown. Here we describe a cep55 nonsense mutant zebrafish with which we can study the in vivo physiologic role of Cep55. Homozygous mutants underwent extensive apoptosis by 24 hours postfertilization (hpf) concomitant with cell cycle defects, and heterozygous carriers were indistinguishable from their wild-type siblings. A similar phenotype was also observed in zebrafish injected with a cep55 morpholino, suggesting the mutant is a cep55 loss-of-function model. Further analysis revealed that Akt was destabilized in the homozygous mutants, which partially phenocopied Akt1 and Akt2 knockdown. Expression of either constitutively activated PIK3CA or AKT1 could partially rescue the homozygous mutants. Consistent with a role for Cep55 in regulation of Akt stability, treatment with proteasome inhibitor, MG132, partially rescued the homozygous mutants. Taken together, these results provide the first description of Cep55 in development and underline the importance of Cep55 in the regulation of Pi3k/Akt pathway and in particular Akt stability. © FASEB.

Controlling the response to DNA damage by the APC/C-Cdh1.

PubMed

de Boer, H Rudolf; Guerrero Llobet, S; van Vugt, Marcel A T M

2016-03-01

Proper cell cycle progression is safeguarded by the oscillating activities of cyclin/cyclin-dependent kinase complexes. An important player in the regulation of mitotic cyclins is the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit E3 ubiquitin ligase. Prior to entry into mitosis, the APC/C remains inactive, which allows the accumulation of mitotic regulators. APC/C activation requires binding to either the Cdc20 or Cdh1 adaptor protein, which sequentially bind the APC/C and facilitate targeting of multiple mitotic regulators for proteasomal destruction, including Securin and Cyclin B, to ensure proper chromosome segregation and mitotic exit. Emerging data have indicated that the APC/C, particularly in association with Cdh1, also functions prior to mitotic entry. Specifically, the APC/C-Cdh1 is activated in response to DNA damage in G2 phase cells. These observations are in line with in vitro and in vivo genetic studies, in which cells lacking Cdh1 expression display various defects, including impaired DNA repair and aberrant cell cycle checkpoints. In this review, we summarize the current literature on APC/C regulation in response to DNA damage, the functions of APC/C-Cdh1 activation upon DNA damage, and speculate how APC/C-Cdh1 can control cell fate in the context of persistent DNA damage.

A novel ubiquitin-binding protein ZNF216 functioning in muscle atrophy

PubMed Central

Hishiya, Akinori; Iemura, Shun-ichiro; Natsume, Tohru; Takayama, Shinichi; Ikeda, Kyoji; Watanabe, Ken

2006-01-01

The ubiquitin–proteasome system (UPS) is critical for specific degradation of cellular proteins and plays a pivotal role on protein breakdown in muscle atrophy. Here, we show that ZNF216 directly binds polyubiquitin chains through its N-terminal A20-type zinc-finger domain and associates with the 26S proteasome. ZNF216 was colocalized with the aggresome, which contains ubiquitinylated proteins and other UPS components. Expression of Znf216 was increased in both denervation- and fasting-induced muscle atrophy and upregulated by expression of constitutively active FOXO, a master regulator of muscle atrophy. Mice deficient in Znf216 exhibited resistance to denervation-induced atrophy, and ubiquitinylated proteins markedly accumulated in neurectomized muscle compared to wild-type mice. These data suggest that ZNF216 functions in protein degradation via the UPS and plays a crucial role in muscle atrophy. PMID:16424905

A downy mildew effector attenuates salicylic acid-triggered immunity in Arabidopsis by interacting with the host mediator complex.

PubMed

Caillaud, Marie-Cécile; Asai, Shuta; Rallapalli, Ghanasyam; Piquerez, Sophie; Fabro, Georgina; Jones, Jonathan D G

2013-12-01

Plants are continually exposed to pathogen attack but usually remain healthy because they can activate defences upon perception of microbes. However, pathogens have evolved to overcome plant immunity by delivering effectors into the plant cell to attenuate defence, resulting in disease. Recent studies suggest that some effectors may manipulate host transcription, but the specific mechanisms by which such effectors promote susceptibility remain unclear. We study the oomycete downy mildew pathogen of Arabidopsis, Hyaloperonospora arabidopsidis (Hpa), and show here that the nuclear-localized effector HaRxL44 interacts with Mediator subunit 19a (MED19a), resulting in the degradation of MED19a in a proteasome-dependent manner. The Mediator complex of ∼25 proteins is broadly conserved in eukaryotes and mediates the interaction between transcriptional regulators and RNA polymerase II. We found MED19a to be a positive regulator of immunity against Hpa. Expression profiling experiments reveal transcriptional changes resembling jasmonic acid/ethylene (JA/ET) signalling in the presence of HaRxL44, and also 3 d after infection with Hpa. Elevated JA/ET signalling is associated with a decrease in salicylic acid (SA)-triggered immunity (SATI) in Arabidopsis plants expressing HaRxL44 and in med19a loss-of-function mutants, whereas SATI is elevated in plants overexpressing MED19a. Using a PR1::GUS reporter, we discovered that Hpa suppresses PR1 expression specifically in cells containing haustoria, into which RxLR effectors are delivered, but not in nonhaustoriated adjacent cells, which show high PR1::GUS expression levels. Thus, HaRxL44 interferes with Mediator function by degrading MED19, shifting the balance of defence transcription from SA-responsive defence to JA/ET-signalling, and enhancing susceptibility to biotrophs by attenuating SA-dependent gene expression.

A Downy Mildew Effector Attenuates Salicylic Acid–Triggered Immunity in Arabidopsis by Interacting with the Host Mediator Complex

PubMed Central

Caillaud, Marie-Cécile; Asai, Shuta; Rallapalli, Ghanasyam; Piquerez, Sophie; Fabro, Georgina; Jones, Jonathan D. G.

2013-01-01

Plants are continually exposed to pathogen attack but usually remain healthy because they can activate defences upon perception of microbes. However, pathogens have evolved to overcome plant immunity by delivering effectors into the plant cell to attenuate defence, resulting in disease. Recent studies suggest that some effectors may manipulate host transcription, but the specific mechanisms by which such effectors promote susceptibility remain unclear. We study the oomycete downy mildew pathogen of Arabidopsis, Hyaloperonospora arabidopsidis (Hpa), and show here that the nuclear-localized effector HaRxL44 interacts with Mediator subunit 19a (MED19a), resulting in the degradation of MED19a in a proteasome-dependent manner. The Mediator complex of ∼25 proteins is broadly conserved in eukaryotes and mediates the interaction between transcriptional regulators and RNA polymerase II. We found MED19a to be a positive regulator of immunity against Hpa. Expression profiling experiments reveal transcriptional changes resembling jasmonic acid/ethylene (JA/ET) signalling in the presence of HaRxL44, and also 3 d after infection with Hpa. Elevated JA/ET signalling is associated with a decrease in salicylic acid (SA)–triggered immunity (SATI) in Arabidopsis plants expressing HaRxL44 and in med19a loss-of-function mutants, whereas SATI is elevated in plants overexpressing MED19a. Using a PR1::GUS reporter, we discovered that Hpa suppresses PR1 expression specifically in cells containing haustoria, into which RxLR effectors are delivered, but not in nonhaustoriated adjacent cells, which show high PR1::GUS expression levels. Thus, HaRxL44 interferes with Mediator function by degrading MED19, shifting the balance of defence transcription from SA-responsive defence to JA/ET-signalling, and enhancing susceptibility to biotrophs by attenuating SA-dependent gene expression. PMID:24339748