Sample records for ubiquitin-proteasome degradation pathway

  1. 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…

  2. GSH-MODIFIED GAMMAC-CRYSTALLIN IS SELECTIVELY DEGRADED BY THE UBIQUITIN-PROTEASOME PATHWAY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The purpose of this research was to explore the ubiquitin-proteasome pathway (UPP) as an important protein quality control process by which cells selectively degrade and remove damaged proteins. Gamma-crystallin is susceptible to formation of mixed disulfides with GSH upon oxidative stress. The aim ...

  3. The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner.

    PubMed

    Zhao, J; Tenev, T; Martins, L M; Downward, J; Lemoine, N R

    2000-12-01

    Survivin, a human inhibitor of apoptosis protein (IAP), plays an important role in both cell cycle regulation and inhibition of apoptosis. Survivin is expressed in cells during the G(2)/M phase of the cell cycle, followed by rapid decline of both mRNA and protein levels at the G(1) phase. It has been suggested that cell cycle-dependent expression of survivin is regulated at the transcriptional level. In this study we demonstrate involvement of the ubiquitin-proteasome pathway in post-translational regulation of survivin. Survivin is a short-lived protein with a half-life of about 30 minutes and proteasome inhibitors greatly stabilise survivin in vivo. Expression of the survivin gene under the control of the CMV promoter cannot block cell cycle-dependent degradation of the protein. Proteasome inhibitors can block survivin degradation during the G(1) phase and polyubiquitinated derivatives can be detected in vivo. Mutation of critical amino acid residues within the baculovirus IAP repeat (BIR) domain or truncation of the N terminus or the C terminus sensitises survivin to proteasome degradation. Together, these results indicate that the ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner and structural changes greatly destabilise the survivin protein. PMID:11069780

  4. Ubiquitin-proteasome pathway and cellular responses to oxidative stress

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Subs...

  5. Ubiquitin proteasome pathway-mediated degradation of proteins: effects due to site-specific substrate deamidation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The accumulation, aggregation, and precipitation of proteins are etiologic for age-related diseases, particularly cataract, because the precipitates cloud the lens. Deamidation of crystallins is associated with protein precipitation, aging, and cataract. Among the roles of the ubiquitin proteasome p...

  6. Serogroup-related escape of Yersinia enterocolitica YopE from degradation by the ubiquitin-proteasome pathway.

    PubMed

    Hentschke, Moritz; Trülzsch, Konrad; Heesemann, Jürgen; Aepfelbacher, Martin; Ruckdeschel, Klaus

    2007-09-01

    Pathogenic Yersinia spp. employ a type III protein secretion system that translocates several Yersinia outer proteins (Yops) into the host cell to modify the host immune response. One strategy of the infected host cell to resist the bacterial attack is degradation and inactivation of injected bacterial virulence proteins through the ubiquitin-proteasome pathway. The cytotoxin YopE is a known target protein of this major proteolytic system in eukaryotic cells. Here, we investigated the sensitivity of YopE belonging to different enteropathogenic Yersinia enterocolitica serogroups to ubiquitination and proteasomal degradation. Analysis of the YopE protein levels in proteasome inhibitor-treated versus untreated cells revealed that YopE from the highly pathogenic Y. enterocolitica serotype O8 was subjected to proteasomal destabilization, whereas the YopE isotypes from serogroups O3 and O9 evaded degradation. Accumulation of YopE from serotypes O3 and O9 was accompanied by an enhanced cytotoxic effect. Using Yersinia strains that specifically produced YopE from either Y. enterocolitica O8 or O9, we found that only the YopE protein from serogroup O8 was modified by polyubiquitination, although both YopE isotypes were highly homologous. We determined two unique N-terminal lysines (K62 and K75) in serogroup O8 YopE, not present in serogroup O9 YopE, that served as polyubiquitin acceptor sites. Insertion of either lysine in serotype O9 YopE enabled its ubiquitination and destabilization. These results define a serotype-dependent difference in the stability and activity of the Yersinia effector protein YopE that could influence Y. enterocolitica pathogenesis. PMID:17606597

  7. The Ubiquitin-Proteasome Pathway as a Therapeutic Target for Muscle Wasting

    Microsoft Academic Search

    Michael J. Tisdale

    2005-01-01

    Atrophy of skeletal muscle is common to a number of condi- tions, including cancer, sepsis, AIDS, renal failure, diabetes, severe trauma, and burns. In all cases, protein synthesis in skeletal muscle is depressed, whereas protein degradation is increased through an increase in activity and expression of the ubiquitin-proteasome proteolytic pathway. This pathway is not responsive to simple nutritional intervention. Certain

  8. The Ubiquitin-Proteasome Pathway and Synaptic Plasticity

    ERIC Educational Resources Information Center

    Hegde, Ashok N.

    2010-01-01

    Proteolysis by the ubiquitin-proteasome pathway (UPP) has emerged as a new molecular mechanism that controls wide-ranging functions in the nervous system, including fine-tuning of synaptic connections during development and synaptic plasticity in the adult organism. In the UPP, attachment of a small protein, ubiquitin, tags the substrates for…

  9. Ubiquitin/proteasome pathway impairment in neurodegeneration: therapeutic implications

    PubMed Central

    Huang, Qian; Figueiredo-Pereira, Maria E.

    2010-01-01

    The ubiquitin/proteasome pathway is the major proteolytic quality control system in cells. In this review we discuss the impact of a deregulation of this pathway on neuronal function and its causal relationship to the intracellular deposition of ubiquitin protein conjugates in pathological inclusion bodies in all the major chronic neurodegenerative disorders, such as Alzheimer’s, Parkinson’s and Huntington’s diseases as well as amyotrophic lateral sclerosis. We describe the intricate nature of the ubiquitin/proteasome pathway and discuss the paradox of protein aggregation, i.e. its potential toxic/protective effect in neurodegeneration. The relations between some of the dysfunctional components of the pathway and neurodegeneration are presented. We highlight possible ubiquitin/proteasome pathway-targeting therapeutic approaches, such as activating the proteasome, enhancing ubiquitination and promoting SUMOylation that might be important to slow/treat the progression of neurodegeneration. Finally, a model time line is presented for neurodegeneration starting at the initial injurious events up to protein aggregation and cell death, with potential time points for therapeutic intervention. PMID:20131003

  10. c-Cbl and Cbl-b ligases mediate 17-allylaminodemethoxygeldanamycin-induced degradation of autophosphorylated Flt3 kinase with internal tandem duplication through the ubiquitin proteasome pathway.

    PubMed

    Oshikawa, Gaku; Nagao, Toshikage; Wu, Nan; Kurosu, Tetsuya; Miura, Osamu

    2011-09-01

    The class III receptor-tyrosine kinase Flt3 regulates normal hematopoiesis. An internal tandem duplication (ITD) in the juxtamembrane domain of Flt3 (Flt3-ITD) contributes to transformation and is associated with poor prognosis in acute myeloid leukemia. Here, we demonstrate that, as compared with wild-type Flt3 (Flt3-WT), Flt3-ITD more rapidly undergoes degradation through the proteasomal and lysosomal pathways in model hematopoietic 32D cells and in human leukemic MV4-11 cells. The Hsp90 inhibitor 17-allylaminodemethoxygeldanamycin (17-AAG) preferentially induced the polyubiquitination and proteasomal degradation of Flt3-ITD autophosphorylated on Tyr-591 in these cells. The E3 ubiquitin ligases c-Cbl and to a lesser extent Cbl-b facilitated at least partly Lys-48-linked polyubiquitination of autophosphorylated Flt3-ITD when coexpressed in 293T cells. Moreover, c-Cbl and Cbl-b facilitated degradation of Flt3-ITD in 293T cells and significantly enhanced the 17-AAG-induced decline in autophosphorylated Flt3-ITD. The enhancement of Flt3-ITD degradation was also observed in 32D cells inducibly overexpressing c-Cbl or Cbl-b. Furthermore, overexpression of loss-of-function mutants of both c-Cbl (c-Cbl-R420Q) and Cbl-b (Cbl-b-C373A) together in 32D cells retarded the degradation of autophosphorylated Flt3-ITD and significantly inhibited the 17-AAG-induced degradation of Flt3-ITD to confer the resistance to cytotoxicity of 17-AAG on these cells. These results suggest that c-Cbl as well as Cbl-b may play important roles in Hsp90 inhibitor-induced degradation of Flt3-ITD through the ubiquitin proteasome system and in regulation of the basal expression level of Flt3-ITD in leukemic cells. PMID:21768087

  11. c-Cbl and Cbl-b Ligases Mediate 17-Allylaminodemethoxygeldanamycin-induced Degradation of Autophosphorylated Flt3 Kinase with Internal Tandem Duplication through the Ubiquitin Proteasome Pathway*

    PubMed Central

    Oshikawa, Gaku; Nagao, Toshikage; Wu, Nan; Kurosu, Tetsuya; Miura, Osamu

    2011-01-01

    The class III receptor-tyrosine kinase Flt3 regulates normal hematopoiesis. An internal tandem duplication (ITD) in the juxtamembrane domain of Flt3 (Flt3-ITD) contributes to transformation and is associated with poor prognosis in acute myeloid leukemia. Here, we demonstrate that, as compared with wild-type Flt3 (Flt3-WT), Flt3-ITD more rapidly undergoes degradation through the proteasomal and lysosomal pathways in model hematopoietic 32D cells and in human leukemic MV4-11 cells. The Hsp90 inhibitor 17-allylaminodemethoxygeldanamycin (17-AAG) preferentially induced the polyubiquitination and proteasomal degradation of Flt3-ITD autophosphorylated on Tyr-591 in these cells. The E3 ubiquitin ligases c-Cbl and to a lesser extent Cbl-b facilitated at least partly Lys-48-linked polyubiquitination of autophosphorylated Flt3-ITD when coexpressed in 293T cells. Moreover, c-Cbl and Cbl-b facilitated degradation of Flt3-ITD in 293T cells and significantly enhanced the 17-AAG-induced decline in autophosphorylated Flt3-ITD. The enhancement of Flt3-ITD degradation was also observed in 32D cells inducibly overexpressing c-Cbl or Cbl-b. Furthermore, overexpression of loss-of-function mutants of both c-Cbl (c-Cbl-R420Q) and Cbl-b (Cbl-b-C373A) together in 32D cells retarded the degradation of autophosphorylated Flt3-ITD and significantly inhibited the 17-AAG-induced degradation of Flt3-ITD to confer the resistance to cytotoxicity of 17-AAG on these cells. These results suggest that c-Cbl as well as Cbl-b may play important roles in Hsp90 inhibitor-induced degradation of Flt3-ITD through the ubiquitin proteasome system and in regulation of the basal expression level of Flt3-ITD in leukemic cells. PMID:21768087

  12. Fucoidan inhibition of lung cancer in vivo and in vitro: role of the Smurf2-dependent ubiquitin proteasome pathway in TGF? receptor degradation

    PubMed Central

    Wu, Yu-Chung; Tsao, Shu-Ming; Hwang, Pai-An; Shih, Yu-Wei; Hsu, Jason

    2014-01-01

    Fucoidan, a polysaccharide extracted from brown seaweeds, reduces tumor cell proliferation. In this study, we demonstrate that fucoidan reduces tumor size in LLC1-xenograft male C57BL/6 mice. Moreover, we found that LLC1-bearing mice continuously fed fucoidan showed greater antitumor activity than mice with discontinuous feeding. Fucoidan inhibited the in vitro growth of lung cancer cells. Transforming growth factor ? (TGF?) receptors (TGFRs) play important roles in the regulation of proliferation and progression, and high TGFRI expression in lung cancer specimens is associated with a worse prognosis. Herein, using lung cancer cells, we found that fucoidan effectively reduces TGFRI and TGFRII protein levels in vivo and in vitro. Moreover, fucoidan reduces TGFR downstream signaling events, including those in Smad2/3 and non-Smad pathways: Akt, Erk1/2, and FAK phosphorylation. Furthermore, fucoidan suppresses lung cancer cell mobility upon TGF? stimulation. To elucidate how fucoidan decreases TGFR proteins in lung cancer cells, we found that fucoidan enhances the ubiquitination proteasome pathway (UPP)-mediated degradation of TGFRs in A549 and CL1-5 cells. Mechanistically, fucoidan promotes Smurf2 and Smad7 to conjugate TGFRs, resulting in TGF degradation; however, Smurf2-shRNA abolishes fucoidan-enhanced UPP-mediated TGFR degradation. Our study is the first to identify a novel mechanism for the antitumor activity of fucoidan, namely decreasing tumor growth by modulating the TGFR/Smad7/Smurf2-dependent axis, leading to TGFR protein degradation and inhibition of lung cancer cell progression in vitro and in vivo. Our current findings indicate that fucoidan is a potential therapeutic agent or dietary supplementation for lung cancer, acting via the Smurf2-dependent ubiquitin degradation of TGF? receptors. PMID:25149540

  13. Pristimerin Induces Apoptosis in Prostate Cancer Cells by Down-regulating Bcl-2 through ROS-dependent Ubiquitin-proteasomal Degradation Pathway

    PubMed Central

    Liu, Yong Bo; Gao, Xiaohua; Deeb, Dorrah; Arbab, Ali S; Gautam, Subhash C

    2014-01-01

    Pristimerin is a quinonemethide triterpenoid with the potential of a promising anticancer agent. Pristimerin (PM) has shown anticancer activity against a range of cancer cell lines, but its activity for prostate cancer has not been adequately investigated. In the present study we have examined the underlying mechanisms of the apoptotic response of the hormone-sensitive (LNCaP) and hormone-refractory (PC-3) prostate cancer cell lines to PM. Treatment with PM induced apoptosis in both cell lines as characterized by increased annexin V-binding and cleavage of PARP-1 and procaspases-3 and -9. It also induced mitochondrial depolarization, cytochrome c release from mitochondria and generation of reactive oxygen species (ROS). Response to PM is regulated by Bcl-2 since it down-regulated Bcl-2 expression and overexpression of Bcl-2 rendered prostate cancer cells resistant to PM. ROS plays a role in down-regulation of Bcl-2, since treatment with PM in the presence of various ROS modulators, e.g., n-acetylcysteine (NAC), a general purpose antioxidant; diphenylene iodonium (DPI), a NADPH inhibitor; rotenone (ROT), a mitochondrial electron transport chain interrupter rotenone or MnTBAP, a O2 scavenger, attenuated the down-regulation of Bcl-2. Furthermore, ROS is also involved in the ubiquitination and proteasomal degradation of Bcl-2 as both of these events were blocked by O 2? scavenger MnTBAP. Thus, pristimerin induces apoptosis in prostate cancer cells predominately through the mitochondrial apoptotic pathway by inhibiting antiapoptic Bcl-2 through a ROS-dependent ubiquitin-proteasomal degradation pathway. PMID:24877026

  14. What do we really know about the ubiquitin-proteasome pathway in muscle atrophy?

    NASA Technical Reports Server (NTRS)

    Jagoe, R. T.; Goldberg, A. L.

    2001-01-01

    Studies of many different rodent models of muscle wasting have indicated that accelerated proteolysis via the ubiquitin-proteasome pathway is the principal cause of muscle atrophy induced by fasting, cancer cachexia, metabolic acidosis, denervation, disuse, diabetes, sepsis, burns, hyperthyroidism and excess glucocorticoids. However, our understanding about how muscle proteins are degraded, and how the ubiquitin-proteasome pathway is activated in muscle under these conditions, is still very limited. The identities of the important ubiquitin-protein ligases in skeletal muscle, and the ways in which they recognize substrates are still largely unknown. Recent in-vitro studies have suggested that one set of ubquitination enzymes, E2(14K) and E3(alpha), which are responsible for the 'N-end rule' system of ubiquitination, plays an important role in muscle, especially in catabolic states. However, their functional significance in degrading different muscle proteins is still unclear. This review focuses on the many gaps in our understanding of the functioning of the ubiquitin-proteasome pathway in muscle atrophy, and highlights the strengths and limitations of the different experimental approaches used in such studies.

  15. Coupling caspase cleavage and ubiquitin-proteasome-dependent degradation of SSRP1 during apoptosis.

    PubMed

    Landais, I; Lee, H; Lu, H

    2006-11-01

    Structure-specific recognition protein (SSRP1) is an 87 kDa protein that heterodimerizes with Spt16 to form FACT, a complex initially shown to facilitate chromatin transcription. Despite its crucial roles in transcription and replication, little is known about the dynamics of FACT turnover in vivo. Here, we show that SSRP1 is cleaved during apoptosis by caspase 3 and/or 7 at the DQHD(450) site. Analysis of the resulting fragments suggests that cleavage of SSRP1 generates a truncated, chromatin-associated form of FACT. Furthermore, the N-terminal product is stabilized by proteasome inhibitors and ubiquitylated in cells, suggesting degradation through the ubiquitin-proteasome pathway. These results demonstrate that SSRP1 degradation during apoptosis is a two-step process coupling caspase cleavage and ubiquitin-dependent proteolysis. PMID:16498457

  16. Protein Degradation by Ubiquitin-Proteasome System in Formation and Labilization of Contextual Conditioning Memory

    ERIC Educational Resources Information Center

    Fustiñana, María Sol; de la Fuente, Verónica; Federman, Noel; Freudenthal, Ramiro; Romano, Arturo

    2014-01-01

    The ubiquitin-proteasome system (UPS) of protein degradation has been evaluated in different forms of neural plasticity and memory. The role of UPS in such processes is controversial. Several results support the idea that the activation of this system in memory consolidation is necessary to overcome negative constrains for plasticity. In this…

  17. The Ubiquitin-Proteasome Pathway and Plant Development Jennifer Moon, Geraint Parry, and Mark Estelle1

    E-print Network

    Estelle, Mark

    REVIEW The Ubiquitin-Proteasome Pathway and Plant Development Jennifer Moon, Geraint Parry particle (Groll and Huber, 2003). The 19S regulatory particle can be further divided into lid and base and for removing the Ub chains. The base contains several subunits that work to unfold the substrate. As a whole

  18. PTEN Increases Autophagy and Inhibits the Ubiquitin-Proteasome Pathway in Glioma Cells Independently of its Lipid Phosphatase Activity

    PubMed Central

    Errafiy, Rajaa; Aguado, Carmen; Ghislat, Ghita; Esteve, Juan M.; Gil, Anabel; Loutfi, Mohammed; Knecht, Erwin

    2013-01-01

    Two major mechanisms of intracellular protein degradation, autophagy and the ubiquitin-proteasome pathway, operate in mammalian cells. PTEN, which is frequently mutated in glioblastomas, is a tumor suppressor gene that encodes a dual specificity phosphatase that antagonizes the phosphatidylinositol 3-kinase class I/AKT/mTOR pathway, which is a key regulator of autophagy. Here, we investigated in U87MG human glioma cells the role of PTEN in the regulation of autophagy and the ubiquitin-proteasome pathway, because both are functionally linked and are relevant in cancer progression. Since U87MG glioma cells lack a functional PTEN, we used stable clones that express, under the control of a tetracycline-inducible system (Tet-on), wild-type PTEN and two of its mutants, G129E-PTEN and C124S-PTEN, which, respectively, lack the lipid phosphatase activity only and both the lipid and the protein phosphatase activities of this protein. Expression of PTEN in U87MG glioma cells decreased proteasome activity and also reduced protein ubiquitination. On the contrary, expression of PTEN increased the autophagic flux and the lysosomal mass. Interestingly, and although PTEN negatively regulates the phosphatidylinositol 3-kinase class I/AKT/mTOR signaling pathway by its lipid phosphatase activity, both effects in U87MG cells were independent of this activity. These results suggest a new mTOR-independent signaling pathway by which PTEN can regulate in opposite directions the main mechanisms of intracellular protein degradation. PMID:24349488

  19. PTEN increases autophagy and inhibits the ubiquitin-proteasome pathway in glioma cells independently of its lipid phosphatase activity.

    PubMed

    Errafiy, Rajaa; Aguado, Carmen; Ghislat, Ghita; Esteve, Juan M; Gil, Anabel; Loutfi, Mohammed; Knecht, Erwin

    2013-01-01

    Two major mechanisms of intracellular protein degradation, autophagy and the ubiquitin-proteasome pathway, operate in mammalian cells. PTEN, which is frequently mutated in glioblastomas, is a tumor suppressor gene that encodes a dual specificity phosphatase that antagonizes the phosphatidylinositol 3-kinase class I/AKT/mTOR pathway, which is a key regulator of autophagy. Here, we investigated in U87MG human glioma cells the role of PTEN in the regulation of autophagy and the ubiquitin-proteasome pathway, because both are functionally linked and are relevant in cancer progression. Since U87MG glioma cells lack a functional PTEN, we used stable clones that express, under the control of a tetracycline-inducible system (Tet-on), wild-type PTEN and two of its mutants, G129E-PTEN and C124S-PTEN, which, respectively, lack the lipid phosphatase activity only and both the lipid and the protein phosphatase activities of this protein. Expression of PTEN in U87MG glioma cells decreased proteasome activity and also reduced protein ubiquitination. On the contrary, expression of PTEN increased the autophagic flux and the lysosomal mass. Interestingly, and although PTEN negatively regulates the phosphatidylinositol 3-kinase class I/AKT/mTOR signaling pathway by its lipid phosphatase activity, both effects in U87MG cells were independent of this activity. These results suggest a new mTOR-independent signaling pathway by which PTEN can regulate in opposite directions the main mechanisms of intracellular protein degradation. PMID:24349488

  20. TGF-? Induces Degradation of PTHrP Through Ubiquitin-Proteasome System in Hepatocellular Carcinoma.

    PubMed

    Li, Hao; He, Guangchun; Yao, Hui; Song, Liujiang; Zeng, Liang; Peng, Xiaoning; Rosol, Thomas J; Deng, Xiyun

    2015-01-01

    Both transforming growth factor-? (TGF-?) and parathyroid hormone-related protein (PTHrP) regulate important cellular processes, such as apoptosis in the development of hepatocellular carcinoma. However, the mechanisms of regulation of PTHrP by TGF-? are largely unknown. We hypothesized that TGF-? regulates the expression of PTHrP protein through a post-translational mechanism. Using hepatocellular carcinoma cell lines as the in vitro model, we investigated the effects of TGF-? on protein expression and post-translational processing of PTHrP. We found that TGF-? treatment led to protein degradation of PTHrP through the ubiquitin-proteasome-dependent pathway. We also provided evidence to show that Smurf2 was the E3 ligase responsible for the ubiquitination of PTHrP. Furthermore, using immunohistochemistry on human hepatocellular carcinoma specimens and a tissue array, we found that the expression of PTHrP was predominantly in the cancer cells, whereas the expression of TGF-? was present in non-neoplastic liver tissue adjacent to hepatocellular carcinoma. Our findings reveal a novel mechanism whereby TGF-? may regulate PTHrP in hepatocellular carcinogenesis and lack of TGF-? in hepatocellular carcinoma may promote cancer progression. Promotion of PTHrP degradation provides a novel target of therapeutic intervention to sensitize hepatocellular carcinoma cells to cytostatic and/or pro-apoptotic signals. PMID:26000041

  1. TGF-? Induces Degradation of PTHrP Through Ubiquitin-Proteasome System in Hepatocellular Carcinoma

    PubMed Central

    Li, Hao; He, Guangchun; Yao, Hui; Song, Liujiang; Zeng, Liang; Peng, Xiaoning; Rosol, Thomas J.; Deng, Xiyun

    2015-01-01

    Both transforming growth factor-? (TGF-?) and parathyroid hormone-related protein (PTHrP) regulate important cellular processes, such as apoptosis in the development of hepatocellular carcinoma. However, the mechanisms of regulation of PTHrP by TGF-? are largely unknown. We hypothesized that TGF-? regulates the expression of PTHrP protein through a post-translational mechanism. Using hepatocellular carcinoma cell lines as the in vitro model, we investigated the effects of TGF-? on protein expression and post-translational processing of PTHrP. We found that TGF-? treatment led to protein degradation of PTHrP through the ubiquitin-proteasome-dependent pathway. We also provided evidence to show that Smurf2 was the E3 ligase responsible for the ubiquitination of PTHrP. Furthermore, using immunohistochemistry on human hepatocellular carcinoma specimens and a tissue array, we found that the expression of PTHrP was predominantly in the cancer cells, whereas the expression of TGF-? was present in non-neoplastic liver tissue adjacent to hepatocellular carcinoma. Our findings reveal a novel mechanism whereby TGF-? may regulate PTHrP in hepatocellular carcinogenesis and lack of TGF-? in hepatocellular carcinoma may promote cancer progression. Promotion of PTHrP degradation provides a novel target of therapeutic intervention to sensitize hepatocellular carcinoma cells to cytostatic and/or pro-apoptotic signals. PMID:26000041

  2. Ubiquitin-proteasomal degradation of antiapoptotic survivin facilitates induction of apoptosis in prostate cancer cells by pristimerin

    PubMed Central

    LIU, YONG BO; GAO, XIAOHUA; DEEB, DORAH; BRIGOLIN, CHRIS; ZHANG, YIGUAN; SHAW, JIAJIU; PINDOLIA, KIRIT; GAUTAM, SUBHASH C.

    2014-01-01

    Pristimerin (PM), a quinonemethide triterpenoid, is a promising anticancer agent with potent antiproliferative and apoptosis-inducing activities against cancer cell lines. However, the anticancer activity and mechanisms of PM in prostate cancer cells have not been adequately investigated. Here we report that the degradation of survivin plays an important role in the antiproliferative and proapoptotic effects of PM in carcinoma of the prostate (CaP) cell lines. Treatment with PM inhibited proliferation and induced apoptosis in LNCaP and PC-3 cells as characterized by the loss of cell viability and an increase in Annexin V-binding and cleavage of PARP-1, respectively. The antiproliferative and apoptosis-inducing effects of PM were associated with the inhibition of cell cycle regulatory proteins, antiapoptotic survivin and members of the Bcl-2 family. Data showed that response to PM is regulated by survivin since overexpression of survivin rendered CaP cells resistant to PM. Furthermore, downregulation of survivin by PM was mediated through the ubiquitin-proteasomal degradation. Together, these data demonstrate that pristimerin inhibits proliferation and induces apoptosis in CaP cells by abolishing survivin through the ubiquitin-proteasome pathway. PMID:25175770

  3. Malformed selenoproteins are removed by the ubiquitin--proteasome pathway in Stanleya pinnata.

    PubMed

    Sabbagh, Melissa; Van Hoewyk, Doug

    2012-03-01

    Despite the widely accepted belief that selenium toxicity in plants is manifested by the misincorporation of selenocysteine into selenoproteins, there is a lack of data suggesting that selenoproteins are malformed or misfolded. Plant mechanisms to prevent the formation of selenoproteins are associated with increased selenium tolerance, yet there is no evidence to suggest that selenoproteins are malformed or potentially misfolded. We reasoned that if selenoproteins are malformed, then they might be degraded by the ubiquitin-proteasome pathway. The data demonstrate that selenate treatment induced the accumulation of both oxidized and ubiquitinated proteins, thus implicating both the 20S and 26S proteasome of Stanleya pinnata, a selenium-hyperaccumulating plant, in a selenate response. Inhibition of the proteasome increases the amount of selenium incorporated into protein, but not other elements. Furthermore, a higher percentage of selenium was found in a ubiquitinated protein fraction compared with other elements, suggesting that malformed selenoproteins are preferentially ubiquitinated and removed by the proteasome. Additionally, levels of the 20S and 26S proteasome and two heat shock proteins increase upon selenate treatment. Arabidopsis mutants with defects in the 26S proteasome have decreased selenium tolerance, which further supports the hypothesis that the 26S proteasome probably prevents selenium toxicity by removing selenoproteins. PMID:22323770

  4. Intranuclear Degradation of Polyglutamine Aggregates by the Ubiquitin-Proteasome System*S?

    PubMed Central

    Iwata, Atsushi; Nagashima, Yu; Matsumoto, Lumine; Suzuki, Takahiro; Yamanaka, Tomoyuki; Date, Hidetoshi; Deoka, Ken; Nukina, Nobuyuki; Tsuji, Shoji

    2009-01-01

    Huntington disease and its related autosomal-dominant polyglutamine (pQ) neurodegenerative diseases are characterized by intraneuronal accumulation of protein aggregates. Studies on protein aggregates have revealed the importance of the ubiquitin-proteasome system as the front line of protein quality control (PQC) machinery against aberrant proteins. Recently, we have shown that the autophagy-lysosomal system is also involved in cytoplasmic aggregate degradation, but the nucleus lacked this activity. Consequently, the nucleus relies entirely on the ubiquitin-proteasome system for PQC. According to previous studies, nuclear aggregates possess a higher cellular toxicity than do their cytoplasmic counterparts, however degradation kinetics of nuclear aggregates have been poorly understood. Here we show that nuclear ubiquitin ligases San1p and UHRF-2 each enhance nuclear pQ aggregate degradation and rescued pQ-induced cytotoxicity in cultured cells and primary neurons. Moreover, UHRF-2 is associated with nuclear inclusion bodies in vitro and in vivo. Our data suggest that UHRF-2 is an essential molecule for nuclear pQ degradation as a component of nuclear PQC machinery in mammalian cells. PMID:19218238

  5. Regulation of ubiquitin-proteasome and autophagy pathways after acute LPS and epoxomicin administration in mice

    PubMed Central

    2014-01-01

    Background The ubiquitin-proteasome pathway (UPP) is a major protein degradation pathway that is activated during sepsis and has been proposed as a therapeutic target for preventing skeletal muscle loss due to cachexia. Although several studies have investigated the modulation of proteasome activity in response to LPS administration, none have characterized the overall UPP response to LPS administration in the fate of proteasome inhibition. Methods Here, we determined the modulation pattern of the main key components of the UPP in the gastrocnemius (GAS) of mice during the acute phase of lipopolysaccharide (LPS)-mediated endotoxemia (7.5 mg/kg – 8 h) by measuring all three ?1, ?2 and ?5 activites of the 20S and 26S proteasomes, the levels of steady state polyubiquitinated proteins, mRNA levels of muscle ligases, as well as signaling pathways regulating the UPP. Another goal was to assess the effects of administration of a specific proteasome inhibitor (epoxomicin, 0.5 mg/kg) on UPP response to sepsis. Results The acute phase of LPS-induced endotoxemia lowered GAS/body weight ratio and increased MuRF1 and MAFbx mRNA concomitantly to an activation of the pathways known to regulate their expression. Unexpectedly, we observed a decrease in all 20S and 26S proteasome activities measured in GAS, which might be related to oxidative stress, as oxidized proteins (carbonyl levels) increase with LPS. While significantly inhibiting 20S and 26S proteasome ?5 activities in heart and liver, epoxomicin did not lower proteasome activity in GAS. However, the increase in mRNA expression of the muscle ligases MuRF1 and MAFbx were partially rescued without affecting the other investigated signaling pathways. LPS also strongly activated autophagy, which could explain the observed GAS atrophy with LPS-induced reduction of proteasome activity. Conclusions Our results highlight an opposite regulation of UPP in the early hours of LPS-induced muscle atrophy by showing reduced proteasome activities and increased mRNA expression of muscle specific ligases. Furthermore, our data do not support any preventive effect of epoxomicin in muscle atrophy due to acute cachexia since proteasome activities are not further repressed. PMID:24885455

  6. Ubiquitin-proteasome dependent degradation of GABAA?1 in autism spectrum disorder

    PubMed Central

    2014-01-01

    Background Although the neurobiological basis of autism spectrum disorder (ASD) is not fully understood, recent studies have indicated the potential role of GABAA receptors in the pathophysiology of ASD. GABAA receptors play a crucial role in various neurodevelopmental processes and adult neuroplasticity. However, the mechanism(s) of regulation of GABAA receptors in ASD remains poorly understood. Methods Postmortem middle frontal gyrus tissues (13 ASD and 13 control subjects) were used. In vitro studies were performed in primary cortical neurons at days in vitro (DIV) 14. The protein levels were examined by western blotting. Immunofluorescence studies were employed for cellular localization. The gene expression was determined by RT-PCR array and qRT-PCR. Results A significant decrease in GABAA?1 protein, but not mRNA levels was found in the middle frontal gyrus of ASD subjects indicating a post-translational regulation of GABAA receptors in ASD. At the cellular level, treatment with proteasomal inhibitor, MG132, or lactacystin significantly increased GABAA?1 protein levels and Lys48-linked polyubiquitination of GABAA?1, but reduced proteasome activity in mouse primary cortical neurons (DIV 14 from E16 embryos). Moreover, treatment with betulinic acid, a proteasome activator significantly decreased GABAA?1 protein levels in cortical neurons indicating the role of polyubiquitination of GABAA?1 proteins with their subsequent proteasomal degradation in cortical neurons. Ubiquitination specific RT-PCR array followed by western blot analysis revealed a significant increase in SYVN1, an endoplasmic reticulum (ER)-associated degradation (ERAD) E3 ubiquitin ligase in the middle frontal gyrus of ASD subjects. In addition, the inhibition of proteasomal activity by MG132 increased the expression of GABAA?1 in the ER. The siRNA knockdown of SYVN1 significantly increased GABAA?1 protein levels in cortical neurons. Moreover, reduced association between SYVN1 and GABAA?1 was found in the middle frontal gyrus of ASD subjects. Conclusions SYVN1 plays a critical role as an E3 ligase in the ubiquitin proteasome system (UPS)-mediated GABAA?1 degradation. Thus, inhibition of the ubiquitin-proteasome-mediated GABAA?1 degradation may be an important mechanism for preventing GABAA?1 turnover to maintain GABAA?1 levels and GABA signaling in ASD. PMID:25392730

  7. TLR4 mediates the impairment of ubiquitin-proteasome and autophagy-lysosome pathways induced by ethanol treatment in brain

    PubMed Central

    Pla, A; Pascual, M; Renau-Piqueras, J; Guerri, C

    2014-01-01

    New evidence indicates the involvement of protein degradation dysfunctions in neurodegeneration, innate immunity response and alcohol hepatotoxicity. We recently demonstrated that ethanol increases brain proinflammatory mediators and causes brain damage by activating Toll-like receptor 4 (TLR4) signaling in glia. However, it is uncertain if the ubiquitin-proteasome and autophagy-lysosome pathways are involved in ethanol-induced brain damage and whether the TLR4 response is implicated in proteolytic processes. Using the cerebral cortex of WT and TLR4-knockout mice with and without chronic ethanol treatment, we demonstrate that ethanol induces poly-ubiquitinated proteins accumulation and promotes immunoproteasome activation by inducing the expression of ?2i, ?5i and PA28?, although it decreases the 20S constitutive proteasome subunits (?2, ?5). Ethanol also upregulates mTOR phosphorylation, leading to a downregulation of the autophagy-lysosome pathway (ATG12, ATG5, cathepsin B, p62, LC3) and alters the volume of autophagic vacuoles. Notably, mice lacking TLR4 receptors are protected against ethanol-induced alterations in protein degradation pathways. In summary, the present results provide the first evidence demonstrating that chronic ethanol treatment causes proteolysis dysfunctions in the mouse cerebral cortex and that these events are TLR4 dependent. These findings could provide insight into the mechanisms underlying ethanol-induced brain damage. PMID:24556681

  8. Formation of distinct inclusion bodies by inhibition of ubiquitin-proteasome and autophagy-lysosome pathways

    SciTech Connect

    Lee, Junho; Yang, Kyu-Hwan; Joe, Cheol O. [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejon 305-701 (Korea, Republic of)] [Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejon 305-701 (Korea, Republic of); Kang, Seok-Seong, E-mail: sskang@ivi.int [Laboratory Sciences Division, International Vaccine Institute, Seoul 151-919 (Korea, Republic of)] [Laboratory Sciences Division, International Vaccine Institute, Seoul 151-919 (Korea, Republic of)

    2011-01-14

    Research highlights: {yields} Distinct inclusion bodies are developed by inhibition of UPP and ALP. {yields} The inclusion bodies differ in morphology, localization and formation process. {yields} The inclusion bodies are distinguishable by the localization of TSC2. {yields} Inhibition of both UPP and ALP simultaneously induces those inclusion bodies. -- Abstract: Accumulation of misfolded proteins is caused by the impairment of protein quality control systems, such as ubiquitin-proteasome pathway (UPP) and autophagy-lysosome pathway (ALP). In this study, the formation of inclusion bodies was examined after the blockade of UPP and/or ALP in A549 cells. UPP inhibition induced a single and large inclusion body localized in microtubule-organizing center. Interestingly, however, ALP inhibition generated dispersed small inclusion bodies in the cytoplasm. Tuberous sclerosis complex 2 was selectively accumulated in the inclusion bodies of UPP-inhibited cells, but not those of ALP-inhibited cells. Blockade of transcription and translation entirely inhibited the formation of inclusion body induced by UPP inhibition, but partially by ALP inhibition. Moreover, the simultaneous inhibition of two protein catabolic pathways independently developed two distinct inclusion bodies within a single cell. These findings clearly demonstrated that dysfunction of each catabolic pathway induced formation and accumulation of unique inclusion bodies on the basis of morphology, localization and formation process in A549 cells.

  9. Activation of the Ubiquitin Proteasome Pathway by Silk Fibroin Modified Chitosan Nanoparticles in Hepatic Cancer Cells

    PubMed Central

    Yang, Ming-Hui; Chung, Tze-Wen; Lu, Yi-Shan; Chen, Yi-Ling; Tsai, Wan-Chi; Jong, Shiang-Bin; Yuan, Shyng-Shiou; Liao, Pao-Chi; Lin, Po-Chiao; Tyan, Yu-Chang

    2015-01-01

    Silk fibroin (SF) is a protein with bulky hydrophobic domains and can be easily purified as sericin-free silk-based biomaterial. Silk fibroin modified chitosan nanoparticle (SF-CSNP), a biocompatible material, has been widely used as a potential drug delivery system. Our current investigation studied the bio-effects of the SF-CSNP uptake by liver cells. In this experiment, the characterizations of SF-CSNPs were measured by particle size analysis and protein assay. The average size of the SF-CSNP was 311.9 ± 10.7 nm, and the average zeta potential was +13.33 ± 0.3 mV. The SF coating on the SF-CSNP was 6.27 ± 0.17 ?g/mL. Moreover, using proteomic approaches, several proteins involved in the ubiquitin proteasome pathway were identified by analysis of differential protein expressions of HepG2 cell uptake the SF-CSNP. Our experimental results have demonstrated that the SF-CSNP may be involved in liver cancer cell survival and proliferation. PMID:25588218

  10. Activation of the ubiquitin proteasome pathway by silk fibroin modified chitosan nanoparticles in hepatic cancer cells.

    PubMed

    Yang, Ming-Hui; Chung, Tze-Wen; Lu, Yi-Shan; Chen, Yi-Ling; Tsai, Wan-Chi; Jong, Shiang-Bin; Yuan, Shyng-Shiou; Liao, Pao-Chi; Lin, Po-Chiao; Tyan, Yu-Chang

    2015-01-01

    Silk fibroin (SF) is a protein with bulky hydrophobic domains and can be easily purified as sericin-free silk-based biomaterial. Silk fibroin modified chitosan nanoparticle (SF-CSNP), a biocompatible material, has been widely used as a potential drug delivery system. Our current investigation studied the bio-effects of the SF-CSNP uptake by liver cells. In this experiment, the characterizations of SF-CSNPs were measured by particle size analysis and protein assay. The average size of the SF-CSNP was 311.9 ± 10.7 nm, and the average zeta potential was +13.33 ± 0.3 mV. The SF coating on the SF-CSNP was 6.27 ± 0.17 ?g/mL. Moreover, using proteomic approaches, several proteins involved in the ubiquitin proteasome pathway were identified by analysis of differential protein expressions of HepG2 cell uptake the SF-CSNP. Our experimental results have demonstrated that the SF-CSNP may be involved in liver cancer cell survival and proliferation. PMID:25588218

  11. The acidosis of chronic renal failure activates muscle proteolysis in rats by augmenting transcription of genes encoding proteins of the ATP-dependent ubiquitin-proteasome pathway.

    PubMed Central

    Bailey, J L; Wang, X; England, B K; Price, S R; Ding, X; Mitch, W E

    1996-01-01

    Chronic renal failure (CRF) is associated with negative nitrogen balance and loss of lean body mass. To identify specific proteolytic pathways activated by CRF, protein degradation was measured in incubated epitrochlearis muscles from CRF and sham-operated, pair-fed rats. CRF stimulated muscle proteolysis, and inhibition of lysosomal and calcium-activated proteases did not eliminate this increase. When ATP production was blocked, proteolysis in CRF muscles fell to the same level as that in control muscles. Increased proteolysis was also prevented by feeding CRF rats sodium bicarbonate, suggesting that activation depends on acidification. Evidence that the ATP-dependent ubiquitin-proteasome pathway is stimulated by the acidemia of CRF includes the following findings: (a) An inhibitor of the proteasome eliminated the increase in muscle proteolysis; and (b) there was an increase in mRNAs encoding ubiquitin (324%) and proteasome subunits C3 (137%) and C9 (251%) in muscle. This response involved gene activation since transcription of mRNAs for ubiquitin and the C3 subunit were selectively increased in muscle of CRF rats. We conclude that CRF stimulates muscle proteolysis by activating the ATP-ubiquitin-proteasome-dependent pathway. The mechanism depends on acidification and increased expression of genes encoding components of the system. These responses could contribute to the loss of muscle mass associated with CRF. PMID:8617877

  12. CDK11{sup p58} represses vitamin D receptor-mediated transcriptional activation through promoting its ubiquitin-proteasome degradation

    SciTech Connect

    Chi, Yayun; Hong, Yi; Zong, Hongliang; Wang, Yanlin; Zou, Weiying; Yang, Junwu; Kong, Xiangfei; Yun, Xiaojing [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China)] [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China); Gu, Jianxin, E-mail: jxgu@shmu.edu.cn [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China)] [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China)

    2009-08-28

    Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates transcription of target genes. In this study, we identified CDK11{sup p58} as a novel protein involved in the regulation of VDR. CDK11{sup p58}, a member of the large family of p34cdc2-related kinases, is associated with cell cycle progression, tumorigenesis, and apoptotic signaling. Our study demonstrated that CDK11{sup p58} interacted with VDR and repressed VDR-dependent transcriptional activation. Furthermore, overexpression of CDK11{sup p58} decreased the stability of VDR through promoting its ubiquitin-proteasome-mediated degradation. Taken together, these results suggest that CDK11{sup p58} is involved in the negative regulation of VDR.

  13. Docosahexaenoic acid induces the degradation of HPV E6/E7 oncoproteins by activating the ubiquitin–proteasome system

    PubMed Central

    Jing, K; Shin, S; Jeong, S; Kim, S; Song, K-S; Park, J-H; Heo, J-Y; Seo, K-S; Park, S-K; Kweon, G-R; Wu, T; Park, J-I; Lim, K

    2014-01-01

    The oncogenic human papillomavirus (HPV) E6/E7 proteins are essential for the onset and maintenance of HPV-associated malignancies. Here, we report that activation of the cellular ubiquitin–proteasome system (UPS) by the omega-3 fatty acid, docosahexaenoic acid (DHA), leads to proteasome-mediated degradation of E6/E7 viral proteins and the induction of apoptosis in HPV-infected cancer cells. The increases in UPS activity and degradation of E6/E7 oncoproteins were associated with DHA-induced overproduction of mitochondrial reactive oxygen species (ROS). Exogenous oxidative stress and pharmacological induction of mitochondrial ROS showed effects similar to those of DHA, and inhibition of ROS production abolished UPS activation, E6/E7 viral protein destabilization, and apoptosis. These findings identify a novel role for DHA in the regulation of UPS and viral proteins, and provide evidence for the use of DHA as a mechanistically unique anticancer agent for the chemoprevention and treatment of HPV-associated tumors. PMID:25393480

  14. The ubiquitin–proteasome pathway protects Chlamydomonas reinhardtii against selenite toxicity, but is impaired as reactive oxygen species accumulate

    PubMed Central

    Vallentine, Patrick; Hung, Chiu-Yueh; Xie, Jiahua; Van Hoewyk, Doug

    2014-01-01

    The ubiquitin–proteasome pathway (UPP) coordinates a myriad of physiological processes in higher plants, including abiotic stress responses, but it is less well characterized in algal species. In this study, the green alga Chlamydomonas reinhardtii was used to gain insights into the role of the UPP during moderate and severe selenite stress at three different time points. The data indicate that activity of the UPP in response to selenium (Se) stress was both time and dose dependent. Moderate selenite stress increased proteasome activity, protein ubiquitination and the proteasomal removal of malformed selenoproteins. However, severe Se stress caused by prolonged selenite treatment or high selenite concentration decreased proteasome activity, inhibited protein ubiquitination and prevented the proteasomal removal of selenoproteins. The UPP impairment during severe Se stress was associated with the observed accumulation of reactive oxygen species (ROS), including mitochondrial superoxide. Additionally, proteasomal inhibition decreased the concentration of chlorophyll in cultures challenged with Se. Therefore, although the UPP protects Chlamydomonas against Se stress, severe oxidative stress induced by selenite toxicity likely hinders the UPP's capacity to mediate a stress response. The possibility that stress tolerance in plants is dependent upon optimal UPP activity and maintenance is discussed. PMID:25301821

  15. Regulation of p63 Protein Stability via Ubiquitin-Proteasome Pathway

    PubMed Central

    Li, Chenghua; Xiao, Zhi-Xiong

    2014-01-01

    The p53-related p63 gene encodes multiple protein isoforms, which are involved in a variety of biological activities. p63 protein stability is mainly regulated by the ubiquitin-dependent proteasomal degradation pathway. Several ubiquitin E3 ligases have been identified and some protein kinases as well as other kinds of proteins are involved in regulation of p63 protein stability. These regulators are responsive to diverse extracellular signaling, resulting in changes of the p63 protein levels and impacting different biological processes. PMID:24822180

  16. Regulation of HTLV-1 Tax Stability, Cellular Trafficking and NF-?B Activation by the Ubiquitin-Proteasome Pathway

    PubMed Central

    Lavorgna, Alfonso; Harhaj, Edward William

    2014-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATLL) in 3%–5% of infected individuals after a long latent period. HTLV-1 Tax is a trans-activating protein that regulates viral gene expression and also modulates cellular signaling pathways to enhance T-cell proliferation and cell survival. The Tax oncoprotein promotes T-cell transformation, in part via constitutive activation of the NF-?B transcription factor; however, the underlying mechanisms remain unknown. Ubiquitination is a type of post-translational modification that occurs in a three-step enzymatic cascade mediated by E1, E2 and E3 enzymes and regulates protein stability as well as signal transduction, protein trafficking and the DNA damage response. Emerging studies indicate that Tax hijacks the ubiquitin machinery to activate ubiquitin-dependent kinases and downstream NF-?B signaling. Tax interacts with the E2 conjugating enzyme Ubc13 and is conjugated on C-terminal lysine residues with lysine 63-linked polyubiquitin chains. Tax K63-linked polyubiquitination may serve as a platform for signaling complexes since this modification is critical for interactions with NEMO and IKK. In addition to NF-?B signaling, mono- and polyubiquitination of Tax also regulate its subcellular trafficking and stability. Here, we review recent advances in the diverse roles of ubiquitin in Tax function and how Tax usurps the ubiquitin-proteasome pathway to promote oncogenesis. PMID:25341660

  17. Regulation of HTLV-1 tax stability, cellular trafficking and NF-?B activation by the ubiquitin-proteasome pathway.

    PubMed

    Lavorgna, Alfonso; Harhaj, Edward William

    2014-10-01

    Human T-cell leukemia virus type 1 (HTLV-1) is a complex retrovirus that infects CD4+ T cells and causes adult T-cell leukemia/lymphoma (ATLL) in 3%-5% of infected individuals after a long latent period. HTLV-1 Tax is a trans-activating protein that regulates viral gene expression and also modulates cellular signaling pathways to enhance T-cell proliferation and cell survival. The Tax oncoprotein promotes T-cell transformation, in part via constitutive activation of the NF-?B transcription factor; however, the underlying mechanisms remain unknown. Ubiquitination is a type of post-translational modification that occurs in a three-step enzymatic cascade mediated by E1, E2 and E3 enzymes and regulates protein stability as well as signal transduction, protein trafficking and the DNA damage response. Emerging studies indicate that Tax hijacks the ubiquitin machinery to activate ubiquitin-dependent kinases and downstream NF-?B signaling. Tax interacts with the E2 conjugating enzyme Ubc13 and is conjugated on C-terminal lysine residues with lysine 63-linked polyubiquitin chains. Tax K63-linked polyubiquitination may serve as a platform for signaling complexes since this modification is critical for interactions with NEMO and IKK. In addition to NF-?B signaling, mono- and polyubiquitination of Tax also regulate its subcellular trafficking and stability. Here, we review recent advances in the diverse roles of ubiquitin in Tax function and how Tax usurps the ubiquitin-proteasome pathway to promote oncogenesis. PMID:25341660

  18. Multi-output model with Box-Jenkins operators of linear indices to predict multi-target inhibitors of ubiquitin-proteasome pathway.

    PubMed

    Casañola-Martin, Gerardo M; Le-Thi-Thu, Huong; Pérez-Giménez, Facundo; Marrero-Ponce, Yovani; Merino-Sanjuán, Matilde; Abad, Concepción; González-Díaz, Humberto

    2015-05-01

    The ubiquitin-proteasome pathway (UPP) plays an important role in the degradation of cellular proteins and regulation of different cellular processes that include cell cycle control, proliferation, differentiation, and apoptosis. In this sense, the disruption of proteasome activity leads to different pathological states linked to clinical disorders such as inflammation, neurodegeneration, and cancer. The use of UPP inhibitors is one of the proposed approaches to manage these alterations. On other hand, the ChEMBL database contains >5,000 experimental outcomes for >2,000 compounds tested as possible proteasome inhibitors using a large number of pharmacological assay protocols. All these assays report a large number of experimental parameters of biological activity like EC50, IC50 percent of inhibition, and many others that have been determined under many different conditions, targets, organisms, etc. Although this large amount of data offers new opportunities for the computational discovery of proteasome inhibitors, the complexity of these data represents a bottleneck for the development of predictive models. In this work, we used linear molecular indices calculated with the software TOMOCOMD-CARDD and Box-Jenkins moving average operators to develop a multi-output model that can predict outcomes for 20 experimental parameters in >450 assays carried out under different conditions. This generated multi-output model showed values of accuracy, sensitivity, and specificity above 70% for training and validation series. Finally, this model is considered multi-target and multi-scale, because it predicts the inhibition of the UPP for drugs against 22 molecular or cellular targets of different organisms contained in the ChEMBL database. PMID:25754075

  19. Intracellular Protein Degradation: From a Vague Idea through the Lysosome and the Ubiquitin-Proteasome System and onto Human Diseases and Drug Targeting

    PubMed Central

    Ciechanover, Aaron

    2012-01-01

    Between the 1950s and 1980s, scientists were focusing mostly on how the genetic code was transcribed to RNA and translated to proteins, but how proteins were degraded had remained a neglected research area. With the discovery of the lysosome by Christian de Duve it was assumed that cellular proteins are degraded within this organelle. Yet, several independent lines of experimental evidence strongly suggested that intracellular proteolysis was largely non-lysosomal, but the mechanisms involved have remained obscure. The discovery of the ubiquitin-proteasome system resolved the enigma. We now recognize that degradation of intracellular proteins is involved in regulation of a broad array of cellular processes, such as cell cycle and division, regulation of transcription factors, and assurance of the cellular quality control. Not surprisingly, aberrations in the system have been implicated in the pathogenesis of human disease, such as malignancies and neurodegenerative disorders, which led subsequently to an increasing effort to develop mechanism-based drugs. PMID:23908826

  20. Role of ubiquitin-proteasome in protein quality control and signaling: implication in the pathogenesis of eye diseases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin–proteasome pathway (UPP) plays important roles in many cellular functions, such as protein quality control, cell cycle control, and signal transduction. The selective degradation of aberrant proteins by the UPP is essential for the timely removal of potential cytotoxic damaged or other...

  1. Molecular Genetics of the Ubiquitin-Proteasome System: Lessons from Yeast

    Microsoft Academic Search

    M. Hochstrasser; M. Deng; A. R. Kusmierczyk; X. Li; S. G. Kreft; T. Ravid; M. Funakoshi; M. Kunjappu; Y. Xie

    \\u000a Our studies with the yeast Saccharomyces cerevisiae have uncovered a number of general principles governing substrate selectivity and proteolysis by the ubiquitin-proteasome\\u000a system. The initial work focused on the degradation of a transcription factor, the MAT?2 repressor, but the pathways uncovered\\u000a have a much broader range of targets. At least two distinct ubiquitination mechanisms contribute to ?2 turnover. One of them\\u000a depends on

  2. The role of the ubiquitination-proteasome pathway in breast cancer: Use of mouse models for analyzing ubiquitination processes

    Microsoft Academic Search

    Sabrina Rossi; Massimo Loda

    2003-01-01

    Turnover of several regulatory proteins results from targeted destruction via ubiquitination and subsequent degradation through the proteosome. The timely and irreversible degradation of critical regulators is essential for normal cellular function. The precise biochemical mechanisms that are involved in protein turnover by ubiquitin-mediated degradation have been elucidated using in vitro assays and cell culture systems. However, pathways that lead to

  3. The ubiquitin-proteasome system regulates plant hormone signaling

    PubMed Central

    Santner, Aaron; Estelle, Mark

    2011-01-01

    SUMMARY Plants utilize the ubiquitin-proteasome system (UPS) to modulate nearly every aspect of growth and development. Ubiquitin is covalently attached to target proteins through the action of three enzymes known as E1, E2, and E3. The ultimate outcome of this post-translational modification depends on the nature of the ubiquitin linkage and the extent of polyubiquitination. In most cases, ubiquitination results in degradation of the target protein in the 26S proteasome. During the last 10 years it has become clear that the UPS plays a prominent regulatory role in hormone biology. E3 ubiquitin ligases in particular actively participate in hormone perception, de-repression of hormone signaling pathways, degradation of hormone specific transcription factors, and regulation of hormone biosynthesis. It is certain that additional functions will be discovered as more of the nearly 1200 potential E3s in plants are elucidated. PMID:20409276

  4. Fbp1-Mediated Ubiquitin-Proteasome Pathway Controls Cryptococcus neoformans Virulence by Regulating Fungal Intracellular Growth in Macrophages

    PubMed Central

    Liu, Tong-Bao

    2014-01-01

    Cryptococcus neoformans is a human fungal pathogen that often causes lung and brain infections in immunocompromised patients, with a high fatality rate. Our previous results showed that an F-box protein, Fbp1, is essential for Cryptococcus virulence independent of the classical virulence factors, suggesting a novel virulence control mechanism. In this study, we show that Fbp1 is part of the ubiquitin-proteasome system, and we further investigated the mechanism of Fbp1 function during infection. Time course studies revealed that the fbp1? mutant causes little damage in the infected lung and that the fungal burden in the lung remains at a low but persistent level throughout infection. The fbp1? mutant cannot disseminate to other organs following pulmonary infection in the murine inhalation model of cryptococcosis but still causes brain infection in a murine intravenous injection model, suggesting that the block of dissemination of the fbp1? mutant is due to its inability to leave the lung. The fbp1? mutant showed a defect in intracellular proliferation after phagocytosis in a Cryptococcus-macrophage interaction assay, which likely contributes to its virulence attenuation. To elucidate the molecular basis of the SCF(Fbp1) E3 ligase function, we analyzed potential Fbp1 substrates based on proteomic approaches combined with phenotypic analysis. One substrate, the inositol phosphosphingolipid-phospholipase C1 (Isc1), is required for fungal survival inside macrophage cells, which is consistent with the role of Fbp1 in regulating Cryptococcus-macrophage interaction and fungal virulence. Our results thus reveal a new determinant of fungal virulence that involves the posttranslational regulation of inositol sphingolipid biosynthesis. PMID:24478071

  5. Stuck between a ROS and a hard place: Analysis of the ubiquitin proteasome pathway in selenocysteine treated Brassica napus reveals different toxicities during selenium assimilation.

    PubMed

    Dimkovikj, Aleksandar; Fisher, Brian; Hutchison, Kim; Van Hoewyk, Doug

    2015-06-01

    During the selenium assimilation pathway, inorganic selenate and selenite are reduced to form selenocysteine (Sec). Tolerance to selenium in plants has long been attributable to minimizing the replacement of cysteine with selenocysteine, which can result in nonspecific selenoproteins that are potentially misfolded. Despite this widely accepted assumption, there is no evidence in higher plants demonstrating that selenocysteine induces toxicity by resulting in malformed proteins. In this study, we use Brassica napus to analyze the ubiquitin-proteasome pathway, which is capable of removing misfolded proteins. Sec rapidly increased proteasome activity and levels of ubiquitinated proteins, strongly indicating that selenocysteine induces protein misfolding. Proteasome inhibition increased the amount of selenium in protein in Sec-treated plants. Collectively, these data provide a mechanism that accounts for Sec toxicity. Additionally, Sec did not cause oxidative stress as judged by examining levels of superoxide using fluorescent microscopy. Therefore, the cellular response to Sec is different compared to selenite, which was recently shown to increase antioxidant metabolism in response to elevated mitochondrial superoxide that ultimately impaired proteasome activity. Therefore, plants must contend with two divergent modes of cytotoxicity during selenium assimilation. Selenite can result in oxidative stress, but increased flux of selenite reduction can yield Sec that in turn can cause protein misfolding. PMID:25974369

  6. Protein quality control and elimination of protein waste: the role of the ubiquitin-proteasome system.

    PubMed

    Amm, Ingo; Sommer, Thomas; Wolf, Dieter H

    2014-01-01

    Mistakes are part of our world and constantly occurring. Due to transcriptional and translational failures, genomic mutations or diverse stress conditions like oxidation or heat misfolded proteins are permanently produced in every compartment of the cell. As misfolded proteins in general lose their native function and tend to aggregate several cellular mechanisms have been evolved dealing with such potentially toxic protein species. Misfolded proteins are mostly recognized by chaperones on the basis of their exposed hydrophobic patches and, if unable to refold them to their native state, are targeted to proteolytic pathways. Most prominent are the ubiquitin-proteasome system and the autophagic vacuolar (lysosomal) system, eliminating misfolded proteins from the cellular environment. A major task of this quality control system is the specific recognition and separation of the misfolded from the correctly folded protein species and the folding intermediates, respectively, which are on the way to the correct folded state but exhibit properties of misfolded proteins. In this review we focus on the recognition process and subsequent degradation of misfolded proteins via the ubiquitin-proteasome system in the different cell compartments of eukaryotic cells. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf. PMID:23850760

  7. Does impairment of the ubiquitin-proteasome system or the autophagy-lysosome pathway predispose individuals to neurodegenerative disorders such as Parkinson's disease?

    PubMed

    Matsuda, Noriyuki; Tanaka, Keiji

    2010-01-01

    About twenty years ago, an abnormal enrichment of ubiquitin in the inclusion bodies of various neurodegenerative disorders was reported. To date, this phenotype has been a diagnostic feature of many neurodegenerative disorders including Alzheimer's and Parkinson's diseases (PD). Because ubiquitin tags proteins that must be eliminated from cells, thereby targeting them for proteasomal degradation, many scientists believed that the ubiquitin-proteasome system (UPS) was inactivated in these neurodegenerative disorders. This inactivation would lead to an accumulation of ubiquitylated proteins with their concomitant aggregation into inclusion bodies and subsequent neuronal death. This hypothesis was further fuelled by the discovery that parkin, the causal gene of autosomal recessive juvenile Parkinsonism, functions as a ubiquitin ligase. However, recent findings by several groups demonstrated that ubiquitylation is also relevant to the autophagy system, with parkin promoting autophagy of dysfunctional mitochondria following the loss of mitochondrial membrane potential. These novel topics do not necessarily mean that the proteasome is involved in neurodegeneration of PD. In this review, we describe current evidence and controversies regarding the relationship between UPS and neurodegenerative disorders such as PD, and discuss several scientific discrepancies that await further clarification. PMID:20061621

  8. A leucine-rich diet modulates the tumor-induced down-regulation of the MAPK/ERK and PI3K/Akt/mTOR signaling pathways and maintains the expression of the ubiquitin-proteasome pathway in the placental tissue of NMRI mice.

    PubMed

    Viana, Laís Rosa; Gomes-Marcondes, Maria Cristina Cintra

    2015-02-01

    Placental tissue injury is concomitant with tumor development. We investigated tumor-driven placental damage by tracing certain steps of the protein synthesis and degradation pathways under leucine-rich diet supplementation in MAC16 tumor-bearing mice. Cell signaling and ubiquitin-proteasome pathways were assessed in the placental tissues of pregnant mice, which were distributed into three groups on a control diet (pregnant control, tumor-bearing pregnant, and pregnant injected with MAC-ascitic fluid) and three other groups on a leucine-rich diet (pregnant, tumor-bearing pregnant, and pregnant injected with MAC-ascitic fluid). MAC tumor growth down-regulated the cell-signaling pathways of the placental tissue and decreased the levels of IRS-1, Akt/PKB, Erk/MAPK, mTOR, p70S6K, STAT3, and STAT6 phosphorylated proteins, as assessed by the multiplex Millipore Luminex assay. Leucine supplementation maintained the levels of these proteins within the established cell-signaling pathways. In the tumor-bearing group (MAC) only, the placental tissue showed increased PC5 mRNA expression, as assessed by quantitative RT-PCR, decreased 19S and 20S protein expression, as assessed by Western blot analysis, and decreased placental tyrosine levels, likely reflecting up-regulation of the ubiquitin-proteasome pathway. Similar effects were found in the pregnant injected with MAC-ascitic fluid group, confirming that the effects of the tumor were mimicked by MAC-ascitic fluid injection. Although tumor progression occurred, the degradation pathway-related protein levels were modulated under leucine-supplementation conditions. In conclusion, tumor evolution reduced the protein expression of the cell-signaling pathway associated with elevated protein degradation, thereby jeopardizing placental activity. Under the leucine-rich diet, the impact of cancer on placental function could be minimized by improving the cell-signaling activity and reducing the proteolytic process. PMID:25395678

  9. Role of the ubiquitin proteasome system in Parkinson's disease

    Microsoft Academic Search

    Kah-Leong Lim; Jeanne MM Tan

    2007-01-01

    Parkinson's disease (PD) is the most common neurodegenerative movement disorder. Although a subject of intense research, the etiology of PD remains poorly understood. Recently, several lines of evidence have implicated an intimate link between aberrations in the ubiquitin proteasome system (UPS) and PD pathogenesis. Derangements of the UPS, which normally functions as a type of protein degradation machinery, lead to

  10. The degradation of kinesin-like calmodulin binding protein of D. salina (DsKCBP) is mediated by the ubiquitin-proteasome system.

    PubMed

    Shi, Ke; Li, Jie; Han, Kang; Jiang, Haili; Xue, Lexun

    2013-04-01

    Kinesin-like calmodulin binding protein (KCBP) is a member of kinesin-14 subfamily with unconventional domains distinct from other kinesins. This unique kinesin has the myosin tail homology 4 domain (MyTH4) and band4.1, ezrin, radixin and moesin domain (FERM) at the N-terminal which interact with several cytoskeleton proteins. Although KCBP is implicated in several microtubule-related cellular processes, studies on the KCBP of Dunaliella salina (DsKCBP) have not been reported. In this study, the roles of DsKCBP in flagella and cytoskeleton were investigated and the results showed that DsKCBP was present in flagella and upregulated during flagellar assembly indicting that it may be a flagellar kinesin and plays a role in flagellar assembly. A MyTH4-FERM domain of the DsKCBP was identified as a microtubule and actin interacting site. The interaction of DsKCBP with both microtubules and actin microfilaments suggests that this kinesin may be employed to coordinate these two cytoskeleton elements in algal cells. To gain more insights into the cellular function of the kinesin, DsKCBP-interacting proteins were examined using yeast two-hybrid screen. A 26S proteasome subunit Rpn8 was identified as a novel interacting partner of DsKCBP and the MyTH4-FERM domain was necessary for the interaction of DsKCBP with Rpn8. Furthermore, the DsKCBP was polyubiquitinated and up-regulated by proteasome inhibitor and degraded by ubiquitin-proteasome system indicating that proteasome is related to kinesin degradation. PMID:23271117

  11. 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.

  12. Impairment of the ubiquitin-proteasome pathway by methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl)carbamate leads to a potent cytotoxic effect in tumor cells: a novel antiproliferative agent with a potential therapeutic implication.

    PubMed

    Dogra, Nilambra; Mukhopadhyay, Tapas

    2012-08-31

    In recent years, there has been a great deal of interest in proteasome inhibitors as a novel class of anticancer drugs. We report that fenbendazole (FZ) (methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl)carbamate) exhibits a potent growth-inhibitory activity against cancer cell lines but not normal cells. We show here, using fluorogenic substrates, that FZ treatment leads to the inhibition of proteasomal activity in the cells. Succinyl-Leu-Leu-Val-Tyr-methylcoumarinamide (MCA), benzyloxycarbonyl-Leu-Leu-Glu-7-amido-4-MCA, and t-butoxycarbonyl-Gln-Ala-Arg-7-amido-4-MCA fluorescent derivatives were used to assess chymotrypsin-like, post-glutamyl peptidyl-hydrolyzing, and trypsin-like protease activities, respectively. Non-small cell lung cancer cells transiently transfected with an expression plasmid encoding pd1EGFP and treated with FZ showed an accumulation of the green fluorescent protein in the cells due to an increase in its half-life. A number of apoptosis regulatory proteins that are normally degraded by the ubiquitin-proteasome pathway like cyclins, p53, and I?B? were found to be accumulated in FZ-treated cells. In addition, FZ induced distinct ER stress-associated genes like GRP78, GADD153, ATF3, IRE1?, and NOXA in these cells. Thus, treatment of human NSCLC cells with fenbendazole induced endoplasmic reticulum stress, reactive oxygen species production, decreased mitochondrial membrane potential, and cytochrome c release that eventually led to cancer cell death. This is the first report to demonstrate the inhibition of proteasome function and induction of endoplasmic reticulum stress/reactive oxygen species-dependent apoptosis in human lung cancer cell lines by fenbendazole, which may represent a new class of anticancer agents showing selective toxicity against cancer cells. PMID:22745125

  13. Identification of a polymorphism in the RING finger of human Bmi-1 that causes its degradation by the ubiquitin-proteasome system

    PubMed Central

    Zhang, Jie; Sarge, Kevin D.

    2009-01-01

    Bmi-1 is a polycomb protein that plays an important role in tumor cell development and maintaining stem cell populations of many cell lineages. Here we identify a polymorphism in human Bmi-1 that changes a cysteine within its RING domain to tyrosine. This C18Y polymorphism is associated with a significant decrease in Bmi-1 level and its elevated ubiquitination, suggesting that it is being destroyed by the ubiquitin-proteasome system. Consistent with this, treating cells with the proteasome inhibitor MG-132 significantly increases C18Y Bmi-1 levels. This is the first example of a polymorphism in Bmi-1 that reduces levels of this important protein. PMID:19233177

  14. Lack of muscle recovery after immobilization in old rats does not result from a defect in normalization of the ubiquitin–proteasome and the caspase-dependent apoptotic pathways

    PubMed Central

    Magne, Hugues; Savary-Auzeloux, Isabelle; Vazeille, Emilie; Claustre, Agnès; Attaix, Didier; Anne, Listrat; Véronique, Santé-Lhoutellier; Philippe, Gatellier; Dardevet, Dominique; Combaret, Lydie

    2011-01-01

    Immobilization periods increase with age because of decreased mobility and/or increased pathological episodes that require bed-rest. Sarcopaenia might be partially explained by an impaired recovery of skeletal muscle mass after a catabolic state due to an imbalance of muscle protein metabolism, apoptosis and cellular regeneration. Mechanisms involved in muscle recovery have been poorly investigated, and remain almost unknown in the elderly. This study aimed at studying the regulation of the capsase-dependent apoptotic and the ubiquitin–proteasome-dependent proteolytic pathways during immobilization and subsequent recovery during ageing. Old rats (22–24-months old) were subjected to unilateral hindlimb casting for 8 days (I8) and allowed to recover for 10 to 40 days (R10 to R40). Immobilized gastrocnemius muscles atrophied by 21%, and did not recover even at R40. Apoptotic index, amount of polyubiquitinated conjugates, proteasome chymotrypsin- and trypsin-like, apoptosome-linked caspase-9, -3, and -8 activities increased at I8. Conversely, the amount of the myogenic factor myf-5 decreased at I8. These changes paralleled the increase of intramuscular inflammation and oxidative stress. All these parameters normalized as soon as R10. The XIAP/Smac-DIABLO protein ratio decreased by half in immobilized muscles and remained low during recovery. Surprisingly, the non-immobilized leg also atrophied from R20, concomitantly with a decreased XIAP/Smac-DIABLO protein ratio. Altogether, this suggests that the impaired recovery following immobilization in ageing does not result from a lack of normalization of the caspase-dependent apoptotic and the ubiquitin–proteasome-dependent pathways, and also that immobilization could induce a general muscle loss and then contribute to the development of sarcopaenia in elderly. PMID:21115641

  15. Local ubiquitin-proteasome-mediated proteolysis and long-term synaptic plasticity

    PubMed Central

    Hegde, Ashok N.; Haynes, Kathryn A.; Bach, Svitlana V.

    2014-01-01

    The ubiquitin-proteasome pathway (UPP) of protein degradation has many roles in synaptic plasticity that underlies memory. Work on both invertebrate and vertebrate model systems has shown that the UPP regulates numerous substrates critical for synaptic plasticity. Initial research took a global view of ubiquitin-protein degradation in neurons. Subsequently, the idea of local protein degradation was proposed a decade ago. In this review, we focus on the functions of the UPP in long-term synaptic plasticity and discuss the accumulated evidence in support of the idea that the components of the UPP often have disparate local roles in different neuronal compartments rather than a single cell-wide function. PMID:25520617

  16. Livin promotes Smac\\/DIABLO degradation by ubiquitin–proteasome pathway

    Microsoft Academic Search

    L Ma; Y Huang; Z Song; S Feng; X Tian; W Du; X Qiu; K Heese; M Wu

    2006-01-01

    Livin, a member of the inhibitor of apoptosis protein (IAP) family, encodes a protein containing a single baculoviral IAP repeat (BIR) domain and a COOH-terminal RING finger domain. It has been reported that Livin directly interacts with caspase-3 and -7 in vitro and caspase-9 in vivo via its BIR domain and is negatively regulated by Smac\\/DIABLO. Nonetheless, the detailed mechanism

  17. Roles and potential therapeutic targets of the ubiquitin proteasome system in muscle wasting

    Microsoft Academic Search

    David Nury; Christine Doucet; Olivier Coux

    2007-01-01

    Muscle wasting, characterized by the loss of protein mass in myofibers, is in most cases largely due to the activation of intracellular protein degradation by the ubiquitin proteasome system (UPS). During the last decade, mechanisms contributing to this activation have been unraveled and key mediators of this process identified. Even though much remains to be understood, the available information already

  18. Regulation of Akt-mTOR, ubiquitin-proteasome and autophagy-lysosome pathways in response to formoterol administration in rat skeletal muscle.

    PubMed

    Joassard, Olivier Roger; Amirouche, Adel; Gallot, Yann Simon; Desgeorges, Marine Maud; Castells, Josiane; Durieux, Anne-Cécile; Berthon, Phanélie; Freyssenet, Damien Gilles

    2013-11-01

    Administration of ?2-agonists triggers skeletal muscle anabolism and hypertrophy. We investigated the time course of the molecular events responsible for rat skeletal muscle hypertrophy in response to 1, 3 and 10 days of formoterol administration (i.p. 2000?g/kg/day). A marked hypertrophy of rat tibialis anterior muscle culminated at day 10. Phosphorylation of Akt, ribosomal protein S6, 4E-BP1 and ERK1/2 was increased at day 3, but returned to control level at day 10. This could lead to a transient increase in protein translation and could explain previous studies that reported increase in protein synthesis following ?2-agonist administration. Formoterol administration was also associated with a significant reduction in MAFbx/atrogin-1 mRNA level (day 3), suggesting that formoterol can also affect protein degradation of MAFbx/atrogin1 targeted substrates, including MyoD and eukaryotic initiation factor-3f (eIF3-f). Surprisingly, mRNA level of autophagy-related genes, light chain 3 beta (LC3b) and gamma-aminobutyric acid receptor-associated protein-like 1 (Gabarapl1), as well as lysosomal hydrolases, cathepsin B and cathepsin L, was significantly and transiently increased after 1 and/or 3 days, suggesting that autophagosome formation would be increased in response to formoterol administration. However, this has to be relativized since the mRNA level of Unc-51-like kinase1 (Ulk1), BCL2/adenovirus E1B interacting protein3 (Bnip3), and transcription factor EB (TFEB), as well as the protein content of Ulk1, Atg13, Atg5-Atg12 complex and p62/Sqstm1 remained unchanged or was even decreased in response to formoterol administration. These results demonstrate that the effects of formoterol are mediated, in part, through the activation of Akt-mTOR pathway and that other signaling pathways become more important in the regulation of skeletal muscle mass with chronic administration of ?2-agonists. PMID:23916784

  19. Arabidopsis CIPK26 interacts with KEG, components of the ABA signalling network and is degraded by the ubiquitin–proteasome system

    PubMed Central

    Stone, Sophia L.

    2013-01-01

    The RING-type E3 ligase, Keep on Going (KEG), is required for early seedling establishment in Arabidopsis thaliana. Post-germination, KEG negatively regulates abscisic acid (ABA) signalling by targeting Abscisic Acid Insensitive 5 (ABI5) for ubiquitination and subsequent degradation. Previous reports suggest that the role of KEG during early seedling development is not limited to regulation of ABI5 abundance. Using a yeast two-hybrid screen, this study identified Calcineurin B-like Interacting Protein Kinase (CIPK) 26 as a KEG-interacting protein. In vitro pull-down and in planta bimolecular fluorescence complementation assays confirmed the interactions between CIPK26 and KEG. In planta experiments demonstrated that CIPK26 was ubiquitinated and degraded via the 26S proteasome. It was also found that turnover of CIPK26 was increased when KEG protein levels were elevated, suggesting that the RING-type E3 ligase is involved in targeting CIPK26 for degradation. CIPK26 was found to interact with the ABA signalling components ABI1, ABI2, and ABI5. In addition, CIPK26 was capable of phosphorylating ABI5 in vitro. Consistent with a role in ABA signalling, overexpression of CIPK26 increased the sensitivity of germinating seeds to the inhibitory effects of ABA. The data presented in this report suggest that KEG mediates the proteasomal degradation of CIPK26 and that CIPK26 is part of the ABA signalling network. PMID:23658427

  20. Intersecting pathways to neurodegeneration in Parkinson's disease: Effects of the pesticide rotenone on DJ1, ?-synuclein, and the ubiquitin–proteasome system

    Microsoft Academic Search

    Ranjita Betarbet; Rosa M. Canet-Aviles; Todd B. Sherer; Pier G. Mastroberardino; Chris McLendon; Jin-Ho Kim; Serena Lund; Hye-Mee Na; Georgia Taylor; Neil F. Bence; Ron Kopito; Byoung Boo Seo; Takao Yagi; Akemi Yagi; Gary Klinefelter; Mark R. Cookson; J. Timothy Greenamyre

    2006-01-01

    Sporadic Parkinson's disease (PD) is most likely caused by a combination of environmental exposures and genetic susceptibilities, although there are rare monogenic forms of the disease. Mitochondrial impairment at complex I, oxidative stress, ?-synuclein aggregation, and dysfunctional protein degradation, have been implicated in PD pathogenesis, but how they are related to each other is unclear. To further evaluated PD pathogenesis

  1. Ligand-switchable Substrates for a Ubiquitin-Proteasome System*

    PubMed Central

    Egeler, Emily L.; Urner, Lorenz M.; Rakhit, Rishi; Liu, Corey W.; Wandless, Thomas J.

    2011-01-01

    Cellular maintenance of protein homeostasis is essential for normal cellular function. The ubiquitin-proteasome system (UPS) plays a central role in processing cellular proteins destined for degradation, but little is currently known about how misfolded cytosolic proteins are recognized by protein quality control machinery and targeted to the UPS for degradation in mammalian cells. Destabilizing domains (DDs) are small protein domains that are unstable and degraded in the absence of ligand, but whose stability is rescued by binding to a high affinity cell-permeable ligand. In the work presented here, we investigate the biophysical properties and cellular fates of a panel of FKBP12 mutants displaying a range of stabilities when expressed in mammalian cells. Our findings correlate observed cellular instability to both the propensity of the protein domain to unfold in vitro and the extent of ubiquitination of the protein in the non-permissive (ligand-free) state. We propose a model in which removal of stabilizing ligand causes the DD to unfold and be rapidly ubiquitinated by the UPS for degradation at the proteasome. The conditional nature of DD stability allows a rapid and non-perturbing switch from stable protein to unstable UPS substrate unlike other methods currently used to interrogate protein quality control, providing tunable control of degradation rates. PMID:21768107

  2. The Role of the Ubiquitin Proteasome System in Ischemia and Ischemic Tolerance

    PubMed Central

    Meller, Robert

    2010-01-01

    Ubiquitin modification targets a protein for rapid degradation by the proteasome. However, poly-ubiquitination of proteins can result in multiple functions depending on the topology of the ubiquitin chain. Therefore ubiquitin signaling offers a more complex and versatile biology compared to many other post translational modifications. One area of potential for the application of this knowledge is the field of ischemia-induced brain damage, as occurs following a stroke. The ubiquitin proteasome system may exert a dual role on neuronal outcome following ischemia. Harmful ischemia results in an overload of the ubiquitin proteasome system, and blocking the proteasome reduces brain infarction following ischemia. However, the rapid and selective degradation of proteins following brief ischemia results in endogenous protection against ischemia. Therefore further understanding of the molecular signaling mechanisms which regulate the ubiquitin proteasome system may reveal novel therapeutic targets to reduce brain damage when ischemia is predicted, or to reduce the activation of the cell death mechanisms and the inflammatory response following stroke. The aim of this review is to discuss some of the recent advances in the understanding of protein ubiquitination and its implications for novel stroke therapies. PMID:19181875

  3. Measuring activity in the ubiquitin-proteasome system: From large scale discoveries to single cells analysis

    PubMed Central

    Melvin, Adam T.; Woss, Gregery S.; Park, Jessica H.; Waters, Marcey L.; Allbritton, Nancy L.

    2013-01-01

    The ubiquitin proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS have provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington’s disease. These reporters, usually consisting of a recognition sequences fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes (DUBs). This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, recent work is presented highlighting the development of novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples. PMID:23686610

  4. Role of the Ubiquitin-Proteasome Systems in the Biology and Virulence of Protozoan Parasites

    PubMed Central

    Muñoz, Christian; San Francisco, Juan; Gutiérrez, Bessy; González, Jorge

    2015-01-01

    In eukaryotic cells, proteasomes perform crucial roles in many cellular pathways by degrading proteins to enforce quality control and regulate many cellular processes such as cell cycle progression, signal transduction, cell death, immune responses, metabolism, protein-quality control, and development. The catalytic heart of these complexes, the 20S proteasome, is highly conserved in bacteria, yeast, and humans. However, until a few years ago, the role of proteasomes in parasite biology was completely unknown. Here, we summarize findings about the role of proteasomes in protozoan parasites biology and virulence. Several reports have confirmed the role of proteasomes in parasite biological processes such as cell differentiation, cell cycle, proliferation, and encystation. Proliferation and cell differentiation are key steps in host colonization. Considering the importance of proteasomes in both processes in many different parasites such as Trypanosoma, Leishmania, Toxoplasma, and Entamoeba, parasite proteasomes might serve as virulence factors. Several pieces of evidence strongly suggest that the ubiquitin-proteasome pathway is also a viable parasitic therapeutic target. Research in recent years has shown that the proteasome is a valid drug target for sleeping sickness and malaria. Then, proteasomes are a key organelle in parasite biology and virulence and appear to be an attractive new chemotherapeutic target.

  5. Differential roles of the ubiquitin proteasome system and autophagy in the clearance of soluble and aggregated TDP-43 species

    PubMed Central

    Scotter, Emma L.; Vance, Caroline; Nishimura, Agnes L.; Lee, Youn-Bok; Chen, Han-Jou; Urwin, Hazel; Sardone, Valentina; Mitchell, Jacqueline C.; Rogelj, Boris; Rubinsztein, David C.; Shaw, Christopher E.

    2014-01-01

    ABSTRACT TAR DNA-binding protein (TDP-43, also known as TARDBP) is the major pathological protein in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Large TDP-43 aggregates that are decorated with degradation adaptor proteins are seen in the cytoplasm of remaining neurons in ALS and FTD patients post mortem. TDP-43 accumulation and ALS-linked mutations within degradation pathways implicate failed TDP-43 clearance as a primary disease mechanism. Here, we report the differing roles of the ubiquitin proteasome system (UPS) and autophagy in the clearance of TDP-43. We have investigated the effects of inhibitors of the UPS and autophagy on the degradation, localisation and mobility of soluble and insoluble TDP-43. We find that soluble TDP-43 is degraded primarily by the UPS, whereas the clearance of aggregated TDP-43 requires autophagy. Cellular macroaggregates, which recapitulate many of the pathological features of the aggregates in patients, are reversible when both the UPS and autophagy are functional. Their clearance involves the autophagic removal of oligomeric TDP-43. We speculate that, in addition to an age-related decline in pathway activity, a second hit in either the UPS or the autophagy pathway drives the accumulation of TDP-43 in ALS and FTD. Therapies for clearing excess TDP-43 should therefore target a combination of these pathways. PMID:24424030

  6. Targeting the ubiquitin-proteasome system for cancer therapy

    PubMed Central

    Yang, Yili; Kitagaki, Jirouta; Wang, Honghe; Hou, Dexing; Perantoni, Alan O.

    2009-01-01

    Summary The ubiquitin-proteasome system plays a critical role in controlling the level, activity, and location of various cellular proteins. Significant progress has been made in investigating the molecular mechanisms of ubiquitination, particularly in understanding the structure of the ubiquitination machinery and identifying ubiquitin protein ligases, the primary specificity-determining enzymes. Therefore, it is now possible to target specific molecules involved in the ubiquitination and proteasomal degradation to regulate many cellular processes such as signal transduction, proliferation and apoptosis. In particular, alterations in ubiquitination are observed in most, if not all, cancer cells. This is manifested by destabilization of tumor suppressors, such as p53, and overexpression of oncogenes such as c-Myc and c-Jun. In addition to the development and clinical validation of proteasome inhibitor Bortezomib in myeloma therapy, recent studies have demonstrated that it is possible to develop inhibitors for specific ubiquitination and deubiquitination enzymes. With the help of structural studies, rational design, and chemical synthesis, it is conceivable that we will be able to use “druggable” inhibitors of the ubiquitin system to evaluate their effects in animal tumor models in the not-so-distant future. PMID:19037995

  7. The ubiquitin-proteasome system in spongiform degenerative disorders

    PubMed Central

    Whatley, Brandi R.; Li, Lian; Chin, Lih-Shen

    2008-01-01

    Summary Spongiform degeneration is characterized by vacuolation in nervous tissue accompanied by neuronal death and gliosis. Although spongiform degeneration is a hallmark of prion diseases, this pathology is also present in the brains of patients suffering from Alzheimer's disease, diffuse Lewy body disease, human immunodeficiency virus (HIV) infection, and Canavan's spongiform leukodystrophy. The shared outcome of spongiform degeneration in these diverse diseases suggests that common cellular mechanisms must underlie the processes of spongiform change and neurodegeneration in the central nervous system. Immunohistochemical analysis of brain tissues reveals increased ubiquitin immunoreactivity in and around areas of spongiform change, suggesting the involvement of ubiquitin-proteasome system dysfunction in the pathogenesis of spongiform neurodegeneration. The link between aberrant ubiquitination and spongiform neurodegeneration has been strengthened by the discovery that a null mutation in the E3 ubiquitin-protein ligase mahogunin ring finger-1 (Mgrn1) causes an autosomal recessively inherited form of spongiform neurodegeneration in animals. Recent studies have begun to suggest that abnormal ubiquitination may alter intracellular signaling and cell functions via proteasome-dependent and proteasome-independent mechanisms, leading to spongiform degeneration and neuronal cell death. Further elucidation of the pathogenic pathways involved in spongiform neurodegeneration should facilitate the development of novel rational therapies for treating prion diseases, HIV infection, and other spongiform degenerative disorders. PMID:18790052

  8. Human papillomavirus-induced carcinogenesis and the ubiquitin-proteasome system.

    PubMed

    Scheffner, Martin; Whitaker, Noel J

    2003-02-01

    Certain types of human papillomaviruses have been etiologically associated with malignant lesions, most notably with cervical cancer. The major oncoproteins of these cancer-associated viruses are encoded by the viral E6 and E7 genes. Thorough characterization of these oncoproteins and their interaction with cellular proteins has shown that both E6 and E7 exploit the ubiquitin-proteasome system to degrade and, thus, to functionally inactivate negative cell-regulatory proteins including members of the p110(RB) family and p53. This act of piracy is assumed to contribute to both the efficient propagation of HPVs and HPV-induced carcinogenesis. PMID:12507557

  9. Skeletal Muscle Deregulation of the ubiquitin-proteasome system

    E-print Network

    Paris-Sud XI, Université de

    Skeletal Muscle Deregulation of the ubiquitin-proteasome system is the predominant molecular pathology in OPMD animal models and patients Anvar et al. Anvar et al. Skeletal Muscle 2011, 1:15 http-onset progressive muscle disorder caused by a poly-alanine expansion mutation in the Poly(A) Binding Protein Nuclear

  10. Inhibition of stat3 activation suppresses caspase-3 and the ubiquitin-proteasome system, leading to preservation of muscle mass in cancer cachexia.

    PubMed

    Silva, Kleiton Augusto Santos; Dong, Jiangling; Dong, Yanjun; Dong, Yanlan; Schor, Nestor; Tweardy, David J; Zhang, Liping; Mitch, William E

    2015-04-24

    Cachexia occurs in patients with advanced cancers. Despite the adverse clinical impact of cancer-induced muscle wasting, pathways causing cachexia are controversial, and clinically reliable therapies are not available. A trigger of muscle protein loss is the Jak/Stat pathway, and indeed, we found that conditioned medium from C26 colon carcinoma (C26) or Lewis lung carcinoma cells activates Stat3 (p-Stat3) in C2C12 myotubes. We identified two proteolytic pathways that are activated in muscle by p-Stat3; one is activation of caspase-3, and the other is p-Stat3 to myostatin, MAFbx/Atrogin-1, and MuRF-1 via CAAT/enhancer-binding protein ? (C/EBP?). Using sequential deletions of the caspase-3 promoter and CHIP assays, we determined that Stat3 activation increases caspase-3 expression in C2C12 cells. Caspase-3 expression and proteolytic activity were stimulated by p-Stat3 in muscles of tumor-bearing mice. In mice with cachexia caused by Lewis lung carcinoma or C26 tumors, knock-out of p-Stat3 in muscle or with a small chemical inhibitor of p-Stat3 suppressed muscle mass losses, improved protein synthesis and degradation in muscle, and increased body weight and grip strength. Activation of p-Stat3 stimulates a pathway from C/EBP? to myostatin and expression of MAFbx/Atrogin-1 and increases the ubiquitin-proteasome system. Indeed, C/EBP? KO decreases the expression of MAFbx/Atrogin-1 and myostatin, while increasing muscle mass and grip strength. In conclusion, cancer stimulates p-Stat3 in muscle, activating protein loss by stimulating caspase-3, myostatin, and the ubiquitin-proteasome system. These results could lead to novel strategies for preventing cancer-induced muscle wasting. PMID:25787076

  11. The Ubiquitin-Proteasome System as a Prospective Molecular Target for Cancer Treatment and Prevention

    PubMed Central

    Chen, Di; Dou, Q. Ping

    2012-01-01

    Proteasomes are large multicatalytic proteinase complexes located in the cytosol and the nucleus of eukaryotic cells. The ubiquitin-proteasome system is responsible for the degradation of most intracellular proteins and therefore plays an essential regulatory role in critical cellular processes including cell cycle progression, proliferation, differentiation, angiogenesis and apoptosis. Besides involving in normal cellular functions and homeostasis, the alteration of proteasomal activity contributes to the pathological states of several clinical disorders including inflammation, neurodegeneration and cancer. It has been reported that human cancer cells possess elevated level of proteasome activity and are more sensitive to proteasome inhibitors than normal cells, indicating that the inhibition of the ubiquitin-proteasome system could be used as a novel approach for cancer therapy. In this review we summarize several specific aspects of research for the proteasome complex, including the structure and catalytic activities of the proteasome, properties and mechanisms of action of various proteasome inhibitors, and finally the clinical development of proteasome inhibitors as novel anticancer agents. PMID:20491623

  12. Carbon and nitrogen metabolism regulated by the ubiquitin-proteasome system

    PubMed Central

    Maekawa, Shugo; Yasuda, Shigetaka

    2011-01-01

    The ubiquitin-proteasome system (UPS) is a unique protein degradation mechanism conserved in the eukaryotic cell. In addition to the control of protein quality, UPS regulates diverse cellular signal transduction via the fine-tuning of target protein degradation. Protein ubiquitylation and subsequent degradation by the 26S proteasome are involved in almost all aspects of plant growth and development and response to biotic and abiotic stresses. Recent studies reveal that the UPS plays an essential role in adaptation to carbon and nitrogen availability in plants. Here we highlight ubiquitin ligase ATL31 and the homolog ATL6 target 14-3-3 proteins for ubiquitylation to be degraded, which control signaling for carbon and nitrogen metabolisms and C/N balance response. We also give an overview of the UPS function involved in carbon and nitrogen metabolisms. PMID:21897122

  13. Regulation of mitochondrial genome inheritance by autophagy and ubiquitin-proteasome system: implications for health, fitness, and fertility.

    PubMed

    Song, Won-Hee; Ballard, John William Oman; Yi, Young-Joo; Sutovsky, Peter

    2014-01-01

    Mitochondria, the energy-generating organelles, play a role in numerous cellular functions including adenosine triphosphate (ATP) production, cellular homeostasis, and apoptosis. Maternal inheritance of mitochondria and mitochondrial DNA (mtDNA) is universally observed in humans and most animals. In general, high levels of mitochondrial heteroplasmy might contribute to a detrimental effect on fitness and disease resistance. Therefore, a disposal of the sperm-derived mitochondria inside fertilized oocytes assures normal preimplantation embryo development. Here we summarize the current research and knowledge concerning the role of autophagic pathway and ubiquitin-proteasome-dependent proteolysis in sperm mitophagy in mammals, including humans. Current data indicate that sperm mitophagy inside the fertilized oocyte could occur along multiple degradation routes converging on autophagic clearance of paternal mitochondria. The influence of assisted reproductive therapies (ART) such as intracytoplasmic sperm injection (ICSI), mitochondrial replacement (MR), and assisted fertilization of oocytes from patients of advanced reproductive age on mitochondrial function, inheritance, and fitness and for the development and health of ART babies will be of particular interest to clinical audiences. Altogether, the study of sperm mitophagy after fertilization has implications in the timing of evolution and developmental and reproductive biology and in human health, fitness, and management of mitochondrial disease. PMID:25028670

  14. Roles of the ubiquitin proteasome system in the effects of drugs of abuse

    PubMed Central

    Massaly, Nicolas; Francès, Bernard; Moulédous, Lionel

    2015-01-01

    Because of its ability to regulate the abundance of selected proteins the ubiquitin proteasome system (UPS) plays an important role in neuronal and synaptic plasticity. As a result various stages of learning and memory depend on UPS activity. Drug addiction, another phenomenon that relies on neuroplasticity, shares molecular substrates with memory processes. However, the necessity of proteasome-dependent protein degradation for the development of addiction has been poorly studied. Here we first review evidences from the literature that drugs of abuse regulate the expression and activity of the UPS system in the brain. We then provide a list of proteins which have been shown to be targeted to the proteasome following drug treatment and could thus be involved in neuronal adaptations underlying behaviors associated with drug use and abuse. Finally we describe the few studies that addressed the need for UPS-dependent protein degradation in animal models of addiction-related behaviors. PMID:25610367

  15. Small Molecule Control of Intracellular Protein Levels Through Modulation of the Ubiquitin Proteasome System

    PubMed Central

    Buckley, Dennis L.

    2015-01-01

    Traditionally, biological probes and drugs have targeted the activities of proteins (such as enzymes and receptors) that can be easily controlled by small molecules. The remaining majority of the proteome has been deemed “undruggable”. By using small molecule modulators of the ubiquitin proteasome, protein levels, rather than protein activities can be targeted instead, increasing the number of druggable targets. While targeting the proteasome itself can lead to a global increase in protein levels, targeting other components of the UPS (e.g., the hundreds of E3 ubiquitin ligases) can lead to an increase in protein levels in a more targeted fashion. Alternatively, multiple strategies for inducing protein degradation with small molecule probes are emerging. With the ability to induce and inhibit the degradation of targeted proteins, small molecule modulators of the UPS have the potential to significantly expand the druggable portion of the proteome beyond traditional targets such as enzymes and receptors. PMID:24459094

  16. The Emerging Role of the Ubiquitin Proteasome in Pulmonary Biology and Disease

    PubMed Central

    Weathington, Nathaniel M.; Sznajder, Jacob I.

    2013-01-01

    Derangements in normal cellular homeostasis at the protein level can cause or be the consequence of initiation and progression of pulmonary diseases related to genotype, infection, injury, smoking, toxin exposure, or neoplasm. We discuss one of the fundamental mechanisms of protein homeostasis, the ubiquitin proteasome system (UPS), as it relates to lung disease. The UPS effects selective degradation of ubiquitinated target proteins via ubiquitin ligase activity. Important pathobiological mechanisms relating to the UPS and lung disease have been the focus of research, with inappropriate cellular proteolysis now a validated therapeutic target. We review the contributions of this system in various lung diseases, and discuss the exciting area of UPS-targeting drug development for pulmonary disease. PMID:23713962

  17. Coordinated Regulation of Nuclear Receptor CAR by CCRP/DNAJC7, HSP70 and the Ubiquitin-Proteasome System

    PubMed Central

    Timsit, Yoav E.; Negishi, Masahiko

    2014-01-01

    The constitutive active/androstane receptor (CAR) plays an important role as a coordinate transcription factor in the regulation of various hepatic metabolic pathways for chemicals such as drugs, glucose, fatty acids, bilirubin, and bile acids. Currently, it is known that in its inactive state, CAR is retained in the cytoplasm in a protein complex with HSP90 and the tetratricopeptide repeat protein cytosoplasmic CAR retention protein (CCRP). Upon activation by phenobarbital (PB) or the PB-like inducer 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene (TCPOBOP), CAR translocates into the nucleus. We have identified two new components to the cytoplasmic regulation of CAR: ubiquitin-dependent degradation of CCRP and protein-protein interaction with HSP70. Treatment with the proteasome inhibitor MG132 (5 µM) causes CAR to accumulate in the cytoplasm of transfected HepG2 cells. In the presence of MG132, TCPOBOP increases CCRP ubiquitination in HepG2 cells co-expressing CAR, while CAR ubiquitination was not detected. MG132 treatment of HepG2 also attenuated of TCPOBOP-induced CAR transcriptional activation on reporter constructs which contain CAR-binding DNA elements derived from the human CYP2B6 gene. The elevation of cytoplasmic CAR protein with MG132 correlated with an increase of HSP70, and to a lesser extent HSP60. Both CCRP and CAR were found to interact with endogenous HSP70 in HepG2 cells by immunoprecipitation analysis. Induction of HSP70 levels by heat shock also increased cytoplasmic CAR levels, similar to the effect of MG132. Lastly, heat shock attenuated TCPOBOP-induced CAR transcriptional activation, also similar to the effect of MG132. Collectively, these data suggest that ubiquitin-proteasomal regulation of CCRP and HSP70 are important contributors to the regulation of cytoplasmic CAR levels, and hence the ability of CAR to respond to PB or PB-like inducers. PMID:24789201

  18. Coordinated regulation of nuclear receptor CAR by CCRP/DNAJC7, HSP70 and the ubiquitin-proteasome system.

    PubMed

    Timsit, Yoav E; Negishi, Masahiko

    2014-01-01

    The constitutive active/androstane receptor (CAR) plays an important role as a coordinate transcription factor in the regulation of various hepatic metabolic pathways for chemicals such as drugs, glucose, fatty acids, bilirubin, and bile acids. Currently, it is known that in its inactive state, CAR is retained in the cytoplasm in a protein complex with HSP90 and the tetratricopeptide repeat protein cytosoplasmic CAR retention protein (CCRP). Upon activation by phenobarbital (PB) or the PB-like inducer 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene (TCPOBOP), CAR translocates into the nucleus. We have identified two new components to the cytoplasmic regulation of CAR: ubiquitin-dependent degradation of CCRP and protein-protein interaction with HSP70. Treatment with the proteasome inhibitor MG132 (5 µM) causes CAR to accumulate in the cytoplasm of transfected HepG2 cells. In the presence of MG132, TCPOBOP increases CCRP ubiquitination in HepG2 cells co-expressing CAR, while CAR ubiquitination was not detected. MG132 treatment of HepG2 also attenuated of TCPOBOP-induced CAR transcriptional activation on reporter constructs which contain CAR-binding DNA elements derived from the human CYP2B6 gene. The elevation of cytoplasmic CAR protein with MG132 correlated with an increase of HSP70, and to a lesser extent HSP60. Both CCRP and CAR were found to interact with endogenous HSP70 in HepG2 cells by immunoprecipitation analysis. Induction of HSP70 levels by heat shock also increased cytoplasmic CAR levels, similar to the effect of MG132. Lastly, heat shock attenuated TCPOBOP-induced CAR transcriptional activation, also similar to the effect of MG132. Collectively, these data suggest that ubiquitin-proteasomal regulation of CCRP and HSP70 are important contributors to the regulation of cytoplasmic CAR levels, and hence the ability of CAR to respond to PB or PB-like inducers. PMID:24789201

  19. Integrated Analysis of Microarray Data of Atherosclerotic Plaques: Modulation of the Ubiquitin-Proteasome System

    PubMed Central

    Wang, Jian; Wang, Rong; Wang, Xiaowei; Zhang, Qunye

    2014-01-01

    Atherosclerosis is a typical complex multi-factorial disease and many molecules at different levels and pathways were involved in its development. Some studies have investigated the dysregulation in atherosclerosis at mRNA, miRNA or DNA methylation level, respectively. However, to our knowledge, the studies that integrated these data and revealed the abnormal networks of atherosclerosis have not been reported. Using microarray technology, we analyzed the omics data in atherosclerosis at mRNA, miRNA and DNA methylation levels. Our results demonstrated that the global DNA methylation and expression of miRNA/mRNA were significantly decreased in atherosclerotic plaque than in normal vascular tissue. The interaction network constructed using the integrative data revealed many genes, cellular processes and signaling pathways which were widely considered to play crucial roles in atherosclerosis and also revealed some genes, miRNAs or signaling pathways which have not been investigated in atherosclerosis until now (e.g. miR-519d and SNTB2). Moreover, the overall protein ubiquitination in atherosclerotic plaque was significantly increased. The proteasome activity was increased early but decreased in advanced atherosclerosis. Our study revealed many classic and novel genes and miRNAs involved in atherosclerosis and indicated the effects of ubiquitin-proteasome system on atherosclerosis might be closely related to the course of atherosclerosis. However, the efficacy of proteasome inhibitors in the treatment of atherosclerosis still needs more research. PMID:25333956

  20. Positive Symptoms of Psychosis Correlate With Expression of Ubiquitin Proteasome Genes in Peripheral Blood

    PubMed Central

    Bousman, Chad A.; Chana, Gursharan; Glatt, Stephen J.; Chandler, Sharon D.; May, Todd; Lohr, James; Kremen, William S.; Tsuang, Ming T.; Everall, Ian P.

    2015-01-01

    Several brain- and blood-based gene expression studies in patients with psychotic disorders (e.g., schizophrenia) have identified genes in the ubiquitin proteasome system (UPS) pathway as putative biomarkers. However, to date an examination of the UPS pathway in the broader context of symptom severity in psychosis has not been conducted. The purpose of this study was to investigate the correlation between clinical scores on the Scales for the Assessment of Positive and Negative Symptoms (SAPS–SANS) and expression of 43 highly annotated genes within the UPS pathway in blood from patients with psychosis. A sample of 19 psychotic patients diagnosed with schizophrenia (n = 13) or bipolar disorder (n = 6) were recruited. Pearson's partial correlations, adjusting for gender, ethnicity, age, education, medication, smoking, and past 6-month substance use, were performed between each of the selected UPS genes and both scales. Significant Bonferroni-adjusted positive associations were observed between SAPS scores and two ubiquitin conjugation genes (i.e., UBE2K, SIAH2), while a negative association was observed with one deubiquitination gene (i.e., USP2). No gene expression levels were significantly associated with scores on the SANS after correction for multiple testing. Our findings suggest that dysregulation of the UPS, specifically ubiquitin conjugation and deubiquitination, may point to a possible underlying biological mechanism for severity of positive but not negative symptoms. PMID:20552680

  1. Pyrrolidine Dithiocarbamate Inhibits Herpes Simplex Virus 1 and 2 Replication, and Its Activity May Be Mediated through Dysregulation of the Ubiquitin-Proteasome System

    PubMed Central

    Qiu, Min; Chen, Yu; Cheng, Lin; Chu, Ying; Song, Hong-Yong

    2013-01-01

    Pyrrolidine dithiocarbamate (PDTC) is widely used as an antioxidant or an NF-?B inhibitor. It has been reported to inhibit the replication of human rhinoviruses, poliovirus, coxsackievirus, and influenza virus. In this paper, we report that PDTC could inhibit the replication of herpes simplex virus 1 and 2 (HSV-1 and HSV-2). PDTC suppressed the expression of HSV-1 and HSV-2 viral immediate early (IE) and late (membrane protein gD) genes and the production of viral progeny. This antiviral property was mediated by the dithiocarbamate moiety of PDTC and required the presence of Zn2+. Although PDTC could potently block reactive oxygen species (ROS) generation, it was found that this property did not contribute to its anti-HSV activity. PDTC showed no activity in disrupting the mitogen-activated protein kinase (MAPK) pathway activation induced by viral infection that was vital for the virus's propagation. We found that PDTC modulated cellular ubiquitination and, furthermore, influenced HSV-2-induced I?B-? degradation to inhibit NF-?B activation and enhanced PML stability in the nucleus, resulting in the inhibition of viral gene expression. These results suggested that the antiviral activity of PDTC might be mediated by its dysregulation of the cellular ubiquitin-proteasome system (UPS). PMID:23740985

  2. The ubiquitin proteasome system in glia and its role in neurodegenerative diseases

    PubMed Central

    Jansen, Anne H. P.; Reits, Eric A. J.; Hol, Elly M.

    2014-01-01

    The ubiquitin proteasome system (UPS) is crucial for intracellular protein homeostasis and for degradation of aberrant and damaged proteins. The accumulation of ubiquitinated proteins is a hallmark of many neurodegenerative diseases, including amyotrophic lateral sclerosis, Alzheimer’s, Parkinson’s, and Huntington’s disease, leading to the hypothesis that proteasomal impairment is contributing to these diseases. So far, most research related to the UPS in neurodegenerative diseases has been focused on neurons, while glial cells have been largely disregarded in this respect. However, glial cells are essential for proper neuronal function and adopt a reactive phenotype in neurodegenerative diseases, thereby contributing to an inflammatory response. This process is called reactive gliosis, which in turn affects UPS function in glial cells. In many neurodegenerative diseases, mostly neurons show accumulation and aggregation of ubiquitinated proteins, suggesting that glial cells may be better equipped to maintain proper protein homeostasis. During an inflammatory reaction, the immunoproteasome is induced in glia, which may contribute to a more efficient degradation of disease-related proteins. Here we review the role of the UPS in glial cells in various neurodegenerative diseases, and we discuss how studying glial cell function might provide essential information in unraveling mechanisms of neurodegenerative diseases. PMID:25152710

  3. A Role for the Ubiquitin-Proteasome System in Activity-Dependent Presynaptic Silencing

    PubMed Central

    Jiang, Xiaoping; Litkowski, Patricia E.; Taylor, Amanda A.; Lin, Ying; Snider, B. Joy; Moulder, Krista L.

    2010-01-01

    Chronic changes in electrical excitability profoundly affect synaptic transmission throughout the lifetime of a neuron. We have previously explored persistent presynaptic silencing, a form of synaptic depression at glutamate synapses produced by ongoing neuronal activity and by strong depolarization. Here we investigate the involvement of the ubiquitin-proteasome system (UPS) in the modulation of presynaptic function. We found that proteasome inhibition prevented the induction of persistent presynaptic silencing. Specifically, application of the proteasome inhibitor, MG-132, prevented decreases in the size of the readily releasable pool of vesicles and in the percentage of active synapses. Presynaptic silencing was accompanied by decreases in levels of the priming proteins, Munc13-1 and Rim1. Importantly, overexpression of Rim1? prevented the induction of persistent presynaptic silencing. Furthermore, strong depolarization itself increased proteasome enzymatic activity measured in cell lysates. These results suggest that modulation of the UPS by electrical activity contributes to persistent presynaptic silencing by promoting the degradation of key presynaptic proteins. PMID:20130189

  4. Piperlongumine induces inhibition of the ubiquitin-proteasome system in cancer cells.

    PubMed

    Jarvius, Malin; Fryknäs, Mårten; D'Arcy, Pädraig; Sun, Chao; Rickardson, Linda; Gullbo, Joachim; Haglund, Caroline; Nygren, Peter; Linder, Stig; Larsson, Rolf

    2013-02-01

    Piperlongumine, a natural product from the plant Piperlongum, has demonstrated selective cytotoxicity to tumor cells and to show anti-tumor activity in animal models [1]. Cytotoxicity of piperlongumine has been attributed to increase in reactive oxygen species (ROS) in cancer cells. We here report that piperlongumine is an inhibitor of the ubiquitin-proteasome system (UPS). Exposure of tumor cells to piperlongumine resulted in accumulation of a reporter substrate known to be rapidly degraded by the proteasome, and of accumulation of ubiquitin conjugated proteins. However, no inhibition of 20S proteolytic activity or 19S deubiquitinating activity was observed at concentrations inducing cytotoxicity. Consistent with previous reports, piperlongumine induced strong ROS activation which correlated closely with UPS inhibition and cytotoxicity. Proteasomal blocking could not be mimicked by agents that induce oxidative stress. Our results suggest that the anti-cancer activity of piperlongumine involves inhibition of the UPS at a pre-proteasomal step, prior to deubiquitination of malfolded protein substrates at the proteasome, and that the previously reported induction of ROS is a consequence of this inhibition. PMID:23318177

  5. The ubiquitin proteasome system – Implications for cell cycle control and the targeted treatment of cancer

    PubMed Central

    Bassermann, Florian; Eichner, Ruth; Pagano, Michele

    2013-01-01

    Two families of E3 ubiquitin ligases are prominent in cell cycle regulation and mediate the timely and precise ubiquitin-proteasome-dependent degradation of key cell cycle proteins: the SCF (Skp1/Cul1/F-box protein) complex and the APC/C (Anaphase Promoting Complex or Cyclosome). While certain SCF ligases drive cell cycle progression throughout the cell cycle, APC/C (in complex with either of two substrate recruiting proteins: Cdc20 and Cdh1) orchestrates exit from mitosis (APC/CCdc20) and establishes a stable G1 phase (APC/CCdh1). Upon DNA damage or perturbation of the normal cell cycle, both ligases are involved in checkpoint activation. Mechanistic insight into these processes has significantly improved over the last ten years, largely due to a better understanding of APC/C and the functional characterization of multiple F-box proteins, the variable substrate recruiting components of SCF ligases. Here, we review the role of SCF- and APC/C-mediated ubiquitylation in the normal and perturbed cell cycle and discuss potential clinical implications of SCF and APC/C functions. PMID:23466868

  6. Modulation of adipocyte differentiation by omega-3 polyunsaturated fatty acids involves the ubiquitin-proteasome system

    PubMed Central

    Wójcik, Cezary; Lohe, Kimberly; Kuang, Chenzhong; Xiao, Yan; Jouni, Zeida; Poels, Eduard

    2014-01-01

    We have evaluated the effects of three different omega-3 polyunsaturated fatty acids (?-3 PUFAs) – docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA) on fat accumulation and expression of adipogenic and inflammatory markers using both 3T3-L1 pre-adipocytes and differentiated 3T3-L1 adipocytes. Our results indicate that ?-3 PUFAs induce the degradation of fatty acid synthase through the ubiquitin-proteasome system, which is likely to have beneficial metabolic effect on adipose cells. Omega-3 PUFAs also increase overall levels of polyubiquitinated proteins, at least in part through decreasing the expression of proteasome subunits. Moreover, adipocytes are resistant to proteasome inhibition, which induces adipophilin while decreasing perilipin expression. On the other hand, ?-3 PUFAs decrease expression of SREBP1 while inducing expression of adipophilin and GLUT4. Moreover, all three ?-3 PUFAs appear to induce tumour necrosis factor-? without affecting NF?B levels. All three ?-3 PUFAs appear to have overall similar effects. Further research is needed to elucidate their mechanism of action. PMID:24834523

  7. UCHL1 deficiency exacerbates human islet amyloid polypeptide toxicity in ?-cells: evidence of interplay between the ubiquitin/proteasome system and autophagy.

    PubMed

    Costes, Safia; Gurlo, Tatyana; Rivera, Jacqueline F; Butler, Peter C

    2014-06-01

    The islet in type 2 diabetes mellitus (T2DM) is characterized by a deficit in ?-cells and increased ?-cell apoptosis attributable at least in part to intracellular toxic oligomers of IAPP (islet amyloid polypeptide). ?-cells of individuals with T2DM are also characterized by accumulation of polyubiquitinated proteins and deficiency in the deubiquitinating enzyme UCHL1 (ubiquitin carboxyl-terminal esterase L1 [ubiquitin thiolesterase]), accounting for a dysfunctional ubiquitin/proteasome system. In the present study, we used mouse genetics to elucidate in vivo whether a partial deficit in UCHL1 enhances the vulnerability of ?-cells to human-IAPP (hIAPP) toxicity, and thus accelerates diabetes onset. We further investigated whether a genetically induced deficit in UCHL1 function in ?-cells exacerbates hIAPP-induced alteration of the autophagy pathway in vivo. We report that a deficit in UCHL1 accelerated the onset of diabetes in hIAPP transgenic mice, due to a decrease in ?-cell mass caused by increased ?-cell apoptosis. We report that UCHL1 dysfunction aggravated the hIAPP-induced defect in the autophagy/lysosomal pathway, illustrated by the marked accumulation of autophagosomes and cytoplasmic inclusions positive for SQSTM1/p62 and polyubiquitinated proteins with lysine 63-specific ubiquitin chains. Collectively, this study shows that defective UCHL1 function may be an early contributor to vulnerability of pancreatic ?-cells for protein misfolding and proteotoxicity, hallmark defects in islets of T2DM. Also, given that deficiency in UCHL1 exacerbated the defective autophagy/lysosomal degradation characteristic of hIAPP proteotoxicity, we demonstrate a previously unrecognized role of UCHL1 in the function of the autophagy/lysosomal pathway in ?-cells. PMID:24879150

  8. The possible role of the ubiquitin proteasome system in the development of atherosclerosis in diabetes

    Microsoft Academic Search

    Raffaele Marfella; Michele D' Amico; Clara Di Filippo; Mario Siniscalchi; Ferdinando Carlo sasso; Franca Ferraraccio; Francesco Rossi; Giuseppe Paolisso

    2007-01-01

    We have reviewed the impact of the ubiquitin proteasome system (UPS) on atherosclerosis progression of diabetic patients. A puzzle of many pieces of evidence suggests that UPS, in addition to its role in the removal of damaged proteins, is involved in a number of biological processes including inflammation, proliferation and apoptosis, all of which constitute important characteristics of atherosclerosis. From

  9. ATF4 Degradation Relies on a Phosphorylation-Dependent Interaction with the SCF TrCP Ubiquitin Ligase

    Microsoft Academic Search

    IRINA LASSOT; EMMANUEL SEGERAL; CLARISSE BERLIOZ-TORRENT; HERVE DURAND; LIONEL GROUSSIN; TSONWIN HAI; RICHARD BENAROUS; FLORENCE MARGOTTIN-GOGUET

    2001-01-01

    The ubiquitin-proteasome pathway regulates gene expression through protein degradation. Here we show that the F-box protein bTrCP, the receptor component of the SCF E3 ubiquitin ligase responsible for IkBa and b-catenin degradation, is colocalized in the nucleus with ATF4, a member of the ATF-CREB bZIP family of transcription factors, and controls its stability. Association between the two proteins depends on

  10. E6AP Ubiquitin Ligase Mediates Ubiquitylation and Degradation of Hepatitis C Virus Core Protein

    Microsoft Academic Search

    Masayuki Shirakura; Kyoko Murakami; Tohru Ichimura; Ryosuke Suzuki; Tetsu Shimoji; Kouichirou Fukuda; Katsutoshi Abe; Shigeko Sato; Masayoshi Fukasawa; Yoshio Yamakawa; Masahiro Nishijima; Kohji Moriishi; Yoshiharu Matsuura; Takaji Wakita; Tetsuro Suzuki; Peter M. Howley; Tatsuo Miyamura; Ikuo Shoji

    2007-01-01

    Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding

  11. Degradation of Phosphorylated p53 by Viral Protein-ECS E3 Ligase Complex

    Microsoft Academic Search

    Yoshitaka Sato; Takumi Kamura; Noriko Shirata; Takayuki Murata; Ayumi Kudoh; Satoko Iwahori; Sanae Nakayama; Hiroki Isomura; Yukihiro Nishiyama; Tatsuya Tsurumi

    2009-01-01

    p53-signaling is modulated by viruses to establish a host cellular environment advantageous for their propagation. The Epstein-Barr virus (EBV) lytic program induces phosphorylation of p53, which prevents interaction with MDM2. Here, we show that induction of EBV lytic program leads to degradation of p53 via an ubiquitin-proteasome pathway independent of MDM2. The BZLF1 protein directly functions as an adaptor component

  12. Dysfunction of the ubiquitin-proteasome system in atherosclerotic cardiovascular disease

    PubMed Central

    Wang, Feilong; Lerman, Amir; Herrmann, Joerg

    2015-01-01

    The ubiquitin-proteasome system (UPS) is an integral part of the protein metabolism and protein quality control in eukaryotic cells. It is involved in a number of biological processes of significance for vascular biology and pathology such as oxidative stress, inflammation, foam cell formation, and apoptosis. This review summarizes both indirect and direct lines of evidence for a role of the UPS in atherosclerosis from the initiation to the progression and complication stage and concludes with a future perspective.

  13. Defective regulation of the ubiquitin/proteasome system in the hypothalamus of obese male mice.

    PubMed

    Ignacio-Souza, Leticia M; Bombassaro, Bruna; Pascoal, Livia B; Portovedo, Mariana A; Razolli, Daniela S; Coope, Andressa; Victorio, Sheila C; de Moura, Rodrigo F; Nascimento, Lucas F; Arruda, Ana P; Anhe, Gabriel F; Milanski, Marciane; Velloso, Licio A

    2014-08-01

    In both human and experimental obesity, inflammatory damage to the hypothalamus plays an important role in the loss of the coordinated control of food intake and energy expenditure. Upon prolonged maintenance of increased body mass, the brain changes the defended set point of adiposity, and returning to normal weight becomes extremely difficult. Here we show that in prolonged but not in short-term obesity, the ubiquitin/proteasome system in the hypothalamus fails to maintain an adequate rate of protein recycling, leading to the accumulation of ubiquitinated proteins. This is accompanied by an increased colocalization of ubiquitin and p62 in the arcuate nucleus and reduced expression of autophagy markers in the hypothalamus. Genetic protection from obesity is accompanied by the normal regulation of the ubiquitin/proteasome system in the hypothalamus, whereas the inhibition of proteasome or p62 results in the acceleration of body mass gain in mice exposed for a short period to a high-fat diet. Thus, the defective regulation of the ubiquitin/proteasome system in the hypothalamus may be an important mechanism involved in the progression and autoperpetuation of obesity. PMID:24892821

  14. The Differential Profiling of Ubiquitin-Proteasome and Autophagy Systems in Different Tissues before the Onset of Huntington's Disease Models.

    PubMed

    Her, Lu-Shiun; Lin, Jian-Yu; Fu, Mu-Hui; Chang, Yu-Fan; Li, Chia-Ling; Tang, Ting-Yu; Jhang, Yu-Ling; Chang, Chih-Yi; Shih, Meng-Chi; Cheng, Pei-Hsun; Yang, Shang-Hsun

    2015-07-01

    Huntington's disease (HD) is a genetic and neurodegenerative disease, leading to motor and cognitive dysfunction in HD patients. At cellular level, this disease is caused by the accumulation of mutant huntingtin (HTT) in different cells, and finally results in the dysfunction of different cells. To clean these mutant proteins, ubiquitin-proteasome system (UPS) and autophagy system are two critical pathways in the brain; however, little is known in other peripheral tissues. As mutant HTT affects different tissues progressively and might influence the UPS and autophagy pathways at early stages, we attempted to examine two clearance systems in HD models before the onset. Here, in vitro results showed that the accumulation of UPS signals with time was observed obviously in neuroblastoma and kidney cells, not in other cells. In HD transgenic mice, we observed the impairment of UPS, but not autophagy, over time in the cortex and striatum. In heart and muscle tissues, disturbance of autophagy was observed, whereas dysfunction of UPS was displayed in liver and lung. These results suggest that two protein clearance pathways are disturbed differentially in different tissues before the onset of HD, and enhancement of protein clearance at early stages might provide a potential stratagem to alleviate the progression of HD. PMID:25178567

  15. The F-Box Protein Skp2 Participates in c-Myc Proteosomal Degradation and Acts as a Cofactor for c-Myc-Regulated Transcription

    Microsoft Academic Search

    Natalie von der Lehr; Sara Johansson; Siqin Wu; Fuad Bahram; Alina Castell; Cihan Cetinkaya; Per Hydbring; Ingrid Weidung; Keiko Nakayama; Keiichi I Nakayama; Ola Söderberg; Tom K Kerppola; Lars-Gunnar Larsson

    2003-01-01

    The transcription regulatory oncoprotein c-Myc controls genes involved in cell growth, apoptosis, and oncogenesis. c-Myc is turned over very quickly through the ubiquitin\\/proteasome pathway. The proteins involved in this process are still unknown. We have found that Skp2 interacts with c-Myc and participates in its ubiquitylation and degradation. The interaction between Skp2 and c-Myc occurs during the G1 to S

  16. A Chaperone-Assisted Degradation Pathway Targets Kinetochore Proteins to Ensure Genome Stability

    PubMed Central

    Kriegenburg, Franziska; Jakopec, Visnja; Poulsen, Esben G.; Nielsen, Sofie Vincents; Roguev, Assen; Krogan, Nevan; Gordon, Colin; Fleig, Ursula; Hartmann-Petersen, Rasmus

    2014-01-01

    Cells are regularly exposed to stress conditions that may lead to protein misfolding. To cope with this challenge, molecular chaperones selectively target structurally perturbed proteins for degradation via the ubiquitin-proteasome pathway. In mammals the co-chaperone BAG-1 plays an important role in this system. BAG-1 has two orthologues, Bag101 and Bag102, in the fission yeast Schizosaccharomyces pombe. We show that both Bag101 and Bag102 interact with 26S proteasomes and Hsp70. By epistasis mapping we identify a mutant in the conserved kinetochore component Spc7 (Spc105/Blinkin) as a target for a quality control system that also involves, Hsp70, Bag102, the 26S proteasome, Ubc4 and the ubiquitin-ligases Ubr11 and San1. Accordingly, chromosome missegregation of spc7 mutant strains is alleviated by mutation of components in this pathway. In addition, we isolated a dominant negative version of the deubiquitylating enzyme, Ubp3, as a suppressor of the spc7-23 phenotype, suggesting that the proteasome-associated Ubp3 is required for this degradation system. Finally, our data suggest that the identified pathway is also involved in quality control of other kinetochore components and therefore likely to be a common degradation mechanism to ensure nuclear protein homeostasis and genome integrity. PMID:24497846

  17. Regulation of ubiquitin-proteasome system, caspase enzyme activities, and extracellular proteinases in rat soleus muscle in response to unloading.

    PubMed

    Berthon, P; Duguez, S; Favier, F B; Amirouche, A; Feasson, L; Vico, L; Denis, C; Freyssenet, D

    2007-07-01

    In the present study, we determined the impact of 5 and 10 days of muscle deconditioning induced by hindlimb suspension (HS) on the ubiquitin-proteasome system of protein degradation and caspase enzyme activities in rat soleus muscles. A second goal was to determine whether activities of matrix metalloproteinase-2/9 (MMP-2/9) and urokinase-type/tissue-type plasminogen activator (PAs) were responsive to HS. As expected, HS led to a pronounced atrophy of soleus muscle. Level of ubiquitinated proteins, chymotrypsin-like activity of 20S proteasome, and Bcl-2-associated gene product-1 protein level were all transitory increased in response to 5 days of HS. These changes may thus potentially account for the decrease in muscle mass observed in response to 5 days of HS. Caspase-3 activity was significantly increased throughout the experimental period, whereas activities of caspase-6, another effector caspase, and caspase-9, the mitochondrial-dependent activator of both caspase-3 and -6, were only increased in response to 10 days of HS. This suggests that caspase-3 may be regulated through mitochondrial-independent and mitochondrial-dependent mechanisms in response to HS. Finally, MMP-2/9 activities remained unchanged, whereas PAs activities were increased after 5 days of HS. Overall, these data suggest that time-dependent regulation of intracellular and extracellular proteinases are important in setting the new phenotype of rat soleus muscle in response to HS. PMID:17334780

  18. Protein Degradation Pathways Regulate the Functions of Helicases in the DNA Damage Response and Maintenance of Genomic Stability

    PubMed Central

    Sommers, Joshua A.; Suhasini, Avvaru N.; Brosh, Robert M.

    2015-01-01

    Degradation of helicases or helicase-like proteins, often mediated by ubiquitin-proteasomal pathways, plays important regulatory roles in cellular mechanisms that respond to DNA damage or replication stress. The Bloom’s syndrome helicase (BLM) provides an example of how helicase degradation pathways, regulated by post-translational modifications and protein interactions with components of the Fanconi Anemia (FA) interstrand cross-link (ICL) repair pathway, influence cell cycle checkpoints, DNA repair, and replication restart. The FANCM DNA translocase can be targeted by checkpoint kinases that exert dramatic effects on FANCM stability and chromosomal integrity. Other work provides evidence that degradation of the F-box DNA helicase (FBH1) helps to balance translesion synthesis (TLS) and homologous recombination (HR) repair at blocked replication forks. Degradation of the helicase-like transcription factor (HLTF), a DNA translocase and ubiquitylating enzyme, influences the choice of post replication repair (PRR) pathway. Stability of the Werner syndrome helicase-nuclease (WRN) involved in the replication stress response is regulated by its acetylation. Turning to transcription, stability of the Cockayne Syndrome Group B DNA translocase (CSB) implicated in transcription-coupled repair (TCR) is regulated by a CSA ubiquitin ligase complex enabling recovery of RNA synthesis. Collectively, these studies demonstrate that helicases can be targeted for degradation to maintain genome homeostasis. PMID:25906194

  19. Narrative Review: Protein Degradation and Human Diseases: The Ubiquitin Connection

    NSDL National Science Digital Library

    MD/PhD Eyal Reinstein (Tel-Aviv University Deparrment of Internal Medicine)

    2006-11-07

    Physiology in Medicine review article. When researchers discovered the organelle lysosome, they assumed that cellular proteins were degraded within it. However, several independent lines of evidence strongly suggested that intracellular proteolysis was largely nonlysosomal. It is now recognized that degradation of intracellular proteins by the ubiquitin proteasome system (UPS) is involved in the regulation of a broad array of cellular processes,drugs that target the degradation pathways of a single or a few proteins without affecting other proteins. It seems that pharmacologic manipulation of the UPS might alter the outcome of many diseases, especially malignant conditions and possibly neurodegenerative and chronic inflammatory diseases.

  20. Cadmium interferes with the degradation of ATF5 via a post-ubiquitination step of the proteasome degradation pathway

    SciTech Connect

    Uekusa, Hiroyuki; Namimatsu, Mihoko; Hiwatashi, Yusuke; Akimoto, Takuya [Laboratory of Environmental Molecular Physiology, School of Life Science, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Nishida, Tamotsu [Department of Human Functional Genomics, Life Science Research Center, Mie University, 1577 Kurima-machiya, Tsu 514-8507 (Japan); Takahashi, Shigeru [Laboratory of Environmental Molecular Physiology, School of Life Science, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan)], E-mail: shigeru@ls.toyaku.ac.jp; Takahashi, Yuji [Laboratory of Environmental Molecular Physiology, School of Life Science, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan)

    2009-03-13

    ATF5 is a member of the CREB/ATF family of transcription factors. In the current study, using a transient transfection system to express FLAG epitope fusion proteins of ATF5, we have shown that CdCl{sub 2} or NaAsO{sub 3} increases the protein levels of ATF5 in cells, and that cadmium stabilizes the ATF5 protein. Proteasome inhibitors had a similar effect to cadmium on the cellular accumulation of ATF5. Proteasome inhibition led to an increase in ubiquitinated ATF5, while cadmium did not appear to reduce the extent of ATF5 ubiquitination. ATF5 contains a putative nuclear export signal within its N-terminus. We demonstrated that whereas deletion of N-terminal region resulted in a increase of ATF5 levels, this region does not appear to be involved in the ubiquitination of ATF5. These results indicate that ATF5 is targeted for degradation by the ubiquitin-proteasome pathway, and that cadmium slows the rate of ATF5 degradation via a post-ubiquitination mechanism.

  1. Epithelial to mesenchymal transition in the pathogenesis of uterine malignant mixed Müllerian tumours: the role of ubiquitin proteasome system and therapeutic opportunities.

    PubMed

    Voutsadakis, I A

    2012-04-01

    Malignant mixed Müllerian tumours (malignant mixed mesodermal tumours, MMMT) of the uterus are metaplastic carcinomas with a sarcomatous component and thus they are also called carcinosarcomas. It has now been accepted that the sarcomatous component is derived from epithelial elements that have undergone metaplasia. The process that produces this metaplasia is epithelial to mesenchymal transition (EMT), which has recently been described as a neoplasia-associated programme shared with embryonic development and enabling neoplastic cells to move and metastasise. The ubiquitin proteasome system (UPS) regulates the turnover and functions of hundreds of cellular proteins. It plays important roles in EMT by being involved in the regulation of several pathways participating in the execution of this metastasis-associated programme. In this review the specifi c role of UPS in EMT of MMMT is discussed and therapeutic opportunities from UPS manipulations are proposed. PMID:22484631

  2. Mechanisms Stimulating Muscle Wasting in Chronic Kidney Disease: The Roles of the Ubiquitin-Proteasome System and Myostatin

    PubMed Central

    Thomas, Sandhya S.; Mitch, William E.

    2013-01-01

    Catabolic conditions including chronic kidney disease (CKD), cancer, and diabetes cause muscle atrophy. The loss of muscle mass worsens the burden of disease because it is associated with increased morbidity and mortality. To avoid these problems or to develop treatment strategies, the mechanisms leading to muscle wasting must be identified. Specific mechanisms uncovered in CKD generally occur in other catabolic conditions. These include stimulation of protein degradation in muscle arising from activation of caspase-3 and the ubiquitin-proteasome system (UPS). These proteases act in a coordinated fashion with caspase-3 initially cleaving the complex structure of proteins in muscle yielding fragments that are substrates which are degraded by the UPS. Fortunately, the UPS exhibits remarkable specificity for proteins to be degraded because it is the major intracellular proteolytic system. Without a high level of specificity cellular functions would be disrupted. The specificity is accomplished by complex reactions that depend on recognition of a protein substrate by specific E3 ubiquitin ligases. In muscle, the specific ligases are Atrogin-1 and MuRF1 and their expression has characteristics of a biomarker of accelerated muscle proteolysis. Specific complications of CKD (metabolic acidosis, insulin resistance, inflammation, and angiotensin II) activate caspase-3 and the UPS through mechanisms that include glucocorticoids and impaired insulin or IGF-1 signaling. Mediators activate myostatin which functions as a negative growth factor in muscle. In models of cancer or CKD, strategies that block myostatin prevent muscle wasting suggesting that therapies which block myostatin could prevent muscle wasting in catabolic conditions. PMID:23292175

  3. Replication of the Rotavirus Genome Requires an Active Ubiquitin-Proteasome System?

    PubMed Central

    López, Tomás; Silva-Ayala, Daniela; López, Susana; Arias, Carlos F.

    2011-01-01

    Here we show that the ubiquitin-proteasome system is required for the efficient replication of rotavirus RRV in MA104 cells. The proteasome inhibitor MG132 decreased the yield of infectious virus under conditions where it severely reduces the synthesis of not only viral but also cellular proteins. Addition of nonessential amino acids to the cell medium restored both viral protein synthesis and cellular protein synthesis, but the production of progeny viruses was still inhibited. In medium supplemented with nonessential amino acids, we showed that MG132 does not affect rotavirus entry but inhibits the replication of the viral genome. It was also shown that it prevents the efficient incorporation into viroplasms of viral polymerase VP1 and the capsid proteins VP2 and VP6, which could explain the inhibitory effect of MG132 on genome replication and infectious virus yield. We also showed that ubiquitination is relevant for rotavirus replication since the yield of rotavirus progeny in cells carrying a temperature-sensitive mutation in the E1 ubiquitin-activating enzyme was reduced at the restrictive temperature. In addition, overexpression of ubiquitin in MG132-treated MA104 cells partially reversed the effect of the inhibitor on virus yield. Altogether, these data suggest that the ubiquitin-proteasome (UP) system has a very complex interaction with the rotavirus life cycle, with both the ubiquitination and proteolytic activities of the system being relevant for virus replication. PMID:21900156

  4. Activation of the ubiquitin-proteasome system against arsenic trioxide cardiotoxicity involves ubiquitin ligase Parkin for mitochondrial homeostasis.

    PubMed

    Watanabe, Mayumi; Funakoshi, Takeshi; Unuma, Kana; Aki, Toshihiko; Uemura, Koichi

    2014-08-01

    Parkin is an E3 ubiquitin ligase involved in the elimination of damaged mitochondria. Ubiquitination of mitochondrial substrates by Parkin results in proteasomal as well as lysosomal degradation of mitochondria, the latter of which is executed by the autophagy machinery and is called as mitophagy (mitochondrial autophagy). The aim of this study is to examine the possible role of Parkin against cardiotoxicity elicited by arsenic trioxide (ATO) exposure in HL-1 mouse atrial cardiomyocytes. HL-1 cells were administered 1-10?M ATO for up to 24h, and the involvements of apoptosis, and the ubiquitin-proteasome and autophagy-lysosome systems (UPS and ALS) were examined. ATO dose-dependently reduced mitochondrial membrane potentials (??m) in HL-1 cells, indicating that ATO works as a mitochondrial toxin in these cells. Apoptosis was evident in cells exposed to more than 6?M ATO for 24h. Levels of Parkin in mitochondria-rich fractions were increased, suggesting the recruitment of Parkin to mitochondria. Ubiquitination of the voltage-dependent anion channel1 (VDAC1), a substrate of Parkin, was also proved by immunoprecipitation. Accumulation of ubiquitinated proteins including both K48- and K63-lineages was observed in HL-1 cells after ATO exposure, implying an increased demand for proteasomal as well as lysosomal degradation of cellular proteins. Although UPS was activated by ATO as proved by increased proteasomal activity, only slight activation of the ALS marker LC3 was observed, suggesting differential reactions of UPS and ALS to ATO toxicity. The abrogation of UPS by the proteasome inhibitor bortezomib significantly sensitized HL-1 cells to ATO toxicity, showing the contribution of UPS to the maintenance of cellular homeostasis during ATO exposure. Taken together, our results reveal the activation of Parkin as well as UPS during ATO exposure in HL-1 cardiomyocytes, which contributes to the maintenance of mitochondrial as well as cellular homeostasis. PMID:24801902

  5. Effect of lysosomal and ubiquitin-proteasome system dysfunction on the abnormal aggregation of ?-synuclein in PC12 cells

    PubMed Central

    WANG, RUNQING; ZHAO, JIE; ZHANG, JIEWEN; LIU, WEI; ZHAO, MEIYING; LI, JIANGTAO; LV, JUAN; LI, YANAN

    2015-01-01

    The aim of this study was to investigate the effect of lysosomal and ubiquitin-proteasome system dysfunction on the abnormal aggregation of ?-synuclein, and to analyze its role in the pathogenesis of Parkinson's disease (PD). PC12 cells subjected to nerve growth factor-induced differentiation were used as the cell model to study the dopaminergic neurons, and the lysosomal and proteasomal inhibitors trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E64) and, respectively, were used exclusively and in combination to treat the PC12 cells. The viability and metabolic state of the cells was assessed using the MTT assay; flow cytometry was used to measure the rate of cell apoptosis; and the double immunofluorescence method was applied to observe the formation of thioflavin S- and ?-synuclein protein-positive aggregates and inclusion bodies in the PC12 cells. In addition, the Hoechst 33258 staining method was used to observe the apoptosis of the ?-synuclein protein and thioflavin-S double-labeled cells. Following the administration of the lysosomal and proteasomal pathway inhibitors, the cell viability decreased in a concentration-dependent manner and the cell apoptosis rate increased. The proportion of PC12 cells with ?-synuclein protein-positive aggregates and inclusion bodies in the E64 group was 7.94%, compared with 20.33 and 36.77% in the lactacystin and combination treatment groups, respectively. Statistical analysis indicated that the number of inclusion body-positive cells in the treatment groups was significantly higher than that in the control group (3.78%) (P<0.05). Apoptosis was evident in the double-positive cells with ?-synuclein protein-positive inclusion bodies (17.29±1.54%). In conclusion, lysosomal and proteasomal dysfunction may play an important role in the pathogenesis of PD through the induction of abnormal ?-synuclein protein aggregation in dopaminergic neurons.

  6. Pathogenesis of human mitochondrial diseases is modulated by reduced activity of the ubiquitin/proteasome system.

    PubMed

    Segref, Alexandra; Kevei, Éva; Pokrzywa, Wojciech; Schmeisser, Kathrin; Mansfeld, Johannes; Livnat-Levanon, Nurit; Ensenauer, Regina; Glickman, Michael H; Ristow, Michael; Hoppe, Thorsten

    2014-04-01

    Mitochondria maintain cellular homeostasis by coordinating ATP synthesis with metabolic activity, redox signaling, and apoptosis. Excessive levels of mitochondria-derived reactive oxygen species (ROS) promote mitochondrial dysfunction, triggering numerous metabolic disorders. However, the molecular basis for the harmful effects of excessive ROS formation is largely unknown. Here, we identify a link between mitochondrial stress and ubiquitin-dependent proteolysis, which supports cellular surveillance both in Caenorhabditis elegans and humans. Worms defective in respiration with elevated ROS levels are limited in turnover of a GFP-based substrate protein, demonstrating that mitochondrial stress affects the ubiquitin/proteasome system (UPS). Intriguingly, we observed similar proteolytic defects for disease-causing IVD and COX1 mutations associated with mitochondrial failure in humans. Together, these results identify a conserved link between mitochondrial metabolism and ubiquitin-dependent proteostasis. Reduced UPS activity during pathological conditions might potentiate disease progression and thus provides a valuable target for therapeutic intervention. PMID:24703696

  7. A high-coverage shRNA screen identifies TMEM129 as an E3 ligase involved in ER-associated protein degradation | Office of Cancer Genomics

    Cancer.gov

    Misfolded ER proteins are retrotranslocated into the cytosol for degradation via the ubiquitin-proteasome system. The human cytomegalovirus protein US11 exploits this ER-associated protein degradation (ERAD) pathway to downregulate HLA class I molecules in virus-infected cells, thereby evading elimination by cytotoxic T-lymphocytes. US11-mediated degradation of HLA class I has been instrumental in the identification of key components of mammalian ERAD, including Derlin-1, p97, VIMP and SEL1L. Despite this, the process governing retrotranslocation of the substrate is still poorly understood.

  8. A high-coverage shRNA screen identifies TMEM129 as an E3 ligase involved in ER-associated protein degradation

    Cancer.gov

    Misfolded ER proteins are retrotranslocated into the cytosol for degradation via the ubiquitin-proteasome system. The human cytomegalovirus protein US11 exploits this ER-associated protein degradation (ERAD) pathway to downregulate HLA class I molecules in virus-infected cells, thereby evading elimination by cytotoxic T-lymphocytes. US11-mediated degradation of HLA class I has been instrumental in the identification of key components of mammalian ERAD, including Derlin-1, p97, VIMP and SEL1L. Despite this, the process governing retrotranslocation of the substrate is still poorly understood.

  9. Functions of plastid protein import and the ubiquitin-proteasome system in plastid development.

    PubMed

    Ling, Qihua; Jarvis, Paul

    2015-09-01

    Plastids, such as chloroplasts, are widely distributed endosymbiotic organelles in plants and algae. Apart from their well-known functions in photosynthesis, they have roles in processes as diverse as signal sensing, fruit ripening, and seed development. As most plastid proteins are produced in the cytosol, plastids have developed dedicated translocon machineries for protein import, comprising the TOC (translocon at the outer envelope membrane of chloroplasts) and TIC (translocon at the inner envelope membrane of chloroplasts) complexes. Multiple lines of evidence reveal that protein import via the TOC complex is actively regulated, based on the specific interplay between distinct receptor isoforms and diverse client proteins. In this review, we summarize recent advances in our understanding of protein import regulation, particularly in relation to control by the ubiquitin-proteasome system (UPS), and how such regulation changes plastid development. The diversity of plastid import receptors (and of corresponding preprotein substrates) has a determining role in plastid differentiation and interconversion. The controllable turnover of TOC components by the UPS influences the developmental fate of plastids, which is fundamentally linked to plant development. Understanding the mechanisms by which plastid protein import is controlled is critical to the development of breakthrough approaches to increase the yield, quality and stress tolerance of important crop plants, which are highly dependent on plastid development. This article is part of a Special Issue entitled: Chloroplast Biogenesis. PMID:25762164

  10. Unfolded protein response and activated degradative pathways regulation in GNE myopathy.

    PubMed

    Li, Honghao; Chen, Qi; Liu, Fuchen; Zhang, Xuemei; Li, Wei; Liu, Shuping; Zhao, Yuying; Gong, Yaoqin; Yan, Chuanzhu

    2013-01-01

    Although intracellular beta amyloid (A?) accumulation is known as an early upstream event in the degenerative course of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) myopathy, the process by which A?deposits initiate various degradative pathways, and their relationship have not been fully clarified. We studied the possible secondary responses after amyloid beta precursor protein (A?PP) deposition including unfolded protein response (UPR), ubiquitin proteasome system (UPS) activation and its correlation with autophagy system. Eight GNE myopathy patients and five individuals with normal muscle morphology were included in this study. We performed immunofluorescence and immunoblotting to investigate the expression of A?PP, phosphorylated tau (p-tau) and endoplasmic reticulum molecular chaperones. Proteasome activities were measured by cleavage of fluorogenic substrates. The expression of proteasome subunits and linkers between proteasomal and autophagy systems were also evaluated by immunoblotting and relative quantitative real-time RT-PCR. Four molecular chaperones, glucose-regulated protein 94 (GRP94), glucose-regulated protein 78 (GRP78), calreticulin and calnexin and valosin containing protein (VCP) were highly expressed in GNE myopathy. 20S proteasome subunits, three main proteasome proteolytic activities, and the factors linking UPS and autophagy system were also increased. Our study suggests that A?PP deposition results in endoplasmic reticulum stress (ERS) and highly expressed VCP deliver unfolded proteins from endoplasmic reticulum to proteosomal system which is activated in endoplasmic reticulum associated degradation (ERAD) in GNE myopathy. Excessive ubiquitinated unfolded proteins are exported by proteins that connect UPS and autophagy to autophagy system, which is activated as an alternative pathway for degradation. PMID:23472144

  11. The N-end rule pathway catalyzes a major fraction of the protein degradation in skeletal muscle

    NASA Technical Reports Server (NTRS)

    Solomon, V.; Lecker, S. H.; Goldberg, A. L.

    1998-01-01

    In skeletal muscle, overall protein degradation involves the ubiquitin-proteasome system. One property of a protein that leads to rapid ubiquitin-dependent degradation is the presence of a basic, acidic, or bulky hydrophobic residue at its N terminus. However, in normal cells, substrates for this N-end rule pathway, which involves ubiquitin carrier protein (E2) E214k and ubiquitin-protein ligase (E3) E3alpha, have remained unclear. Surprisingly, in soluble extracts of rabbit muscle, we found that competitive inhibitors of E3alpha markedly inhibited the 125I-ubiquitin conjugation and ATP-dependent degradation of endogenous proteins. These inhibitors appear to selectively inhibit E3alpha, since they blocked degradation of 125I-lysozyme, a model N-end rule substrate, but did not affect the degradation of proteins whose ubiquitination involved other E3s. The addition of several E2s or E3alpha to the muscle extracts stimulated overall proteolysis and ubiquitination, but only the stimulation by E3alpha or E214k was sensitive to these inhibitors. A similar general inhibition of ubiquitin conjugation to endogenous proteins was observed with a dominant negative inhibitor of E214k. Certain substrates of the N-end rule pathway are degraded after their tRNA-dependent arginylation. We found that adding RNase A to muscle extracts reduced the ATP-dependent proteolysis of endogenous proteins, and supplying tRNA partially restored this process. Finally, although in muscle extracts the N-end rule pathway catalyzes most ubiquitin conjugation, it makes only a minor contribution to overall protein ubiquitination in HeLa cell extracts.

  12. The ubiquitin proteasome system is required for cell proliferation of the lens epithelium and for differentiation of lens fiber cells in zebrafish.

    PubMed

    Imai, Fumiyasu; Yoshizawa, Asuka; Fujimori-Tonou, Noriko; Kawakami, Koichi; Masai, Ichiro

    2010-10-01

    In the developing vertebrate lens, epithelial cells differentiate into fiber cells, which are elongated and flat in shape and form a multilayered lens fiber core. In this study, we identified the zebrafish volvox (vov) mutant, which shows defects in lens fiber differentiation. In the vov mutant, lens epithelial cells fail to proliferate properly. Furthermore, differentiating lens fiber cells do not fully elongate, and the shape and position of lens fiber nuclei are affected. We found that the vov mutant gene encodes Psmd6, the subunit of the 26S proteasome. The proteasome regulates diverse cellular functions by degrading polyubiquitylated proteins. Polyubiquitylated proteins accumulate in the vov mutant. Furthermore, polyubiquitylation is active in nuclei of differentiating lens fiber cells, suggesting roles of the proteasome in lens fiber differentiation. We found that an E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) is involved in lens defects in the vov mutant. These data suggest that the ubiquitin proteasome system is required for cell proliferation of lens epithelium and for the differentiation of lens fiber cells in zebrafish. PMID:20724448

  13. Ubiquitin Proteasome System in Parkinson Disease: a keeper or a witness?

    PubMed Central

    Martins-Branco, Diogo; Esteves, Ana R.; Santos, Daniel; Arduino, Daniela M.; Swerdlow, Russell H.; Oliveira, Catarina R.; Januario, Cristina; Cardoso, Sandra M.

    2014-01-01

    Objective The aim of this work was to evaluate the role of Ubiquitin-Proteasome System (UPS) on mitochondrial-driven alpha-synuclein (aSN) clearance in in vitro, ex vivo and in vivo Parkinson disease (PD) cellular models. Method We used SH-SY5Y ndufa2 knock-down (KD) cells, PD cybrids and peripheral blood mononuclear cells (PBMC) from patients meeting the diagnostic criteria for PD. We quantified aSN aggregation, proteasome activity and protein ubiquitination levels. In PBMC of PD patients population we evaluated aSN levels in plasma and the influence of several demographic characteristics in the above mentioned determinations. Results We found that ubiquitin-independent proteasome activity was up-regulated in SH-SY5Y ndufa2 KD cells while a down regulation was observed in PD cybrids and PBMC. Moreover, we observed an increase in protein ubiquitination that correlates with a decrease in ubiquitin-dependent proteasome activity. Accordingly, proteasome inhibition prevented ubiquitin-dependent aSN clearance. Ubiquitin-independent proteasome activity was positively correlated with ubiquitination in PBMC. We also report a negative correlation of chymotrypsin-like activity with age in control and late-onset PD groups. Total ubiquitin content is positively correlated with aSN oligomers levels, which leads to an age-dependent increase of aSN ubiquitination in LOPD. Moreover, aSN levels are increased in the plasma of PD patients. Interpretation aSN oligomers are ubiquitinated and we identified an ubiquitin-dependent clearance insufficiency with accumulation of both aSN and ubiquitin. However, SH-SY5Y ndufa2 KD cells showed a significant up-regulation of ubiquitin-independent proteasomal enzymatic activity that could mean a cell rescue attempt. Moreover, we identified that UPS function is age-dependent in PBMC. PMID:22921536

  14. Altered ubiquitin-proteasome signaling in right ventricular hypertrophy and failure

    PubMed Central

    Rajagopalan, Viswanathan; Zhao, Mingming; Reddy, Sushma; Fajardo, Giovanni; Wang, Xuejun; Dewey, Shannamar; Gomes, Aldrin V.

    2013-01-01

    Alterations in the ubiquitin-proteasome system (UPS) have been described in left ventricular hypertrophy and failure, although results have been inconsistent. The role of the UPS in right ventricular (RV) hypertrophy (RVH) and RV failure (RVF) is unknown. Given the greater percent increase in RV mass associated with RV afterload stress, as present in many congenital heart lesions, we hypothesized that alterations in the UPS could play an important role in RVH/RVF. UPS expression and activity were measured in the RV from mice with RVH/RVF secondary to pulmonary artery constriction (PAC). Epoxomicin and MG132 were used to inhibit the proteasome, and overexpression of the 11S PA28? subunit was used to activate the proteasome. PAC mice developed RVH (109.3% increase in RV weight to body weight), RV dilation with septal shift, RV dysfunction, and clinical RVF. Proteasomal function (26S ?5 chymotrypsin-like activity) was decreased 26% (P < 0.05). Protein expression of 19S subunit Rpt5 (P < 0.05), UCHL1 deubiquitinase (P < 0.0001), and Smurf1 E3 ubiquitin ligase (P < 0.01) were increased, as were polyubiquitinated proteins (P < 0.05) and free-ubiquitins (P = 0.05). Pro-apoptotic Bax was increased (P < 0.0001), whereas anti-apoptotic Bcl-2 decreased (P < 0.05), resulting in a sixfold increase in the Bax/Bcl-2 ratio. Proteasomal inhibition did not accelerate RVF. However, proteasome enhancement by cardiac-specific proteasome overexpression partially improved survival. Proteasome activity is decreased in RVH/RVF, associated with upregulation of key UPS regulators and pro-apoptotic signaling. Enhancement of proteasome function partially attenuates RVF, suggesting that UPS dysfunction contributes to RVF. PMID:23729213

  15. The ubiquitin proteasome system plays a role in venezuelan equine encephalitis virus infection.

    PubMed

    Amaya, Moushimi; Keck, Forrest; Lindquist, Michael; Voss, Kelsey; Scavone, Lauren; Kehn-Hall, Kylene; Roberts, Brian; Bailey, Charles; Schmaljohn, Connie; Narayanan, Aarthi

    2015-01-01

    Many viruses have been implicated in utilizing or modulating the Ubiquitin Proteasome System (UPS) to enhance viral multiplication and/or to sustain a persistent infection. The mosquito-borne Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is an important biodefense pathogen and select agent. There are currently no approved vaccines or therapies for VEEV infections; therefore, it is imperative to identify novel targets for therapeutic development. We hypothesized that a functional UPS is required for efficient VEEV multiplication. We have shown that at non-toxic concentrations Bortezomib, a FDA-approved inhibitor of the proteasome, proved to be a potent inhibitor of VEEV multiplication in the human astrocytoma cell line U87MG. Bortezomib inhibited the virulent Trinidad donkey (TrD) strain and the attenuated TC-83 strain of VEEV. Additional studies with virulent strains of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV) demonstrated that Bortezomib is a broad spectrum inhibitor of the New World alphaviruses. Time-of-addition assays showed that Bortezomib was an effective inhibitor of viral multiplication even when the drug was introduced many hours post exposure to the virus. Mass spectrometry analyses indicated that the VEEV capsid protein is ubiquitinated in infected cells, which was validated by confocal microscopy and immunoprecipitation assays. Subsequent studies revealed that capsid is ubiquitinated on K48 during early stages of infection which was affected by Bortezomib treatment. This study will aid future investigations in identifying host proteins as potential broad spectrum therapeutic targets for treating alphavirus infections. PMID:25927990

  16. Coupling caspase cleavage and ubiquitin–proteasome-dependent degradation of SSRP1 during apoptosis

    Microsoft Academic Search

    I Landais; H Lee; H Lu

    2006-01-01

    Structure-specific recognition protein (SSRP1) is an 87 kDa protein that heterodimerizes with Spt16 to form FACT, a complex initially shown to facilitate chromatin transcription. Despite its crucial roles in transcription and replication, little is known about the dynamics of FACT turnover in vivo. Here, we show that SSRP1 is cleaved during apoptosis by caspase 3 and\\/or 7 at the DQHD450

  17. Contribution of the autophagy-lysosomal and ubiquitin-proteasomal proteolytic systems to total proteolysis in rainbow trout (Oncorhynchus mykiss) myotubes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two major proteolytic systems are thought to (co-) operate in the skeletal muscle of vertebrates, the ubiquitin-proteasomal system (UPS) and the autophagic/lysosomal system (ALS). While their relative contribution to muscle loss has been already well documented in mammals, little is known in fish sp...

  18. The Ubiquitin-Proteasome Pathway and the Regulation of Growth Hormone Receptor Availability

    Microsoft Academic Search

    P. J. M. van Kerkhof

    2001-01-01

    Growth hormone (GH) promotes postnatal longitudinal growth in children and is active throughout an individual?s live in protein, fat and carbohydrate metabolism. GH is an anabolic hormone, inducing a positive nitrogen balance and protein synthesis in muscle. The multiple actions of GH start when GH binds to the growth hormone receptor (GHR). The effectiveness of the peptide hormone depends on

  19. 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), GSK3?, and phospho-GSK3?(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 GSK3? and phospho-GSK3?, 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-GSK3?, and did not alter the expression of total Akt, total GSK3?, 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. PMID:25626892

  20. Cbl-b and c-Cbl negatively regulate osteoblast differentiation by enhancing ubiquitination and degradation of Osterix.

    PubMed

    Choi, You Hee; Han, Younho; Lee, Sung Ho; Jin, Yun-Hye; Bahn, Minjin; Hur, Kyu Chung; Yeo, Chang-Yeol; Lee, Kwang Youl

    2015-06-01

    E3 ubiquitin ligase Cbl-b and c-Cbl play important roles in bone formation and maintenance. Cbl-b and c-Cbl regulate the activity of various receptor tyrosine kinases and intracellular protein tyrosine kinases mainly by regulating the degradation of target proteins. However, the precise mechanisms of how Cbl-b and c-Cbl regulate osteoblast differentiation are not well known. In this study, we investigated potential targets of Cbl-b and c-Cbl. We found that Cbl-b and c-Cbl inhibit BMP2-induced osteoblast differentiation in mesenchymal cells. Among various osteogenic transcription factors, we identified that Cbl-b and c-Cbl suppress the protein stability and transcriptional activity of Osterix. Our results suggest that Cbl-b and c-Cbl inhibit the function of Osterix by enhancing the ubiquitin-proteasome-mediated degradation of Osterix. Taken together, we propose novel regulatory roles of Cbl-b and c-Cbl during osteoblast differentiation in which Cbl-b and c-Cbl regulate the degradation of Osterix through the ubiquitin-proteasome pathway. PMID:25744063

  1. Lipotoxic Stress Induces Pancreatic ?-Cell Apoptosis through Modulation of Bcl-2 Proteins by the Ubiquitin-Proteasome System

    PubMed Central

    Litwak, Sara A.; Wali, Jibran A.; Pappas, Evan G.; Saadi, Hamdi; Stanley, William J.; Varanasi, L. Chitra; Kay, Thomas W. H.; Thomas, Helen E.; Gurzov, Esteban N.

    2015-01-01

    Pancreatic ?-cell loss induced by saturated free fatty acids (FFAs) is believed to contribute to type 2 diabetes. Previous studies have shown induction of endoplasmic reticulum (ER) stress, increased ubiquitinated proteins, and deregulation of the Bcl-2 family in the pancreas of type 2 diabetic patients. However, the precise mechanism of ?-cell death remains unknown. In the present study we demonstrate that the FFA palmitate blocks the ubiquitin-proteasome system (UPS) and causes apoptosis through induction of ER stress and deregulation of Bcl-2 proteins. We found that palmitate and the proteasome inhibitor MG132 induced ER stress in ?-cells, resulting in decreased expression of the prosurvival proteins Bcl-2, Mcl-1, and Bcl-XL, and upregulation of the prodeath BH3-only protein PUMA. On the other hand, pharmacological activation of the UPS by sulforaphane ameliorated ER stress, upregulated prosurvival Bcl-2 proteins, and protected ?-cells from FFA-induced cell death. Furthermore, transgenic overexpression of Bcl-2 protected islets from FFA-induced cell death in vitro and improved glucose-induced insulin secretion in vivo. Together our results suggest that targeting the UPS and Bcl-2 protein expression may be a valuable strategy to prevent ?-cell demise in type 2 diabetes.

  2. Degradation and aggresome formation of the Gn tail of the apathogenic Tula hantavirus.

    PubMed

    Wang, Hao; Strandin, Tomas; Hepojoki, Jussi; Lankinen, Hilkka; Vaheri, Antti

    2009-12-01

    The cytoplasmic tails of envelope glycoprotein Gn of pathogenic hantaviruses but not of the apathogenic Prospect Hill virus (PHV) were recently reported to be proteasomally degraded in simian COS7 cells. Here, we show that the cytoplasmic tails of the glycoproteins of the apathogenic hantaviruses Tula virus (TULV) and PHV are also degraded through the ubiquitin-proteasome pathway, both in human HEK-293 and in simian Vero E6 cells. TULV Gn tails formed aggresomes in cells with proteasomal inhibitors. We conclude that degradation upon aggregation of Gn tails, which may represent a general cellular response to misfolded protein used by hantaviruses to control maturation of virions, is unrelated to pathogenicity. PMID:19675185

  3. Cellular senescence and protein degradation

    PubMed Central

    Deschênes-Simard, Xavier; Lessard, Frédéric; Gaumont-Leclerc, Marie-France; Bardeesy, Nabeel; Ferbeyre, Gerardo

    2014-01-01

    Autophagy and the ubiquitin–proteasome pathway (UPP) are the major protein degradation systems in eukaryotic cells. Whereas the former mediate a bulk nonspecific degradation, the UPP allows a rapid degradation of specific proteins. Both systems have been shown to play a role in tumorigenesis, and the interest in developing therapeutic agents inhibiting protein degradation is steadily growing. However, emerging data point to a critical role for autophagy in cellular senescence, an established tumor suppressor mechanism. Recently, a selective protein degradation process mediated by the UPP was also shown to contribute to the senescence phenotype. This process is tightly regulated by E3 ubiquitin ligases, deubiquitinases, and several post-translational modifications of target proteins. Illustrating the complexity of UPP, more than 600 human genes have been shown to encode E3 ubiquitin ligases, a number which exceeds that of the protein kinases. Nevertheless, our knowledge of proteasome-dependent protein degradation as a regulated process in cellular contexts such as cancer and senescence remains very limited. Here we discuss the implications of protein degradation in senescence and attempt to relate this function to the protein degradation pattern observed in cancer cells. PMID:24866342

  4. Aggresome-like structure induced by isothiocyanates is novel proteasome-dependent degradation machinery

    SciTech Connect

    Mi, Lixin, E-mail: lm293@georgetown.edu [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States)] [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States); Gan, Nanqin; Chung, Fung-Lung [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States)] [Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057 (United States)

    2009-10-16

    Unwanted or misfolded proteins are either refolded by chaperones or degraded by the ubiquitin-proteasome system (UPS). When UPS is impaired, misfolded proteins form aggregates, which are transported along microtubules by motor protein dynein towards the juxta-nuclear microtubule-organizing center to form aggresome, a single cellular garbage disposal complex. Because aggresome formation results from proteasome failure, aggresome components are degraded through the autophagy/lysosome pathway. Here we report that small molecule isothiocyanates (ITCs) can induce formation of aggresome-like structure (ALS) through covalent modification of cytoplasmic {alpha}- and {beta}-tubulin. The formation of ALS is related to neither proteasome inhibition nor oxidative stress. ITC-induced ALS is a proteasome-dependent assembly for emergent removal of misfolded proteins, suggesting that the cell may have a previously unknown strategy to cope with misfolded proteins.

  5. Dysregulation of the Ubiquitin-Proteasome System by Curcumin Suppresses Coxsackievirus B3 Replication

    Microsoft Academic Search

    Xiaoning Si; Yahong Wang; Jerry Wong; Jingchun Zhang; Bruce M. McManus; Honglin Luo

    2007-01-01

    Curcumin (diferuloylmethane), a natural polyphenolic compound extracted from the spice turmeric, has been reported to have anti-inflammatory, antioxidant, and antiproliferative properties by modulating multiple cellular machineries. It inhibits several intracellular signaling pathways, including the mitogen-activated protein kinases (MAPKs), casein kinase II (CKII), and the COP9 signalosome (CSN), in various cell types. It has also been recently demonstrated that exposure to

  6. Cbl-b-dependent degradation of FLIP(L) is involved in ATO-induced autophagy in leukemic K562 and gastric cancer cells.

    PubMed

    Zhang, Guodong; Liu, Jing; Zhang, Ye; Qu, Jinglei; Xu, Ling; Zheng, Huachuan; Liu, Yunpeng; Qu, Xiujuan

    2012-09-21

    Various molecular mechanisms are involved in the efficacy of arsenic trioxide (ATO) against malignant hematologic and some solid tumors. FLICE-like inhibitory protein (FLIP) is an inhibitor of apoptosis mediated by death receptors. In this study, we identified a new link between the down-regulation of cellular FLIP(L) and ATO-induced autophagy. ATO induced the degradation of FLIP(L) in K562 and MGC803 cells, which was mediated by the ubiquitin-proteasome pathway. Moreover, the casitas B-lineage lymphoma-b (Cbl-b) was involved in this process, which interacted with FLIP(L) and promoted proteasomal degradation of FLIP(L). Our findings lead to a better understanding of the mechanism of action of ATO, and suggest that a novel signaling pathway is required for ATO-induced autophagy in K562 and MGC803 cells. PMID:22884570

  7. Computer Modeling Illuminates Degradation Pathways of

    E-print Network

    Computer Modeling Illuminates Degradation Pathways of Cations in Alkaline Membrane Fuel Cells Cation degradation insights obtained by computational modeling could result in better performance are effective in increasing cation stability. With the help of computational modeling, more cations are being

  8. The phosphorylation of the androgen receptor by TFIIH directs the ubiquitin/proteasome process.

    PubMed

    Chymkowitch, Pierre; Le May, Nicolas; Charneau, Pierre; Compe, Emmanuel; Egly, Jean-Marc

    2011-02-01

    In response to hormonal stimuli, a cascade of hierarchical post-translational modifications of nuclear receptors are required for the correct expression of target genes. Here, we show that the transcription factor TFIIH, via its cdk7 kinase, phosphorylates the androgen receptor (AR) at position AR/S515. Strikingly, this phosphorylation is a key step for an accurate transactivation that includes the cyclic recruitment of the transcription machinery, the MDM2 E3 ligase, the subsequent ubiquitination of AR at the promoter of target genes and its degradation by the proteasome machinery. Impaired phosphorylation disrupts the transactivation, as observed in cells either overexpressing the non-phosphorylated AR/S515A, isolated from xeroderma pigmentosum patient (bearing a mutation in XPD subunit of TFIIH), or in which cdk7 kinase was silenced. Indeed, besides affecting the cyclic recruitment of the transcription machinery, the AR phosphorylation defect favourizes to the recruitment of the E3 ligase CHIP instead of MDM2, at the PSA promoter, that will further attract the proteasome machinery. These observations illustrate how the TFIIH phosphorylation might participate to the transactivation by regulating the nuclear receptors turnover. PMID:21157430

  9. Sigma-1 receptor is involved in degradation of intranuclear inclusions in a cellular model of Huntington's disease.

    PubMed

    Miki, Yasuo; Tanji, Kunikazu; Mori, Fumiaki; Wakabayashi, Koichi

    2015-02-01

    The sigma-1 receptor (SIGMAR1) is one of the endoplasmic reticulum (ER) chaperones, which participate in the degradation of misfolded proteins via the ER-related degradation machinery linked to the ubiquitin-proteasome pathway. ER dysfunction in the formation of inclusion bodies in various neurodegenerative diseases has also become evident. Recently, we demonstrated that accumulation of SIGMAR1 was common to neuronal nuclear inclusions in polyglutamine diseases including Huntington's disease. Our study also indicated that SIGMAR1 might shuttle between the cytoplasm and the nucleus. In the present study, we investigated the role of SIGMAR1 in nuclear inclusion (NI) formation, using HeLa cells transfected with N-terminal mutant huntingtin. Cell harboring the mutant huntingtin produced SIGMAR1-positive NIs. SIGMAR1 siRNA and a specific inhibitor of the proteasome (epoxomicin) caused significant accumulation of aggregates in the cytoplasm and nucleus. A specific inhibitor of exportin 1 (leptomycin B) also caused NIs. Huntingtin became insolubilized in Western blot analysis after treatments with SIGMAR1 siRNA and epoxomicin. Furthermore, proteasome activity increased chronologically along with the accumulation of mutant huntingtin, but was significantly reduced in cells transfected with SIGMAR1 siRNA. By contrast, overexpression of SIGMAR1 reduced the accumulation of NIs containing mutant huntingtin. Although the LC3-I level was decreased in cells treated with both SIGMAR1 siRNA and control siRNA, the levels of LC3-II and p62 were unchanged. SIGMAR1 agonist and antagonist had no effect on cellular viability and proteasome activity. These findings suggest that the ubiquitin-proteasome pathway is implicated in NI formation, and that SIGMAR1 degrades aberrant proteins in the nucleus via the ER-related degradation machinery. SIGMAR1 might be a promising candidate for therapy of Huntington's disease. PMID:25449906

  10. Distinct effects of methamphetamine on autophagy-lysosome and ubiquitin-proteasome systems in HL-1 cultured mouse atrial cardiomyocytes.

    PubMed

    Funakoshi-Hirose, Izumi; Aki, Toshihiko; Unuma, Kana; Funakoshi, Takeshi; Noritake, Kanako; Uemura, Koichi

    2013-10-01

    The aim of this study is to investigate the molecular mechanism underling the cardiotoxicity of methamphetamine, a psychostimulant drug that is currently abused in the world. A mouse atrial cardiac cell line, HL-1, which retains phenotypes of cardiac cells and serves as a useful model for examining cardiac pathophysiology, was used for this purpose. During treatment with 1mM methamphetamine (MAP) for 3-48h, massive but transient cytoplasmic vacuolization (3-12h) followed by an intracellular accumulation of granules (24-48h) was observed under light microscopy. The vacuoles were surrounded by the lysosome membrane marker LAMP1, while the granules colocalized with the autophagy markers LC3 and p62 as well as ubiquitinated proteins. Western blot analysis showed that LC3 was activated during MAP administration, although p62 was not degraded but rather accumulated. Concordant with p62 accumulation, the nuclear translocation of an anti-oxidative transcription factor, Nrf2, and the subsequent induction of its target gene, HO-1, was observed, suggesting an impairment of autophagic protein degradation and the subsequent activation of the p62/Nrf2/HO-1 pathway. In addition, proteomic analysis revealed a reduction in myosin heavy chain (MHC) protein levels during MAP administration. The ubiquitination of MHC and the induction of the muscle sarcomere protein-specific E3 ubiquitin ligases MuRF1 and atrogin-1 were proved by immunoprecipitation and quantitative real-time PCR, respectively. Taken together, the vacuolization of lysosomes and the subsequent accumulation of autophagosomes indicate an impairment of autophagic protein degradation during MAP administration; on the other hand, the ubiquitination and subsequent degradation of MHC indicate the proper progression of proteasomal degradation. PMID:23933405

  11. Interactions of Xanthomonas type-III effector proteins with the plant ubiquitin and ubiquitin-like pathways

    PubMed Central

    Üstün, Suayib; Börnke, Frederik

    2014-01-01

    In eukaryotes, regulated protein turnover is required during many cellular processes, including defense against pathogens. Ubiquitination and degradation of ubiquitinated proteins via the ubiquitin–proteasome system (UPS) is the main pathway for the turnover of intracellular proteins in eukaryotes. The extensive utilization of the UPS in host cells makes it an ideal pivot for the manipulation of cellular processes by pathogens. Like many other Gram-negative bacteria, Xanthomonas species secrete a suite of type-III effector proteins (T3Es) into their host cells to promote virulence. Some of these T3Es exploit the plant UPS to interfere with immunity. This review summarizes T3E examples from the genus Xanthomonas with a proven or suggested interaction with the host UPS or UPS-like systems and also discusses the apparent paradox that arises from the presence of T3Es that inhibit the UPS in general while others rely on its activity for their function. PMID:25566304

  12. Transforming growth factor-?3 regulates cell junction restructuring via MAPK-mediated mRNA destabilization and Smad-dependent protein degradation of junctional adhesion molecule B (JAM-B).

    PubMed

    Zhang, Xu; Lui, Wing-Yee

    2015-06-01

    Junctional adhesion molecule-B (JAM-B) is found between Sertoli cells at the blood-testis barrier (BTB) as well as between Sertoli and germ cells at the apical ectoplasmic specializations (ES) in the testis. The expression of JAM-B is tightly regulated to modulate the passage of spermatocytes across the BTB as well as the release of mature spermatozoa from the seminiferous epithelium. Transforming growth factor beta (TGF-?) family is implicated in the regulation of testicular cell junction dynamics during spermatogenesis. This study aims to investigate the effects of TGF-?3 on the expression of JAM-B as well as the underlying mechanisms on how TGF-?3 regulates JAM-B expression to facilitate the disassembly of the BTB and apical ES. Our results revealed that TGF-?3 suppresses JAM-B at post-transcriptional and post-translational levels. Inhibitor, siRNA knockdown and co-immunoprecipitation have shown that TGF-?3 induces JAM-B protein degradation via ubiquitin-proteasome pathway. Immunofluorescence staining further confirmed that blockage of ubiquitin-proteasome pathway could abrogate TGF-?3-induced loss of JAM-B at the cell-cell interface. siRNA knockdown and immunofluorescence staining also demonstrated that activation of Smad signaling is required for TGF-?3-induced JAM-B protein degradation. In addition, TGF-?3 reduces JAM-B mRNA levels, at least in part, via post-transcriptional regulation. mRNA stability assay has confirmed that TGF-?3 promotes the degradation of JAM-B transcript and TGF-?3-mediated mRNA destabilization requires the activation of ERK1/2 and p54 JNK signal cascades. Taken together, TGF-?3 significantly downregulates JAM-B expression via post-transcriptional and post-translational modulation and results in the disruption of BTB and apical ES. PMID:25817991

  13. Protein recycling pathways in neurodegenerative diseases.

    PubMed

    Fecto, Faisal; Esengul, Y Taylan; Siddique, Teepu

    2014-01-01

    Many progressive neurodegenerative diseases, including Alzheimer disease, Parkinson disease, Huntington disease, amyotrophic lateral sclerosis, and frontotemporal lobe dementia, are associated with the formation of insoluble intracellular proteinaceous inclusions. It is therefore imperative to understand the factors that regulate normal, as well as abnormal, protein recycling in neurons. Dysfunction of the ubiquitin-proteasome or autophagy pathways might contribute to the pathology of various neurodegenerative diseases. Induction of these pathways may offer a rational therapeutic strategy for a number of these diseases. PMID:25031631

  14. Induction of Caspase-3-like activity in Rice following release of cytochrome-f from the chloroplast and subsequent interaction with the Ubiquitin-Proteasome System

    PubMed Central

    Wang, Hongjuan; Zhu, Xiaonan; Li, Huan; Cui, Jing; Liu, Cheng; Chen, Xi; Zhang, Wei

    2014-01-01

    It has been known that the process of leaf senescence is accompanied by programmed cell death (PCD), and the previous study indicated that dark-induced senescence in detached leaves from rice led to the release of cytochrome f (Cyt f) from chloroplast into the cytoplasm. In this study, the effects of Cyt f on PCD were studied both in vitro and in vivo. In a cell-free system, purified Cyt f activated caspase-3-like protease and endonuclease OsNuc37, and induced DNA fragmentation. Furthermore, Cyt f-induced caspase-3-like activity could be inhibited by MG132, which suggests that the activity was attributed to the 26S proteasome. Conditional expression of Cyt f in the cytoplasm could also activate caspase-3-like activity and DNA fragmentation. Fluorescein diacetate staining and annexin V-FITC/PI double staining demonstrated that Cyt f expression in cytoplasm significantly increased the percentage of PCD protoplasts. Yeast two-hybrid screening showed that Cyt f might interact with E3-ubiquitin ligase and RPN9b, the subunits of the ubiquitin proteasome system (UPS), and other PCD-related proteins. Taken together, these results suggest that the released Cyt f from the chloroplast into the cytoplasm might activate or rescue caspase-3-like activity by interacting with the UPS, ultimately leading to the induction of PCD. PMID:25103621

  15. Ubiquitin-Mediated Proteasomal Degradation of Oleosins is Involved in Oil Body Mobilization During Post-Germinative Seedling Growth in Arabidopsis.

    PubMed

    Deruyffelaere, Carine; Bouchez, Isabelle; Morin, Halima; Guillot, Alain; Miquel, Martine; Froissard, Marine; Chardot, Thierry; D'Andrea, Sabine

    2015-07-01

    In oleaginous seeds, lipids-stored in organelles called oil bodies (OBs)-are degraded post-germinatively to provide carbon and energy for seedling growth. To date, little is known about how OB coat proteins, known as oleosins, control OB dynamics during seed germination. Here, we demonstrated that the sequential proteolysis of the five Arabidopsis thaliana oleosins OLE1-OLE5 begins just prior to lipid degradation. Several post-translational modifications (e.g. phosphorylation and ubiquination) of oleosins were concomitant with oleosin degradation. Phosphorylation occurred only on the minor OLE5 and on an 8 kDa proteolytic fragment of OLE2. A combination of immunochemical and proteomic approaches revealed ubiquitination of the four oleosins OLE1-OLE4 at the onset of OB mobilization. Ubiquitination topology was surprisingly complex. OLE1 and OLE2 were modified by three distinct and predominantly exclusive motifs: monoubiquitin, K48-linked diubiquitin (K48Ub2) and K63-linked diubiquitin. Ubiquitinated oleosins may be channeled towards specific degradation pathways according to ubiquitination type. One of these pathways was identified as the ubiquitin-proteasome pathway. A proteasome inhibitor (MG132) reduced oleosin degradation and induced cytosolic accumulation of K48Ub2-oleosin aggregates. These results indicate that K48Ub2-modified oleosins are selectively extracted from OB coat and degraded by the proteasome. Proteasome inhibition also reduced lipid hydrolysis, providing in vivo evidence that oleosin degradation is required for lipid mobilization. PMID:25907570

  16. Prion degradation pathways: Potential for therapeutic intervention

    PubMed Central

    Goold, Rob; McKinnon, Chris; Tabrizi, Sarah J.

    2015-01-01

    Prion diseases are fatal neurodegenerative disorders. Pathology is closely linked to the misfolding of native cellular PrPC into the disease-associated form PrPSc that accumulates in the brain as disease progresses. Although treatments have yet to be developed, strategies aimed at stimulating the degradation of PrPSc have shown efficacy in experimental models of prion disease. Here, we describe the cellular pathways that mediate PrPSc degradation and review possible targets for therapeutic intervention. This article is part of a Special Issue entitled ‘Neuronal Protein’. PMID:25584786

  17. Regulation of Subtilase Cytotoxin-Induced Cell Death by an RNA-Dependent Protein Kinase-Like Endoplasmic Reticulum Kinase-Dependent Proteasome Pathway in HeLa Cells

    PubMed Central

    Tsutsuki, Hiroyasu; Ogura, Kohei; Nagasawa, Sayaka; Moss, Joel; Noda, Masatoshi

    2012-01-01

    Shiga-toxigenic Escherichia coli (STEC) produces subtilase cytotoxin (SubAB), which cleaves the molecular chaperone BiP in the endoplasmic reticulum (ER), leading to an ER stress response and then activation of apoptotic signaling pathways. Here, we show that an early event in SubAB-induced apoptosis in HeLa cells is mediated by RNA-dependent protein kinase (PKR)-like ER kinase (PERK), not activating transcription factor 6 (ATF6) or inositol-requiring enzyme 1(Ire1), two other ER stress sensors. PERK knockdown suppressed SubAB-induced eIF2? phosphorylation, activating transcription factor 4 (ATF4) expression, caspase activation, and cytotoxicity. Knockdown of eIF2? by small interfering RNA (siRNA) or inhibition of eIF2? dephosphorylation by Sal003 enhanced SubAB-induced caspase activation. Treatment with proteasome inhibitors (i.e., MG132 and lactacystin), but not a general caspase inhibitor (Z-VAD) or a lysosome inhibitor (chloroquine), suppressed SubAB-induced caspase activation and poly(ADP-ribose) polymerase (PARP) cleavage, suggesting that the ubiquitin-proteasome system controls events leading to caspase activation, i.e., Bax/Bak conformational changes, followed by cytochrome c release from mitochondria. Levels of ubiquitinated proteins in HeLa cells were significantly decreased by SubAB treatment. Further, in an early event, some antiapoptotic proteins, which normally turn over rapidly, have their synthesis inhibited, and show enhanced degradation via the proteasome, resulting in apoptosis. In PERK knockdown cells, SubAB-induced loss of ubiquitinated proteins was inhibited. Thus, SubAB-induced ER stress is caused by BiP cleavage, leading to PERK activation, not by accumulation of ubiquitinated proteins, which undergo PERK-dependent degradation via the ubiquitin-proteasome system. PMID:22354021

  18. Ubiquitination Regulates PSD95 Degradation and AMPA Receptor Surface Expression

    Microsoft Academic Search

    Marcie Colledge; Eric M. Snyder; Robert A. Crozier; Jacquelyn A. Soderling; Yetao Jin; Lorene K. Langeberg; Hua Lu; Mark F. Bear; John D. Scott

    2003-01-01

    PSD-95 is a major scaffolding protein of the postsynaptic density, tethering NMDA- and AMPA-type glutamate receptors to signaling proteins and the neuronal cytoskeleton. Here we show that PSD-95 is regulated by the ubiquitin-proteasome pathway. PSD-95 interacts with and is ubiquitinated by the E3 ligase Mdm2. In response to NMDA receptor activation, PSD-95 is ubiquitinated and rapidly removed from synaptic sites

  19. The ubiquitin-proteasome system is responsible for cysteine-responsive regulation of cysteine dioxygenase concentration in liver.

    PubMed

    Stipanuk, Martha H; Hirschberger, Lawrence L; Londono, Monica P; Cresenzi, Carrie L; Yu, Anthony F

    2004-03-01

    Hepatic cysteine dioxygenase (CDO) activity is a critical regulator of cellular cysteine concentration and availability of cysteine for anabolic processes and is markedly higher in animals fed diets containing excess sulfur amino acids compared with those fed levels at or below the requirement. Rat hepatocytes responded to a deficiency or excess of cysteine in the culture medium with a decrease or increase in CDO level but no change in CDO mRNA level. The cysteine analog, cysteamine, but not cysteine metabolites or thiol reagents, was also effective in increasing CDO. Inhibitors of the 26S proteasome blocked CDO degradation in cysteine-deficient cells but had little or no effect on CDO concentration in hepatocytes cultured with excess cysteine. High-molecular-mass CDO-ubiquitin conjugates were observed in cells cultured in cysteine-deficient medium, whether or not proteasome inhibitor was present, but these CDO-ubiquitin conjugates were not observed in cells cultured in cysteine-supplemented medium with or without proteasome inhibitor. Similar results were observed for degradation of recombinant CDO expressed in human heptocarcinoma cells cultured in cysteine-deficient or cysteine-supplemented medium. CDO is an example of a mammalian enzyme that is robustly regulated via its substrate, with the presence of substrate blocking the ubiquitination of CDO and, hence, the targeting of CDO for proteasomal degradation. This regulation occurs in primary hepatocytes in a manner that corresponds with changes observed in intact animals. PMID:14644768

  20. The E3 ubiquitin ligase specificity subunit ASB2? targets filamins for proteasomal degradation by interacting with the filamin actin-binding domain

    PubMed Central

    Razinia, Ziba; Baldassarre, Massimiliano; Bouaouina, Mohamed; Lamsoul, Isabelle; Lutz, Pierre G.; Calderwood, David A.

    2011-01-01

    Filamins are an important family of actin-binding and crosslinking proteins that mediate remodeling of the actin cytoskeleton and maintain extracellular matrix connections by anchoring transmembrane proteins to actin filaments and linking them to intracellular signaling cascades. We recently found that filamins are targeted for proteasomal degradation by the E3 ubiquitin ligase specificity subunit ASB? and that acute degradation of filamins through this ubiquitin–proteasome pathway correlates with cell differentiation. Specifically, in myeloid leukemia cells retinoic-acid-induced expression of ASB2? triggers filamin degradation and recapitulates early events crucial for cell differentiation. ASB2? is thought to link substrates to the ubiquitin transferase machinery; however, the mechanism by which ASB2? interacts with filamin to induce degradation remained unknown. Here, we use cell-based and biochemical assays to show that the subcellular localization of ASB2? to actin-rich structures is dependent on filamin and that the actin-binding domain (ABD) of filamin mediates the interaction with ASB2?. Furthermore, we show that the ABD is necessary and sufficient for ASB2?-mediated filamin degradation. We propose that ASB2? exerts its effect by binding the ABD and mediating its polyubiquitylation, so targeting filamins for degradation. These studies provide the molecular basis for ASB2?-mediated filamin degradation and unravel an important mechanism by which filamin levels can be acutely regulated. PMID:21750192

  1. A comprehensive framework of E2–RING E3 interactions of the human ubiquitin–proteasome system

    PubMed Central

    van Wijk, Sjoerd J L; de Vries, Sjoerd J; Kemmeren, Patrick; Huang, Anding; Boelens, Rolf; Bonvin, Alexandre M J J; Timmers, H Th Marc

    2009-01-01

    Covalent attachment of ubiquitin to substrates is crucial to protein degradation, transcription regulation and cell signalling. Highly specific interactions between ubiquitin-conjugating enzymes (E2) and ubiquitin protein E3 ligases fulfil essential roles in this process. We performed a global yeast-two hybrid screen to study the specificity of interactions between catalytic domains of the 35 human E2s with 250 RING-type E3s. Our analysis showed over 300 high-quality interactions, uncovering a large fraction of new E2–E3 pairs. Both within the E2 and the E3 cohorts, several members were identified that are more versatile in their interaction behaviour than others. We also found that the physical interactions of our screen compare well with reported functional E2–E3 pairs in in vitro ubiquitination experiments. For validation we confirmed the interaction of several versatile E2s with E3s in in vitro protein interaction assays and we used mutagenesis to alter the E3 interactions of the E2 specific for K63 linkages, UBE2N(Ubc13), towards the K48-specific UBE2D2(UbcH5B). Our data provide a detailed, genome-wide overview of binary E2–E3 interactions of the human ubiquitination system. PMID:19690564

  2. PML Activates Transcription by Protecting HIPK2 and p300 from SCFFbx3-Mediated Degradation? †

    PubMed Central

    Shima, Yutaka; Shima, Takito; Chiba, Tomoki; Irimura, Tatsuro; Pandolfi, Pier Paolo; Kitabayashi, Issay

    2008-01-01

    PML, a nuclear protein, interacts with several transcription factors and their coactivators, such as HIPK2 and p300, resulting in the activation of transcription. Although PML is thought to achieve transcription activation by stabilizing the transcription factor complex, little is known about the underlying molecular mechanism. To clarify the role of PML in transcription regulation, we purified the PML complex and identified Fbxo3 (Fbx3), Skp1, and Cullin1 as novel components of this complex. Fbx3 formed SCFFbx3 ubiquitin ligase and promoted the degradation of HIPK2 and p300 by the ubiquitin-proteasome pathway. PML inhibited this degradation through a mechanism that unexpectedly did not involve inhibition of the ubiquitination of HIPK2. PML, Fbx3, and HIPK2 synergistically activated p53-induced transcription. Our findings suggest that PML stabilizes the transcription factor complex by protecting HIPK2 and p300 from SCFFbx3-induced degradation until transcription is completed. In contrast, the leukemia-associated fusion PML-RAR? induced the degradation of HIPK2. We discuss the roles of PML and PML-retinoic acid receptor ?, as well as those of HIPK2 and p300 ubiquitination, in transcriptional regulation and leukemogenesis. PMID:18809579

  3. PML activates transcription by protecting HIPK2 and p300 from SCFFbx3-mediated degradation.

    PubMed

    Shima, Yutaka; Shima, Takito; Chiba, Tomoki; Irimura, Tatsuro; Pandolfi, Pier Paolo; Kitabayashi, Issay

    2008-12-01

    PML, a nuclear protein, interacts with several transcription factors and their coactivators, such as HIPK2 and p300, resulting in the activation of transcription. Although PML is thought to achieve transcription activation by stabilizing the transcription factor complex, little is known about the underlying molecular mechanism. To clarify the role of PML in transcription regulation, we purified the PML complex and identified Fbxo3 (Fbx3), Skp1, and Cullin1 as novel components of this complex. Fbx3 formed SCF(Fbx3) ubiquitin ligase and promoted the degradation of HIPK2 and p300 by the ubiquitin-proteasome pathway. PML inhibited this degradation through a mechanism that unexpectedly did not involve inhibition of the ubiquitination of HIPK2. PML, Fbx3, and HIPK2 synergistically activated p53-induced transcription. Our findings suggest that PML stabilizes the transcription factor complex by protecting HIPK2 and p300 from SCF(Fbx3)-induced degradation until transcription is completed. In contrast, the leukemia-associated fusion PML-RARalpha induced the degradation of HIPK2. We discuss the roles of PML and PML-retinoic acid receptor alpha, as well as those of HIPK2 and p300 ubiquitination, in transcriptional regulation and leukemogenesis. PMID:18809579

  4. N-Terminal Ubiquitination of Extracellular Signal-Regulated Kinase 3 and p21 Directs Their Degradation by the Proteasome

    PubMed Central

    Coulombe, Philippe; Rodier, Geneviève; Bonneil, Eric; Thibault, Pierre; Meloche, Sylvain

    2004-01-01

    Extracellular signal-regulated kinase 3 (ERK3) is an unstable mitogen-activated protein kinase homologue that is constitutively degraded by the ubiquitin-proteasome pathway in proliferating cells. Here we show that a lysineless mutant of ERK3 is still ubiquitinated in vivo and requires a functional ubiquitin conjugation pathway for its degradation. Addition of N-terminal sequence tags of increasing size stabilizes ERK3 by preventing its ubiquitination. Importantly, we identified a fusion peptide between the N-terminal methionine of ERK3 and the C-terminal glycine of ubiquitin in vivo by tandem mass spectrometry analysis. These findings demonstrate that ERK3 is conjugated to ubiquitin via its free NH2 terminus. We found that large N-terminal tags also stabilize the expression of the cell cycle inhibitor p21 but not that of substrates ubiquitinated on internal lysine residues. Consistent with this observation, lysineless p21 is ubiquitinated and degraded in a ubiquitin-dependent manner in intact cells. Our results suggests that N-terminal ubiquitination is a more prevalent modification than originally recognized. PMID:15226418

  5. Preventing p38 MAPK-Mediated MafA Degradation Ameliorates ?-Cell Dysfunction under Oxidative Stress

    PubMed Central

    El Khattabi, Ilham

    2013-01-01

    The reduction in the expression of glucose-responsive insulin gene transcription factor MafA accompanies the development of ?-cell dysfunction under oxidative stress/diabetic milieu. Humans with type 2 diabetes have reduced MafA expression, and thus preventing this reduction could overcome ?-cell dysfunction and diabetes. We previously showed that p38 MAPK, but not glycogen synthase kinase 3 (GSK3), is a major regulator of MafA degradation under oxidative stress. Here, we examined the mechanisms of this degradation and whether preventing MafA degradation under oxidative stress will overcome ?-cell dysfunction. We show that under oxidative and nonoxidative conditions p38 MAPK directly binds to MafA and triggers MafA degradation via ubiquitin proteasomal pathway. However, unlike nonoxidative conditions, MafA degradation under oxidative stress depended on p38 MAPK-mediated phosphorylation at threonine (T) 134, and not T57. Furthermore the expression of alanine (A) 134-MafA, but not A57-MafA, reduced the oxidative stress-mediated loss of glucose-stimulated insulin secretion, which was independent of p38 MAPK action on protein kinase D, a regulator of insulin secretion. Interestingly, the expression of proteasomal activator PA28? that degrades GSK3-phosphorylated (including T57) MafA was reduced under oxidative stress, explaining the dominance of p38 MAPK over the GSK3 pathway in regulating MafA stability under oxidative stress. These results identify two distinct pathways mediating p38 MAPK-dependent MafA degradation under oxidative and nonoxidative conditions and show that inhibiting MafA degradation under oxidative stress ameliorates ?-cell dysfunction and could lead to novel therapies for diabetes. PMID:23660596

  6. Isoform-specific proteasomal degradation of Rbfox3 during chicken embryonic development.

    PubMed

    Kim, Kee K; Adelstein, Robert S; Kawamoto, Sachiyo

    2014-08-01

    Rbfox3, a neuron-specific RNA-binding protein, plays an important role in neuronal differentiation during development. An isoform Rbfox3-d31, which excludes the 93-nucleotide cassette exon within the RNA recognition motif of chicken Rbfox3, has been previously identified. However, the cellular functions of Rbfox3-d31 remain largely unknown. Here we find that Rbfox3-d31 mRNA is highly expressed during the early developmental stages of the chicken embryo, while Rbfox3-d31 protein is barely detected during the same stage due to its rapid degradation mediated by the ubiquitin-proteasome pathway. Importantly, this degradation is specific to the Rbfox3-d31 isoform and it does not occur with full-length Rbfox3. Furthermore, suppression of Rbfox3-d31 protein degradation with the proteasome inhibitor MG132 attenuates the splicing activity of another Rbfox family member Rbfox2 by altering the subcellular localization of Rbfox2. These results suggest that Rbfox3-d31 functions as a repressor for the splicing activity of the Rbfox family and its protein level is regulated in an isoform-specific manner in vivo. PMID:25044120

  7. The proteasome activity reporter GFP-Cl1 is degraded by autophagy in the aging model Podospora anserina

    PubMed Central

    Wiemer, Matthias; Osiewacz, Heinz D.

    2014-01-01

    The degradation of damaged proteins is an important vital function especially during aging and stress. The ubiquitin proteasome system is one of the major cellular machineries for protein degradation. Health and longevity are associated with high proteasome activity. To demonstrate such a role in aging of Podospora anserina, we first analyzed the transcript and protein abundance of selected proteasome components in wild-type cultures of different age. No significant differences were observed. Next, in order to increase the overall proteasome abundance we generated strains overexpressing the catalytic proteasome subunits PaPRE2 and PaPRE3. Although transcript levels were strongly increased, no substantial effect on the abundance of the corresponding proteins was observed. Finally, the analysis of the P. anserina strains expressing the sequence coding for the CL1 degron fused to the Gfp gene revealed no evidence for degradation of the GFP-CL1 fusion protein by the proteasome. Instead, our results demonstrate the degradation of the CL1-degron sequence via autophagy, indicating that basal autophagy appears to be a very effective protein quality control pathway in P. anserina. PMID:25520781

  8. Negative regulation of DAB2IP by Akt and SCFFbw7 pathways

    PubMed Central

    Inuzuka, Hiroyuki

    2014-01-01

    Deletion of ovarian carcinoma 2/disabled homolog 2 (DOC-2/DAB2) interacting protein (DAB2IP), is a tumor suppressor that serves as a scaffold protein involved in coordinately regulating cell proliferation, survival and apoptotic pathways. DAB2IP is epigenetically down-regulated in a variety of tumors through the action of the histone methyltransferase EZH2. Although DAB2IP is transcriptionally down-regulated in a variety of tumors, it remains unclear if other mechanisms contribute to functional inactivation of DAB2IP. Here we demonstrate that DAB2IP can be functionally down-regulated by two independent mechanisms. First, we identified that Akt1 can phosphorylate DAB2IP on S847, which regulates the interaction between DAB2IP and its effector molecules H-Ras and TRAF2. Second, we demonstrated that DAB2IP can be degraded in part through ubiquitin-proteasome pathway by SCFFbw7. DAB2IP harbors two Fbw7 phosho-degron motifs, which can be regulated by the kinase, CK1?. Our data hence indicate that in addition to epigenetic down-regulation, two additional pathways can functional inactivate DAB2IP. Given that DAB2IP has previously been identified to possess direct causal role in tumorigenesis and metastasis, our data indicate that a variety of pathways may pass through DAB2IP to govern cancer development, and therefore highlight DAB2IP agonists as potential therapeutic approaches for future anti-cancer drug development. PMID:24912918

  9. Ubiquitin-protein ligase activity of X-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in Fas-induced cell death

    PubMed Central

    Suzuki, Yasuyuki; Nakabayashi, Yui; Takahashi, Ryosuke

    2001-01-01

    The inhibitor of apoptosis (IAP) family of anti-apoptotic proteins regulate programmed cell death and/or apoptosis. One such protein, X-linked IAP (XIAP), inhibits the activity of the cell death proteases, caspase-3, -7, and -9. In this study, using constitutively active mutants of caspase-3, we found that XIAP promotes the degradation of active-form caspase-3, but not procaspase-3, in living cells. The XIAP mutants, which cannot interact with caspase-3, had little or no activity of promoting the degradation of caspase-3. RING finger mutants of XIAP also could not promote the degradation of caspase-3. A proteasome inhibitor suppressed the degradation of caspase-3 by XIAP, suggesting the involvement of a ubiquitin-proteasome pathway in the degradation. An in vitro ubiquitination assay revealed that XIAP acts as a ubiquitin-protein ligase for caspase-3. Caspase-3 was ubiquitinated in the presence of XIAP in living cells. Both the association of XIAP with caspase-3 and the RING finger domain of XIAP were essential for ubiquitination. Finally, the RING finger mutants of XIAP were less effective than wild-type XIAP at preventing apoptosis induced by overexpression of either active-form caspase-3 or Fas. These results demonstrate that the ubiquitin-protein ligase activity of XIAP promotes the degradation of caspase-3, which enhances its anti-apoptotic effect. PMID:11447297

  10. Targeted degradation of the AML1/MDS1/EVI1 oncoprotein by arsenic trioxide.

    PubMed

    Shackelford, David; Kenific, Candia; Blusztajn, Agnieszka; Waxman, Samuel; Ren, Ruibao

    2006-12-01

    Arsenic trioxide (ATO) has been found to be an effective treatment for acute promyelocytic leukemia patients and is being tested for treating other hematologic malignancies. We have previously shown that AML1/MDS1/EVI1 (AME), a fusion gene generated by a t(3;21)(q26;q22) translocation found in patients with chronic myelogenous leukemia during blast phase, myelodysplastic syndrome, or acute myelogenous leukemia (AML), impairs hematopoiesis and eventually induces an AML in mice. Both fusion partners of AME, AML1 and MDS1/EVI1, encode transcription factors and are also targets of a variety of genetic abnormalities in human hematologic malignancies. In addition, aberrant expression of ectopic viral integration site 1 (EVI1) has also been found in solid tumors, such as ovarian and colon cancers. In this study, we examined whether ATO could target AME and related oncoproteins. We found that ATO used at therapeutic levels degrades AME. The ATO treatment induces differentiation and apoptosis in AME leukemic cells in vitro as well as reduces tumor load and increases the survival of mice transplanted with these cells. We further found that ATO targets AME via both myelodysplastic syndrome 1 (MDS1) and EVI1 moieties and degrades EVI1 via the ubiquitin-proteasome pathway and MDS1 in a proteasome-independent manner. Our results suggest that ATO could be used as a part of targeted therapy for AME-, AML1/MDS1-, MDS1/EVI1-, and EVI1-positive human cancers. PMID:17145882

  11. Regulating the 20S Proteasome Ubiquitin-Independent Degradation Pathway

    PubMed Central

    Ben-Nissan, Gili; Sharon, Michal

    2014-01-01

    For many years, the ubiquitin-26S proteasome degradation pathway was considered the primary route for proteasomal degradation. However, it is now becoming clear that proteins can also be targeted for degradation by the core 20S proteasome itself. Degradation by the 20S proteasome does not require ubiquitin tagging or the presence of the 19S regulatory particle; rather, it relies on the inherent structural disorder of the protein being degraded. Thus, proteins that contain unstructured regions due to oxidation, mutation, or aging, as well as naturally, intrinsically unfolded proteins, are susceptible to 20S degradation. Unlike the extensive knowledge acquired over the years concerning degradation by the 26S proteasome, relatively little is known about the means by which 20S-mediated proteolysis is controlled. Here, we describe our current understanding of the regulatory mechanisms that coordinate 20S proteasome-mediated degradation, and highlight the gaps in knowledge that remain to be bridged. PMID:25250704

  12. Roles for the ubiquitin-proteasome pathway in protein quality control and signaling in the retina: implications in the pathogenesis of age-related macular degeneration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The accumulation of damaged or postsynthetically modified proteins and dysregulation of inflammatory responses and angiogenesis in the retina/RPE are thought be etiologically related to formation of drusen and choroidal neovascularization (CNV), hallmarks of age-related macular degeneration (AMD). T...

  13. Mutations in the tumor suppressors Smad2 and Smad4 inactivate transforming growth factor signaling by targeting Smads to the ubiquitin-proteasome pathway

    Microsoft Academic Search

    Jing Xu; Liliana Attisano

    2000-01-01

    Biological signals for transforming growth factor (TGF-) are transduced through transmembrane serine\\/threonine kinase receptors that signal to a family of intracellular mediators known as Smads. Smad2 and Smad4 are important for transcriptional and antiproliferative responses to TGF-, and their inactivation in human cancers indicates that they are tumor suppressors. A missense mutation at a conserved arginine residue in the amino-terminal

  14. Role of the ubiquitin-proteasome pathway in the inner ear : identification of an E3 ubiquitin ligase for Atoh1

    E-print Network

    Cheng, Yen-Fu

    2014-01-01

    Atoh1, the proneural basic-helix-loop-helix transcription factor, is critical for the differentiation of inner ear hair cells. Hair cells do not develop in mice that lack Atoh1, and overexpression of the transcription ...

  15. Ubiquitin-protein ligase E3C promotes glioma progression by mediating the ubiquitination and degrading of Annexin A7

    PubMed Central

    Pan, Si-Jian; Zhan, Shi-Kun; Ji, Wei-Zhong; Pan, Yi-Xin; Liu, Wei; Li, Dian-You; Huang, Peng; Zhang, Xiao-Xiao; Cao, Chun-Yan; Zhang, Jing; Bian, Liu-Guan; Sun, Bomin; Sun, Qing-Fang

    2015-01-01

    The ubiquitin-protein ligase E3C (UBE3C) belongs to the E3 ligase enzyme family and implicates in the ubiquitin-proteasome pathway, thus regulates physiological and cancer-related processes. Here, we investigated the expression and roles of UBE3C in glioma. We demonstrated that UBE3C was overexpressed in glioma tissues and cell lines. Inhibition of UBE3C expression in glioma cells significantly decreased cell migration and invasion in vitro. Mechanistically, we disclosed that UBE3C physically interacted with and ubiquitinated tumor suppressor gene annexin A7 (ANXA7), resulting in ubiquitination and degradation of ANXA7. Our results also revealed that increased UBE3C expression was accompanied by a reduction in ANXA7 protein expression in glioma tissues, but not ANXA7 mRNA. Importantly, the inhibition of ANXA7 expression in gliomas cells with UBE3C interference could rescue the cell invasion. Clinically, UBE3C overexpression significantly correlated with high-grade tumors (p?degradation of ANXA7, and thus presents a novel and promising target for glioma therapy. PMID:26067607

  16. Understanding Degradation Pathways in Organic Photovoltaics (Poster)

    SciTech Connect

    Lloyd, M. T.; Olson, D. C.; Garcia, A.; Kauvar, I.; Kopidakis, N.; Reese, M. O.; Berry, J. J.; Ginley, D. S.

    2011-02-01

    Organic Photovoltaics (OPVs) recently attained power conversion efficiencies that are of interest for commercial production. Consequently, one of the most important unsolved issues facing a new industry is understanding what governs lifetime in organic devices and discovering solutions to mitigate degradation mechanisms. Historically, the active organic components are considered vulnerable to photo-oxidation and represent the primary degradation channel. However, we present several (shelf life and light soaking) studies pointing the relative stability of the active layers and instabilities in commonly used electrode materials. We show that engineering of the hole/electron layer at the electrode can lead to environmentally stable devices even without encapsulation.

  17. Redox-Regulated Pathway of Tyrosine Phosphorylation Underlies NF-?B Induction by an Atypical Pathway Independent of the 26S Proteasome

    PubMed Central

    Cullen, Sarah; Ponnappan, Subramaniam; Ponnappan, Usha

    2015-01-01

    Alternative redox stimuli such as pervanadate or hypoxia/reoxygenation, induce transcription factor NF-?B by phospho-tyrosine-dependent and proteasome-independent mechanisms. While considerable attention has been paid to the absence of proteasomal regulation of tyrosine phosphorylated I?B?, there is a paucity of information regarding proteasomal regulation of signaling events distinct from tyrosine phosphorylation of I?B?. To delineate roles for the ubiquitin-proteasome pathway in the phospho-tyrosine dependent mechanism of NF-?B induction, we employed the proteasome inhibitor, Aclacinomycin, and the phosphotyrosine phosphatase inhibitor, pervanadate (PV). Results from these studies demonstrate that phospho-I?B? (Tyr-42) is not subject to proteasomal degradation in a murine stromal epithelial cell line, confirming results previously reported. Correspondingly, proteasome inhibition had no discernable effect on the key signaling intermediaries, Src and ERK1/2, involved in the phospho-tyrosine mechanisms regulating PV-mediated activation of NF-?B. Consistent with previous reports, a significant redox imbalance leading to the activation of tyrosine kinases, as occurs with pervanadate, is required for the induction of NF-?B. Strikingly, our studies demonstrate that proteasome inhibition can potentiate oxidative stress associated with PV-stimulation without impacting kinase activation, however, other cellular implications for this increase in intracellular oxidation remain to be fully delineated. PMID:25671697

  18. Trichloroethylene degradation by two independent aromatic-degrading pathways in alcaligenes eutrophus JMP134

    SciTech Connect

    Harker, A.R.; Kim, Y. (Oklahoma State Univ. Stillwater (USA))

    1990-04-01

    The bacterium Alcaligenes eutrophus JMP134(pJP4) degrades trichloroethylene (TCE) by a chromosomal phenol-dependent pathway and by the plasmid-encoded 2,4-dichlorophenoxyacetic acid pathway. The two pathways were independent and exhibited different rates of removal and capacities for quantity of TCE removed. The phenol-dependent pathway was more rapid (0.2 versus 0.06 nmol of TCE removed per min per mg of protein) and consumed all detectable TCE. The 2,4-dichlorophenoxyacetic acid-dependent pathway removed 40 to 60% of detectable TCE.

  19. Prokaryotic Proteasomes: Nanocompartments of Degradation

    PubMed Central

    Humbard, Matthew A.; Maupin-Furlow, Julie A.

    2014-01-01

    Proteasomes are self-compartmentalized energy-dependent proteolytic machines found in Archaea, Actinobacteria species of bacteria and eukaryotes. Proteasomes consist of two separate protein complexes, the core particle that hydrolyzes peptide bonds and an AAA+ ATPase domain responsible for the binding, unfolding and translocation of protein substrates into the core particle for degradation. Similarly to eukaryotes, proteasomes play a central role in protein degradation and can be essential in Archaea. Core particles associate with and utilize a variety of ATPase complexes to carry out protein degradation in Archaea. In actinobacterial species, such as Mycobacterium tuberculosis, proteasome-mediated degradation is associated with pathogenesis and does not appear to be essential. Interestingly, both actinobacterial species and Archaea use small proteins to covalently modify proteins, prokaryotic ubiquitin-like proteins (Pup) in Actinobacteria and ubiquitin-like small archaeal modifier proteins (SAMP) in Archaea. These modifications may play a role in proteasome targeting similar to the ubiquitin-proteasome system in eukaryotes. PMID:23920495

  20. Validation of microarray data in human lymphoblasts shows a role of the ubiquitin-proteasome system and NF-kB in the pathogenesis of Down syndrome

    PubMed Central

    2013-01-01

    Background Down syndrome (DS) is a complex disorder caused by the trisomy of either the entire, or a critical region of chromosome 21 (21q22.1-22.3). Despite representing the most common cause of mental retardation, the molecular bases of the syndrome are still largely unknown. Methods To better understand the pathogenesis of DS, we analyzed the genome-wide transcription profiles of lymphoblastoid cell lines (LCLs) from six DS and six euploid individuals and investigated differential gene expression and pathway deregulation associated with trisomy 21. Connectivity map and PASS-assisted exploration were used to identify compounds whose molecular signatures counteracted those of DS lymphoblasts and to predict their therapeutic potential. An experimental validation in DS LCLs and fetal fibroblasts was performed for the most deregulated GO categories, i.e. the ubiquitin mediated proteolysis and the NF-kB cascade. Results We show, for the first time, that the level of protein ubiquitination is reduced in human DS cell lines and that proteasome activity is increased in both basal conditions and oxidative microenvironment. We also provide the first evidence that NF-kB transcription levels, a paradigm of gene expression control by ubiquitin-mediated degradation, is impaired in DS due to reduced IkB-alfa ubiquitination, increased NF-kB inhibitor (IkB-alfa) and reduced p65 nuclear fraction. Finally, the DSCR1/DYRK1A/NFAT genes were analysed. In human DS LCLs, we confirmed the presence of increased protein levels of DSCR1 and DYRK1A, and showed that the levels of the transcription factor NFATc2 were decreased in DS along with a reduction of its nuclear translocation upon induction of calcium fluxes. Conclusions The present work offers new perspectives to better understand the pathogenesis of DS and suggests a rationale for innovative approaches to treat some pathological conditions associated to DS. PMID:23830204

  1. Cathodic degradation of antibiotics: characterization and pathway analysis.

    PubMed

    Kong, Deyong; Liang, Bin; Yun, Hui; Cheng, Haoyi; Ma, Jincai; Cui, Minhua; Wang, Aijie; Ren, Nanqi

    2015-04-01

    Antibiotics in wastewaters must be degraded to eliminate their antibacterial activity before discharging into the environment. A cathode can provide continuous electrons for the degradation of refractory pollutants, however the cathodic degradation feasibility, efficiency and pathway for different kinds of antibiotics is poorly understood. Here, we investigated the degradation of four antibiotics, namely nitrofurazone (NFZ), metronidazole (MNZ), chloramphenicol (CAP), and florfenicol (FLO) by a poised cathode in a dual chamber electrochemical reactor. The cyclic voltammetry preliminarily proved the feasibility of the cathodic degradation of these antibiotics. The cathodic reducibility of these antibiotics followed the order of NFZ > MNZ > CAP > FLO. A decreased phosphate buffered solution (PBS) concentration as low as 2 mM or utilization of NaCl buffer solution as catholyte had significant influence on antibiotics degradation rate and efficiency for CAP and FLO but not for NFZ and MNZ. PBS could be replaced by Na2CO3-NaHCO3 buffer solution as catholyte for the degradation of these antibiotics. Reductive dechlorination of CAP proceeded only after the reduction of the nitro group to aromatic amine. The composition of the degradation products depended on the cathode potential except for MNZ. The cathodic degradation process could eliminate the antibacterial activity of these antibiotics. The current study suggests that the electrochemical reduction could serve as a potential pretreatment or advanced treatment unit for the treatment of antibiotics containing wastewaters. PMID:25660806

  2. Fenretinide induces ubiquitin-dependent proteasomal degradation of stearoyl-CoA desaturase in human retinal pigment epithelial cells.

    PubMed

    Samuel, William; Kutty, R Krishnan; Duncan, Todd; Vijayasarathy, Camasamudram; Kuo, Bryan C; Chapa, Krysten M; Redmond, T Michael

    2014-08-01

    Stearoyl-CoA desaturase (SCD, SCD1), an endoplasmic reticulum (ER) resident protein and a rate-limiting enzyme in monounsaturated fatty acid biosynthesis, regulates cellular functions by controlling the ratio of saturated to monounsaturated fatty acids. Increase in SCD expression is strongly implicated in the proliferation and survival of cancer cells, whereas its decrease is known to impair proliferation, induce apoptosis, and restore insulin sensitivity. We examined whether fenretinide, (N-(4-hydroxyphenyl)retinamide, 4HPR), which induces apoptosis in cancer cells and recently shown to improve insulin sensitivity, can modulate the expression of SCD. We observed that fenretinide decreased SCD protein and enzymatic activity in the ARPE-19 human retinal pigment epithelial cell line. Increased expression of BiP/GRP78, ATF4, and GADD153 implicated ER stress. Tunicamycin and thapsigargin, compounds known to induce ER stress, also decreased the SCD protein. This decrease was completely blocked by the proteasome inhibitor MG132. In addition, PYR41, an inhibitor of ubiquitin activating enzyme E1, blocked the fenretinide-mediated decrease in SCD. Immunoprecipitation analysis using anti-ubiquitin and anti-SCD antibodies and the blocking of SCD loss by PYR41 inhibition of ubiquitination further corroborate that fenretinide mediates the degradation of SCD in human RPE cells via the ubiquitin-proteasome dependent pathway. Therefore, the effect of fenretinide on SCD should be considered in its potential therapeutic role against cancer, type-2 diabetes, and retinal diseases. PMID:24357007

  3. Tamoxifen Inhibits ER-negative Breast Cancer Cell Invasion and Metastasis by Accelerating Twist1 Degradation.

    PubMed

    Ma, Gang; He, Jianjun; Yu, Yang; Xu, Yixiang; Yu, Xiaobin; Martinez, Jarrod; Lonard, David M; Xu, Jianming

    2015-01-01

    Twist1 is a transcription factor driving epithelial-mesenchymal transition, invasion and metastasis of breast cancer cells. Mice with germ-line Twist1 knockout are embryonic lethal, while adult mice with inducible Twist1 knockout have no obvious health problems, suggesting that Twist1 is a viable therapeutic target for the inhibition of invasion and metastasis of breast cancer in adult patients. In this study, we expressed a luciferase protein or a Twist1-luciferase fusion protein in HeLa cells as part of a high throughput system to screen 1280 compounds in the Library of Pharmacologically Active Compounds (LOPAC) from Sigma-Aldrich for their effects on Twist1 protein expression. One of the most interesting compounds identified is tamoxifen, a selective estrogen receptor (ER) modulator used to treat ER-positive breast cancer. Tamoxifen treatment significantly accelerated Twist1 degradation in multiple cell lines including HEK293 human kidney cells, 4T1 and 168FARN mouse mammary tumor cells with either ectopically or endogenously expressed Twist1. Tamoxifen-induced Twist1 degradation could be blocked by the MG132 proteasome inhibitor, suggesting that tamoxifen induces Twist1 degradation through the ubiquitination-proteasome pathway. However, tamoxifen-induced Twist1 degradation was independent of Twist1 mRNA expression, estrogen signaling and MAPK-mediated Twist1 phosphorylation in these cells. Importantly, tamoxifen also significantly inhibited invasive behavior in Matrigel and lung metastasis in SCID-bg mice of ER-negative 4T1 mammary tumor cells, which depend on endogenous Twist1 to invade and metastasize. These results indicate that tamoxifen can significantly accelerate Twist1 degradation to suppress cancer cell invasion and metastasis, suggesting that tamoxifen can be used not only to treat ER-positive breast cancers but also to reduce Twist1-mediated invasion and metastasis in ER-negative breast cancers. PMID:25892968

  4. Epoxy Coenzyme A Thioester Pathways for Degradation of Aromatic Compounds

    PubMed Central

    Gescher, Johannes

    2012-01-01

    Aromatic compounds (biogenic and anthropogenic) are abundant in the biosphere. Some of them are well-known environmental pollutants. Although the aromatic nucleus is relatively recalcitrant, microorganisms have developed various catabolic routes that enable complete biodegradation of aromatic compounds. The adopted degradation pathways depend on the availability of oxygen. Under oxic conditions, microorganisms utilize oxygen as a cosubstrate to activate and cleave the aromatic ring. In contrast, under anoxic conditions, the aromatic compounds are transformed to coenzyme A (CoA) thioesters followed by energy-consuming reduction of the ring. Eventually, the dearomatized ring is opened via a hydrolytic mechanism. Recently, novel catabolic pathways for the aerobic degradation of aromatic compounds were elucidated that differ significantly from the established catabolic routes. The new pathways were investigated in detail for the aerobic bacterial degradation of benzoate and phenylacetate. In both cases, the pathway is initiated by transforming the substrate to a CoA thioester and all the intermediates are bound by CoA. The subsequent reactions involve epoxidation of the aromatic ring followed by hydrolytic ring cleavage. Here we discuss the novel pathways, with a particular focus on their unique features and occurrence as well as ecological significance. PMID:22582071

  5. Biochemical pathway and degradation of phthalate ester isomers by bacteria.

    PubMed

    Gu, J D; Li, J; Wang, Y

    2005-01-01

    Degradation of dimethyl isophthalate (DMI) and dimethyl phthalate ester (DMPE) was investigated using microorganisms isolated from mangrove sediment of Hong Kong Mai Po Nature Reserve. One enrichment culture was capable of utilizing DMI as the sole source of carbon and energy, but none of the bacteria in the enrichment culture was capable of degrading DMI alone. In co-culture of two bacteria, degradation was observed proceeding through monomethyl isophthalate (MMI) ester and isophthalic acid (IPA) before the aromatic ring opening. Using DMI as the sole carbon and energy source, Klebsiella oxytoca Sc and Methylobacterium mesophilicum Sr degraded DMI through the biochemical cooperation. The initial hydrolytic reaction of the ester bond was by K. oxytoca Sc and the next step of transformation was by M. mesophilicum Sr, and IPA was degraded by both of them. In another investigation, a novel bacterium, strain MPsc, was isolated for degradation of dimethyl phthalate ester (DMPE) also from the mangrove sediment. On the basis of phenotypic, biochemical and 16S rDNA gene sequence analyses, the strain MPsc should be considered as a new bacterium at the genus level (8% differences). This strain, together with a Rhodococcus zopfii isolated from the same mangrove sediment, was able to degrade DMPE aerobically. The consortium consisting of the two species degraded 450 mg/l DMPE within 3 days as the sole source of carbon and energy, but none of the individual species alone was able to transform DMPE. Furthermore, the biochemical degradation pathway proceeded through monomethyl phthalate (MMP), phthalic acid (PA) and then protocatechuate before aromatic ring cleavage. Our results suggest that degradation of complex organic compounds including DMI and DMPE may be carried out by several members of microorganisms working together in the natural environments. PMID:16312973

  6. New insights into the alternative D-glucarate degradation pathway.

    PubMed

    Aghaie, Asadollah; Lechaplais, Christophe; Sirven, Peggy; Tricot, Sabine; Besnard-Gonnet, Marielle; Muselet, Delphine; de Berardinis, Véronique; Kreimeyer, Annett; Gyapay, Gabor; Salanoubat, Marcel; Perret, Alain

    2008-06-01

    Although the D-glucarate degradation pathway is well characterized in Escherichia coli, genetic and biochemical information concerning the alternative pathway proposed in Pseudomonas species and Bacillus subtilis remains incomplete. Acinetobacter baylyi ADP1 is a Gram-negative soil bacterium possessing the alternative pathway and able to grow using D-glucarate as the only carbon source. Based on the annotation of its sequenced genome (1), we have constructed a complete collection of singlegene deletion mutants (2). High throughput profiling for growth on a minimal medium containing D-glucarate as the only carbon source for approximately 2450 mutants led to the identification of the genes involved in D-glucarate degradation. Protein purification after recombinant production in E. coli allowed us to reconstitute the enzymatic pathway in vitro. We describe here the kinetic characterization of D-glucarate dehydratase, d-5-keto-4-deoxyglucarate dehydratase, and of cooperative alpha-ketoglutarate semialdehyde dehydrogenase. Transcription and expression analyses of the genes involved in D-glucarate metabolism within a single organism made it possible to access information regarding the regulation of this pathway for the first time. PMID:18364348

  7. Aerobic degradation of dinitrotoluenes and pathway for bacterial degradation of 2,6-dinitrotoluene

    SciTech Connect

    Nishino, S.F.; Paoli, G.C.; Spain, J.C.

    2000-05-01

    An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2,4-DNT) by Burkholderia sp. strain DNT has been reported previously. The authors report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2,6-dinitrotoluene (2,6-DNT) by a different pathway. Burkholderia cepacia strain JS850 and Hydrogenophaga palleronii strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen. The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance. 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite. 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid. 2,4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol. Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol.

  8. Identification of the major degradation pathways of ticagrelor.

    PubMed

    Sadou Yaye, Hassane; Secrétan, Philippe-Henri; Henriet, Théo; Bernard, Mélisande; Amrani, Fatma; Akrout, Wiem; Tilleul, Patrick; Yagoubi, Najet; Do, Bernard

    2015-02-01

    Ticagrelor is a direct-acting and reversible P2Y12-adenosine diphosphate (ADP) receptor blocker used as antiplatelet drug. Forced degradation under various stress conditions was carried out. The degradation products have been detected and identified by high-pressure liquid chromatography multistage mass spectrometry (LC-MS(n)) along with high-resolution mass spectrometry. C18 XTerra MS column combined with a linear gradient mobile phase composed of a mixture of 10 mM acetate ammonium/acetonitrile was shown suitable for drug and impurity determinations and validated as a stability indicating method. Structural elucidation of the degradation products relied on MS(n) studies and accurate mass measurements giving access to elemental compositions. Up to nine degradation products resulting from oxidation/auto-oxidation, S-dealkylation and N-dealkylation have been identified, covering a range of possible degradation pathways for derivatives with such functional groups. Kinetics was also studied in order to assess the molecule's shelf-life and to identify the most important degradation factors. PMID:25543285

  9. Down-regulation of Akt/mammalian target of rapamycin signaling pathway in response to myostatin overexpression in skeletal muscle.

    PubMed

    Amirouche, Adel; Durieux, Anne-Cécile; Banzet, Sébastien; Koulmann, Nathalie; Bonnefoy, Régis; Mouret, Catherine; Bigard, Xavier; Peinnequin, André; Freyssenet, Damien

    2009-01-01

    Myostatin, a member of the TGF-beta family, has been identified as a master regulator of embryonic myogenesis and early postnatal skeletal muscle growth. However, cumulative evidence also suggests that alterations in skeletal muscle mass are associated with dysregulation in myostatin expression and that myostatin may contribute to muscle mass loss in adulthood. Two major branches of the Akt pathway are relevant for the regulation of skeletal muscle mass, the Akt/mammalian target of rapamycin (mTOR) pathway, which controls protein synthesis, and the Akt/forkhead box O (FOXO) pathway, which controls protein degradation. Here, we provide further insights into the mechanisms by which myostatin regulates skeletal muscle mass by showing that myostatin negatively regulates Akt/mTOR signaling pathway. Electrotransfer of a myostatin expression vector into the tibialis anterior muscle of Sprague Dawley male rats increased myostatin protein level and decreased skeletal muscle mass 7 d after gene electrotransfer. Using RT-PCR and immunoblot analyses, we showed that myostatin overexpression was ineffective to alter the ubiquitin-proteasome pathway. By contrast, myostatin acted as a negative regulator of Akt/mTOR pathway. This was supported by data showing that the phosphorylation of Akt on Thr308, tuberous sclerosis complex 2 on Thr1462, ribosomal protein S6 on Ser235/236, and 4E-BP1 on Thr37/46 was attenuated 7 d after myostatin gene electrotransfer. The data support the conclusion that Akt/mTOR signaling is a key target that accounts for myostatin function during muscle atrophy, uncovering a novel role for myostatin in protein metabolism and more specifically in the regulation of translation in skeletal muscle. PMID:18801898

  10. A label-free quantitative proteomics strategy to identify E3 ubiquitin ligase substrates targeted to proteasome degradation.

    PubMed

    Burande, Clara F; Heuzé, Mélina L; Lamsoul, Isabelle; Monsarrat, Bernard; Uttenweiler-Joseph, Sandrine; Lutz, Pierre G

    2009-07-01

    The ubiquitin-proteasome system is a central mechanism for controlled proteolysis that regulates numerous cellular processes in eukaryotes. As such, defects in this system can contribute to disease pathogenesis. In this pathway, E3 ubiquitin ligases provide platforms for binding specific substrates, thereby coordinating their ubiquitylation and subsequent degradation by the proteasome. Despite the identification of many E3 ubiquitin ligases, the identities of their specific substrates are still largely unresolved. The ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) gene that we initially identified as a retinoic acid-response gene in acute promyelocytic leukemia cells encodes the specificity subunit of an E3 ubiquitin ligase complex that is involved in hematopoietic cell differentiation. We have recently identified filamin A and filamin B as the first ASB2 targets and shown that ASB2 triggers ubiquitylation and proteasome-mediated degradation of these proteins. Here a global quantitative proteomics strategy is provided to identify substrates of E3 ubiquitin ligases targeted to proteasomal degradation. Indeed we used label-free methods for quantifying proteins identified by shotgun proteomics in extracts of cells expressing wild-type ASB2 or an E3 ubiquitin ligase-defective mutant of ASB2 under the control of an inducible promoter. Measurements of spectral count and mass spectrometric signal intensity demonstrated a drastic decrease of filamin A and filamin B in myeloid leukemia cells expressing wild-type ASB2 compared with cells expressing an E3 ubiquitin ligase-defective mutant of ASB2. Altogether we provide an original strategy that enables identification of E3 ubiquitin ligase substrates that have to be degraded. PMID:19376791

  11. Bacterial Transcriptional Regulators for Degradation Pathways of Aromatic Compounds

    PubMed Central

    Tropel, David; van der Meer, Jan Roelof

    2004-01-01

    Human activities have resulted in the release and introduction into the environment of a plethora of aromatic chemicals. The interest in discovering how bacteria are dealing with hazardous environmental pollutants has driven a large research community and has resulted in important biochemical, genetic, and physiological knowledge about the degradation capacities of microorganisms and their application in bioremediation, green chemistry, or production of pharmacy synthons. In addition, regulation of catabolic pathway expression has attracted the interest of numerous different groups, and several catabolic pathway regulators have been exemplary for understanding transcription control mechanisms. More recently, information about regulatory systems has been used to construct whole-cell living bioreporters that are used to measure the quality of the aqueous, soil, and air environment. The topic of biodegradation is relatively coherent, and this review presents a coherent overview of the regulatory systems involved in the transcriptional control of catabolic pathways. This review summarizes the different regulatory systems involved in biodegradation pathways of aromatic compounds linking them to other known protein families. Specific attention has been paid to describing the genetic organization of the regulatory genes, promoters, and target operon(s) and to discussing present knowledge about signaling molecules, DNA binding properties, and operator characteristics, and evidence from regulatory mutants. For each regulator family, this information is combined with recently obtained protein structural information to arrive at a possible mechanism of transcription activation. This demonstrates the diversity of control mechanisms existing in catabolic pathways. PMID:15353566

  12. Inhibition of UCH-L1 in oligodendroglial cells results in microtubule stabilization and prevents ?-synuclein aggregate formation by activating the autophagic pathway: implications for multiple system atrophy

    PubMed Central

    Pukaß, Katharina; Richter-Landsberg, Christiane

    2015-01-01

    ?-Synuclein (?-syn) positive glial cytoplasmic inclusions (GCI) originating in oligodendrocytes (ODC) are a characteristic hallmark in multiple system atrophy (MSA). Their occurrence may be linked to a failure of the ubiquitin proteasome system (UPS) or the autophagic pathway. For proteasomal degradation, proteins need to be covalently modified by ubiquitin, and deubiquitinated by deubiquitinating enzymes (DUBs) before proteolytic degradation is performed. The DUB ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) is a component of the UPS, it is abundantly expressed in neuronal brain cells and has been connected to Parkinson’s disease (PD). It interacts with ?-syn and tubulin. The present study was undertaken to investigate whether UCH-L1 is a constituent of ODC, the myelin forming cells of the CNS, and is associated with GCIs in MSA. Furthermore, LDN-57444 (LDN), a specific UCH-L1 inhibitor, was used to analyze its effects on cell morphology, microtubule (MT) organization and the proteolytic degradation system. Towards this an oligodendroglial cell line (OLN cells), stably transfected with ?-syn or with ?-syn and GFP-LC3, to monitor the autophagic flux, was used. The data show that UCH-L1 is expressed in ODC derived from the brains of newborn rats and colocalizes with ?-syn in GCIs of MSA brain sections. LDN treatment had a direct impact on the MT network by affecting tubulin posttranslational modifications, i.e., acetylation and tyrosination. An increase in ?-tubulin detyrosination was observed and detyrosinated MT were abundantly recruited to the cellular extensions. Furthermore, small ?-syn aggregates, which are constitutively expressed in OLN cells overexpressing ?-syn, were abolished, and LDN caused the upregulation of the autophagic pathway. Our data add to the knowledge that the UPS and the autophagy-lysosomal pathway are tightly balanced, and that UCH-L1 and its regulation may play a role in neurodegenerative diseases with oligodendroglia pathology. PMID:25999815

  13. Autophagy as a Regulated Pathway of Cellular Degradation

    NSDL National Science Digital Library

    Daniel Klionsky (University of Michigan; Department of Biology)

    2000-12-01

    Macroautophagy is a dynamic process involving the rearrangement of subcellular membranes to sequester cytoplasm and organelles for delivery to the lysosome or vacuole where the sequestered cargo is degraded and recycled. This process takes place in all eukaryotic cells. It is highly regulated through the action of various kinases, phosphatases, and guanosine triphosphatases (GTPases). The core protein machinery that is necessary to drive formation and consumption of intermediates in the macroautophagy pathway includes a ubiquitin-like protein conjugation system and a protein complex that directs membrane docking and fusion at the lysosome or vacuole. Macroautophagy plays an important role in developmental processes, human disease, and cellular response to nutrient deprivation.

  14. The Endoplasmic Reticulum-Associated Degradation Pathways of Budding Yeast

    PubMed Central

    Thibault, Guillaume; Ng, Davis T.W.

    2012-01-01

    Protein misfolding is a common cellular event that can produce intrinsically harmful products. To reduce the risk, quality control mechanisms are deployed to detect and eliminate misfolded, aggregated, and unassembled proteins. In the secretory pathway, it is mainly the endoplasmic reticulum-associated degradation (ERAD) pathways that perform this role. Here, specialized factors are organized to monitor and process the folded states of nascent polypeptides. Despite the complex structures, topologies, and posttranslational modifications of client molecules, the ER mechanisms are the best understood among all protein quality-control systems. This is the result of convergent and sometimes serendipitous discoveries by researchers from diverse fields. Although major advances in ER quality control and ERAD came from all model organisms, this review will focus on the discoveries culminating from the simple budding yeast. PMID:23209158

  15. Arsenic trioxide induces programmed cell death through stimulation of ER stress and inhibition of the ubiquitin-proteasome system in human sarcoma cells.

    PubMed

    Chiu, Hui-Wen; Tseng, Yin-Chiu; Hsu, Yung-Ho; Lin, Yuh-Feng; Foo, Ning-Ping; Guo, How-Ran; Wang, Ying-Jan

    2015-01-28

    Sarcoma is a rare form of cancer that differs from the much more common carcinomas because it occurs in a distinct type of tissue. Many patients of sarcoma have poor response to chemotherapy and an increased risk for local recurrence. Arsenic trioxide (ATO) is used to treat certain types of leukemia. Recently, data have revealed that ATO induces sarcoma cell death in several types of solid tumor cell lines. In the present study, we investigated whether ATO induces cancer cell death and elucidated the underlying anti-cancer mechanisms. Our results showed that ATO caused concentration- and time-dependent cell death in human osteosarcoma and fibrosarcoma cells. The types of cell death that were induced by ATO were primarily autophagy and apoptosis. Furthermore, ATO activated p38, JNK and AMPK and inhibited the Akt/mTOR signaling pathways. Specifically, we found that ATO induced endoplasmic reticulum (ER) stress and suppressed proteasome activation in two types of sarcoma cell lines. However, the level of proteasome inhibition in osteosarcoma cells was lower than in fibrosarcoma cells. Thus, we used combined treatment with ATO and a proteasome inhibitor to examine the antitumor activity in fibrosarcoma cells. The data indicated showed that the combination treatment of ATO and MG132 (a proteasome inhibitor) resulted in synergistic cytotoxicity. In a fibrosarcoma xenograft mouse model, the combined treatment significantly reduced tumor progression. Immunohistochemical studies revealed that combined treatment induced autophagy and apoptosis. In summary, our results suggest a potential clinical application of ATO in sarcoma therapy and that combined treatment with a proteasome inhibitor can increase the therapeutic efficacy. PMID:25449439

  16. Ubiquitin conjugation by the N-end rule pathway and mRNAs for its components increase in muscles of diabetic rats

    NASA Technical Reports Server (NTRS)

    Lecker, S. H.; Solomon, V.; Price, S. R.; Kwon, Y. T.; Mitch, W. E.; Goldberg, A. L.

    1999-01-01

    Insulin deficiency (e.g., in acute diabetes or fasting) is associated with enhanced protein breakdown in skeletal muscle leading to muscle wasting. Because recent studies have suggested that this increased proteolysis is due to activation of the ubiquitin-proteasome (Ub-proteasome) pathway, we investigated whether diabetes is associated with an increased rate of Ub conjugation to muscle protein. Muscle extracts from streptozotocin-induced insulin-deficient rats contained greater amounts of Ub-conjugated proteins than extracts from control animals and also 40-50% greater rates of conjugation of (125)I-Ub to endogenous muscle proteins. This enhanced Ub-conjugation occurred mainly through the N-end rule pathway that involves E2(14k) and E3alpha. A specific substrate of this pathway, alpha-lactalbumin, was ubiquitinated faster in the diabetic extracts, and a dominant negative form of E2(14k) inhibited this increase in ubiquitination rates. Both E2(14k) and E3alpha were shown to be rate-limiting for Ub conjugation because adding small amounts of either to extracts stimulated Ub conjugation. Furthermore, mRNA for E2(14k) and E3alpha (but not E1) were elevated 2-fold in muscles from diabetic rats, although no significant increase in E2(14k) and E3alpha content could be detected by immunoblot or activity assays. The simplest interpretation of these results is that small increases in both E2(14k) and E3alpha in muscles of insulin-deficient animals together accelerate Ub conjugation and protein degradation by the N-end rule pathway, the same pathway activated in cancer cachexia, sepsis, and hyperthyroidism.

  17. Degradation of sulphonated azo dye Red HE7B by Bacillus sp. and elucidation of degradative pathways.

    PubMed

    Thakur, Jyoti Kumar; Paul, Sangeeta; Dureja, Prem; Annapurna, K; Padaria, Jasdeep C; Gopal, Madhuban

    2014-08-01

    Bacteria capable of degrading the sulfonated azo dye Red HE7B were isolated from textile mill effluent contaminated soil. The most efficient isolate was identified as Bacillus sp. Azo1 and the isolate could successfully decolorize up to 89% of the dye. The decolorized cultural extract analyzed by HPLC confirmed degradation. Enzymatic analysis showed twofold and fourfold increase in the activity of azoreductase and laccase enzymes, respectively, indicating involvement of both reductive and oxidative enzymes in biodegradation of Red HE7B. Degraded products which were identified by GC/MS analysis included various metabolites like 8-nitroso 1-naphthol, 2-diazonium naphthalene. Mono azo dye intermediate was initially generated from the parent molecule. This mono azo dye was further degraded by the organism, into additional products, depending on the site of cleavage of R-N=N-R molecule. Based on the degradation products identified, three different pathways have been proposed. The mechanism of degradation in two of these pathways is different from that of the previously reported pathway for azo dye degradation. This is the first report of a microbial isolate following multiple pathways for azo dye degradation. Azo dye Red HE7B was observed to be phytotoxic, leading to decrease in root development, shoot length and seedling fresh weight. However, after biotreatment the resulting degradation products were non-phytotoxic. PMID:24682261

  18. Exploitation of eukaryotic ubiquitin signaling pathways by effectors translocated by bacterial type III and type IV secretion systems.

    PubMed

    Angot, Aurélie; Vergunst, Annette; Genin, Stéphane; Peeters, Nemo

    2007-01-01

    The specific and covalent addition of ubiquitin to proteins, known as ubiquitination, is a eukaryotic-specific modification central to many cellular processes, such as cell cycle progression, transcriptional regulation, and hormone signaling. Polyubiquitination is a signal for the 26S proteasome to destroy earmarked proteins, but depending on the polyubiquitin chain topology, it can also result in new protein properties. Both ubiquitin-orchestrated protein degradation and modification have also been shown to be essential for the host's immune response to pathogens. Many animal and plant pathogenic bacteria utilize type III and/or type IV secretion systems to inject effector proteins into host cells, where they subvert host signaling cascades as part of their infection strategy. Recent progress in the determination of effector function has taught us that playing with the host's ubiquitination system seems a general tactic among bacteria. Here, we discuss how bacteria exploit this system to control the timing of their effectors' action by programming them for degradation, to block specific intermediates in mammalian or plant innate immunity, or to target host proteins for degradation by mimicking specific ubiquitin/proteasome system components. In addition to analyzing the effectors that have been described in the literature, we screened publicly available bacterial genomes for mimicry of ubiquitin proteasome system subunits and detected several new putative effectors. Our understanding of the intimate interplay between pathogens and their host's ubiquitin proteasome system is just beginning. This exciting research field will aid in better understanding this interplay, and may also provide new insights into eukaryotic ubiquitination processes. PMID:17257058

  19. Biotransformation of nitrobenzene by bacteria containing toluene degradative pathways

    SciTech Connect

    Haigler, B.E.; Spain, J.C. (Air Force Civil Engineering Support Agency, Tyndall AFB, FL (United States))

    1991-11-01

    Nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of the electron withdrawing properties of the nitro group. The authors have investigate the ability of seven bacterial strains containing toluene degradative pathways to oxidize nitrobenzene. Cultures were induced with toluene vapor prior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Pseudomonas cepacia G4 and a strain of Pseudomonas harboring the TOL plasmid (pTN2) did not transform nitrobenzene. Cells of Pseudomonas putida F1 and Pseudomonas sp. strain JS150 converted nitrobenzene to 3-nitrocatechol. Transformation of nitrobenzene in the presence of {sup 18}O{sub 2} indicated that the reaction in JS150 involved the incorporation of both atoms of oxygen in the 3-nitrocatechol, which suggests a dioxygenase mechanism. P. putida 39/D, a mutant strain of P. putida F1, converted nitrobenzene to a compound tentatively identified as cis-1, 2-dihydroxy-3-nitrocyclohexa-3, 5-diene. This compound was rapidly converted to 3-nitrocatechol by cells of strain JS150. Cultures of Pseudomonas mendocina KR-1 converted nitrobenzene to a mixture of 3- and 4-nitrophenol (10 and 63%, respectively). Pseudomonas pickettii PKO1 converted nitrobenzene to 3- and 4-nitrocatechol via 3- and 4-nitrophenol. The nitrocatechols were slowly degraded to unidentified metabolites. Nitrobenzene did not serve as an inducer for the enzymes that catalyzed its oxidation.

  20. Mitofusin 1 degradation is induced by a disruptor of mitochondrial calcium homeostasis, CGP37157: a role in apoptosis in prostate cancer cells.

    PubMed

    Choudhary, Vivek; Kaddour-Djebbar, Ismail; Alaisami, Rabei; Kumar, M Vijay; Bollag, Wendy B

    2014-05-01

    Mitochondria constantly divide (mitochondrial fission) and fuse (mitochondrial fusion) in a normal cell. Disturbances in the balance between these two physiological processes may lead to cell dysfunction or to cell death. Induction of cell death is the prime goal of prostate cancer chemotherapy. Our previous study demonstrated that androgens increase the expression of a mitochondrial protein involved in fission and facilitate an apoptotic response to CGP37157 (CGP), an inhibitor of mitochondrial calcium efflux, in prostate cancer cells. However, the regulation and role of mitochondrial fusion proteins in the death of these cells have not been examined. Therefore, our objective was to investigate the effect of CGP on a key mitochondrial fusion protein, mitofusin 1 (Mfn1), and the role of Mfn1 in prostate cancer cell apoptosis. We used various prostate cancer cell lines and western blot analysis, qRT-PCR, siRNA, M30 apoptosis assay and immunoprecipitation techniques to determine mechanisms regulating Mfn1. Treatment of prostate cancer cells with CGP resulted in selective degradation of Mfn1. Mfn1 ubiquitination was detected following immunoprecipitation of overexpressed Myc-tagged Mfn1 protein from CGP-treated cells, and treatment with the proteasomal inhibitor lactacystin, as well as siRNA-mediated knockdown of the E3 ubiquitin ligase March5, protected Mfn1 from CGP-induced degradation. These data indicate the involvement of the ubiquitin-proteasome pathway in CGP-induced degradation of Mfn1. We also demonstrated that downregulation of Mfn1 by siRNA enhanced the apoptotic response of LNCaP cells to CGP, suggesting a likely pro-survival role for Mfn1 in these cells. Our results suggest that manipulation of mitofusins may provide a novel therapeutic advantage in treating prostate cancer. PMID:24626641

  1. Ubiquitination and proteasomal degradation of nucleophosmin-anaplastic lymphoma kinase induced by 17-allylamino-demethoxygeldanamycin: role of the co-chaperone carboxyl heat shock protein 70-interacting protein.

    PubMed

    Bonvini, Paolo; Dalla Rosa, Henry; Vignes, Nadia; Rosolen, Angelo

    2004-05-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a constitutively active fusion tyrosine kinase involved in lymphomagenesis of human anaplastic large cell lymphomas (ALCL), the maturation and activity of which depend on the association with the heat shock protein (hsp) 90 protein chaperone. Targeting hsp90 by the ansamycins geldanamycin and 17-allyl-amino-demethoxygeldanamycin (17-AAG) promotes degradation of several proteins through the ubiquitin-proteasome pathway, including oncogenic Raf, v-Src, erbB2, and BCR-ABL. We have previously shown that 17-AAG prevents hsp90/NPM-ALK complex formation and fosters NPM-ALK turnover, perhaps through its association with the hsp70 chaperone. Here, we show that inhibition of the proteasome activity by the potent and specific compound pyrazylcarbonyl-Phe-Leu-boronate (PS-341) blocks 17-AAG-induced down-regulation of NPM-ALK, which becomes detergent-insoluble and relocates into ubiquitin-rich perinuclear vesicles that represent aggregated polyubiquitinated forms of the protein. Kinase activity was not mandatory for proteasomal degradation of NPM-ALK, because kinase-defective NPM-ALK was even more rapidly degraded upon 17-AAG treatment. Prolonged exposure to the proteasome inhibitor was shown to trigger caspase-3-mediated apoptosis in proliferating ALCL cells at nanomolar concentrations. However, we verified that the accumulation of detergent-insoluble NPM-ALK in ALCL cells was not a spurious consequence of PS341-committed apoptosis, because caspase inhibitors prevented poly(ADP-ribose) polymerase cleavage whereas they did not affect partitioning of aggregated NPM-ALK. In line with these observations, the carboxyl hsp70-interacting ubiquitin ligase (CHIP), was shown to increase basal ubiquitination and turnover of NPM-ALK kinase, supporting a mechanism whereby NPM-ALK proceeds rapidly toward hsp70-assisted ubiquitin-dependent proteasomal degradation, when chaperoning activity of hsp90 is prohibited by 17-AAG. PMID:15126367

  2. c-Myc is targeted to the proteasome for degradation in a SUMOylation-dependent manner, regulated by PIAS1, SENP7 and RNF4.

    PubMed

    González-Prieto, Román; Cuijpers, Sabine Ag; Kumar, Ramesh; Hendriks, Ivo A; Vertegaal, Alfred Co

    2015-06-18

    c-Myc is the most frequently overexpressed oncogene in tumors, including breast cancer, colon cancer and lung cancer. Post-translational modifications comprising phosphorylation, acetylation and ubiquitylation regulate the activity of c-Myc. Recently, it was shown that c-Myc-driven tumors are strongly dependent on the SUMO pathway. Currently, the relevant SUMO target proteins in this pathway are unknown. Here we show that c-Myc is a target protein for SUMOylation, and that SUMOylated c-Myc is subsequently ubiquitylated and degraded by the proteasome. SUMO chains appeared to be dispensable for this process, polymerization-deficient SUMO mutants supported proteolysis of SUMOylated c-Myc. These results indicate that multiple SUMO monomers conjugated to c-Myc could be sufficient to direct SUMOylated c-Myc to the ubiquitin-proteasome pathway. Knocking down the SUMO-targeted ubiquitin ligase RNF4 enhanced the levels of SUMOylated c-Myc, indicating that RNF4 could recognize a multi-SUMOylated protein as a substrate in addition to poly-SUMOylated proteins. Knocking down the SUMO E3 ligase PIAS1 resulted in reduced c-Myc SUMOylation and increased c-Myc transcriptional activity, indicating that PIAS1 mediates c-Myc SUMOylation. Increased SUMOylation of c-Myc was noted upon knockdown of the SUMO protease SENP7, indicating that it also could regulate a multi-SUMOylated protein in addition to poly-SUMOylated proteins. C-Myc lacks KxE-type SUMOylation consensus motifs. We used mass spectrometry to identify 10 SUMO acceptor lysines: K52, K148, K157, K317, K323, K326, K389, K392, K398 and K430. Intriguingly, mutating all 10 SUMO acceptor lysines did not reduce c-Myc SUMOylation, suggesting that SUMO acceptor lysines in c-Myc act promiscuously. Our results provide novel insight into the complexity of c-Myc post-translational regulation. PMID:25895136

  3. Biotransformation of nitrobenzene by bacteria containing toluene degradative pathways.

    PubMed Central

    Haigler, B E; Spain, J C

    1991-01-01

    Nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of the electron withdrawing properties of the nitro group. We have investigated the ability of seven bacterial strains containing toluene degradative pathways to oxidize nitrobenzene. Cultures were induced with toluene vapor prior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Pseudomonas cepacia G4 and a strain of Pseudomonas harboring the TOL plasmid (pTN2) did not transform nitrobenzene. Cells of Pseudomonas putida F1 and Pseudomonas sp. strain JS150 converted nitrobenzene to 3-nitrocatechol. Transformation of nitrobenzene in the presence of 18O2 indicated that the reaction in JS150 involved the incorporation of both atoms of oxygen in the 3-nitrocatechol, which suggests a dioxygenase mechanism. P. putida 39/D, a mutant strain of P. putida F1, converted nitrobenzene to a compound tentatively identified as cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene. This compound was rapidly converted to 3-nitrocatechol by cells of strain JS150. Cultures of Pseudomonas mendocina KR-1 converted nitrobenzene to a mixture of 3- and 4-nitrophenol (10 and 63%, respectively). Pseudomonas pickettii PKO1 converted nitrobenzene to 3- and 4-nitrocatechol via 3- and 4-nitrophenol. The nitrocatechols were slowly degraded to unidentified metabolites. Nitrobenzene did not serve as an inducer for the enzymes that catalyzed its oxidation. These results indicate that the nitrobenzene ring is subject to initial attack by both mono- and dioxygenase enzymes. PMID:1781679

  4. A New 4-Nitrotoluene Degradation Pathway in a Mycobacterium Strain

    PubMed Central

    Spiess, Tilmann; Desiere, Frank; Fischer, Peter; Spain, Jim C.; Knackmuss, Hans-Joachim; Lenke, Hiltrud

    1998-01-01

    Mycobacterium sp. strain HL 4-NT-1, isolated from a mixed soil sample from the Stuttgart area, utilized 4-nitrotoluene as the sole source of nitrogen, carbon, and energy. Under aerobic conditions, resting cells of the Mycobacterium strain metabolized 4-nitrotoluene with concomitant release of small amounts of ammonia; under anaerobic conditions, 4-nitrotoluene was completely converted to 6-amino-m-cresol. 4-Hydroxylaminotoluene was converted to 6-amino-m-cresol by cell extracts and thus could be confirmed as the initial metabolite in the degradative pathway. This enzymatic equivalent to the acid-catalyzed Bamberger rearrangement requires neither cofactors nor oxygen. In the same crucial enzymatic step, the homologous substrate hydroxylaminobenzene was rearranged to 2-aminophenol. Abiotic oxidative dimerization of 6-amino-m-cresol, observed during growth of the Mycobacterium strain, yielded a yellow dihydrophenoxazinone. Another yellow metabolite (?max, 385 nm) was tentatively identified as 2-amino-5-methylmuconic semialdehyde, formed from 6-amino-m-cresol by meta ring cleavage. PMID:9464378

  5. Complete and Integrated Pyrene Degradation Pathway in Mycobacterium vanbaalenii PYR-1 Based on Systems Biology

    Microsoft Academic Search

    Seong-Jae Kim; Ohgew Kweon; Richard C. Jones; James P. Freeman; Ricky D. Edmondson; Carl E. Cerniglia

    2007-01-01

    Mycobacterium vanbaalenii PYR-1 was the first bacterium isolated by virtue of its ability to metabolize the high-molecular-weight polycyclic aromatic hydrocarbon (PAH) pyrene. We used metabolic, genomic, and proteomic approaches in this investigation to construct a complete and integrated pyrene degradation pathway for M. vanbaalenii PYR-1. Genome sequence analyses identified genes involved in the pyrene degradation pathway that we have proposed

  6. UBA domain containing proteins in fission yeast

    Microsoft Academic Search

    Rasmus Hartmann-Petersen; Colin A. M Semple; Chris P Ponting; Klavs B Hendil; Colin Gordon

    2003-01-01

    The ubiquitin–proteasome pathway for intracellular proteolysis is involved in a series of cellular and molecular functions, including the degradation of bulk proteins, cell cycle control, DNA repair, antigen presentation, vesicle transport and the regulation of signal transudation pathways and transcription. Considering this variety of cell biological processes, it is puzzling that until recently only very few proteins were known to

  7. Molecular mechanisms and signaling pathways of angiotensin II-induced muscle wasting: potential therapeutic targets for cardiac cachexia

    PubMed Central

    Yoshida, Tadashi; Tabony, A. Michael; Galvez, Sarah; Mitch, William E.; Higashi, Yusuke; Sukhanov, Sergiy; Delafontaine, Patrice

    2013-01-01

    Cachexia is a serious complication of many chronic diseases, such as congestive heart failure (CHF) and chronic kidney disease (CKD). Many factors are involved in the development of cachexia, and there is increasing evidence that angiotensin II (Ang II), the main effector molecule of the renin-angiotensin system (RAS), plays an important role in this process. Patients with advanced CHF or CKD often have increased Ang II levels and cachexia, and angiotensin-converting enzyme (ACE) inhibitor treatment improves weight loss. In rodent models, an increase in systemic Ang II leads to weight loss through increased protein breakdown, reduced protein synthesis in skeletal muscle and decreased appetite. Ang II activates the ubiquitin-proteasome system via generation of reactive oxygen species and via inhibition of the insulin-like growth factor-1 signaling pathway. Furthermore, Ang II inhibits 5? AMP-activated protein kinase (AMPK) activity and disrupts normal energy balance. Ang II also increases cytokines and circulating hormones such as tumor necrosis factor-?, interleukin-6, serum amyloid-A, glucocorticoids and myostatin, which regulate muscle protein synthesis and degradation. Ang II acts on hypothalamic neurons to regulate orexigenic/anorexigenic neuropeptides, such as neuropeptide-Y, orexin and corticotropin-releasing hormone, leading to reduced appetite. Also, Ang II may regulate skeletal muscle regenerative processes. Several clinical studies have indicated that blockade of Ang II signaling via ACE inhibitors or Ang II type 1 receptor blockers prevents weight loss and improves muscle strength. Thus the RAS is a promising target for the treatment of muscle atrophy in patients with CHF and CKD. PMID:23769949

  8. Reaction pathways in the electrochemical degradation of thiocarbamate herbicides in NaCl solution

    Microsoft Academic Search

    FERENC MOGYORÓDY

    2006-01-01

    We have identified the intermediates and end products which are formed during the electrolytic degradation of thiocarbamate pesticides in aqueous NaCl solutions and investigated how the intermediate and end product volumes and ratios depend on reaction conditions. Further, we have defined both the reaction pathways leading to intermediate and end product formation and the methods affecting this process. The degradation

  9. Allophanate hydrolase of Oleomonas sagaranensis involved in an ATP-dependent degradation pathway specific to urea

    Microsoft Academic Search

    Takeshi Kanamori; Norihisa Kanou; Shingo Kusakabe; Haruyuki Atomi; Tadayuki Imanaka

    2005-01-01

    The first prokaryotic urea carboxylase has previously been purified and characterized from Oleomonas sagaranensis. As the results indicated the presence of an ATP-dependent urea degradation pathway in Bacteria, the characterization of the second component of this pathway, allophanate hydrolase, was carried out. The gene encoding allophanate hydrolase was found adjacent to the urea carboxylase gene. The purified, recombinant enzyme exhibited

  10. Timely Activation of Budding Yeast APCCdh1 Involves Degradation of Its Inhibitor, Acm1, by an Unconventional Proteolytic Mechanism

    PubMed Central

    Melesse, Michael; Choi, Eunyoung; Hall, Hana; Walsh, Michael J.; Geer, M. Ariel; Hall, Mark C.

    2014-01-01

    Regulated proteolysis mediated by the ubiquitin proteasome system is a fundamental and essential feature of the eukaryotic cell division cycle. Most proteins with cell cycle-regulated stability are targeted for degradation by one of two related ubiquitin ligases, the Skp1-cullin-F box protein (SCF) complex or the anaphase-promoting complex (APC). Here we describe an unconventional cell cycle-regulated proteolytic mechanism that acts on the Acm1 protein, an inhibitor of the APC activator Cdh1 in budding yeast. Although Acm1 can be recognized as a substrate by the Cdc20-activated APC (APCCdc20) in anaphase, APCCdc20 is neither necessary nor sufficient for complete Acm1 degradation at the end of mitosis. An APC-independent, but 26S proteasome-dependent, mechanism is sufficient for complete Acm1 clearance from late mitotic and G1 cells. Surprisingly, this mechanism appears distinct from the canonical ubiquitin targeting pathway, exhibiting several features of ubiquitin-independent proteasomal degradation. For example, Acm1 degradation in G1 requires neither lysine residues in Acm1 nor assembly of polyubiquitin chains. Acm1 was stabilized though by conditional inactivation of the ubiquitin activating enzyme Uba1, implying some requirement for the ubiquitin pathway, either direct or indirect. We identified an amino terminal predicted disordered region in Acm1 that contributes to its proteolysis in G1. Although ubiquitin-independent proteasome substrates have been described, Acm1 appears unique in that its sensitivity to this mechanism is strictly cell cycle-regulated via cyclin-dependent kinase (Cdk) phosphorylation. As a result, Acm1 expression is limited to the cell cycle window in which Cdk is active. We provide evidence that failure to eliminate Acm1 impairs activation of APCCdh1 at mitotic exit, justifying its strict regulation by cell cycle-dependent transcription and proteolytic mechanisms. Importantly, our results reveal that strict cell-cycle expression profiles can be established independent of proteolysis mediated by the APC and SCF enzymes. PMID:25072887

  11. ORGANOPHOSPHORUS PESTICIDE DEGRADATION PATHWAYS DURING DRINKING WATER TREATMENT

    EPA Science Inventory

    The objective of this work was to investigate organophosphorus (OP) pesticide transformation pathways as a class in the presence of aqueous chlorine. Seven priority OP pesticides were examined for their reactivity with aqueous chlorine: chlorpyrifos (CP), parathion (PA), diazino...

  12. In silico prediction of pharmaceutical degradation pathways: a benchmarking study.

    PubMed

    Kleinman, Mark H; Baertschi, Steven W; Alsante, Karen M; Reid, Darren L; Mowery, Mark D; Shimanovich, Roman; Foti, Chris; Smith, William K; Reynolds, Dan W; Nefliu, Marcela; Ott, Martin A

    2014-11-01

    Zeneth is a new software application capable of predicting degradation products derived from small molecule active pharmaceutical ingredients. This study was aimed at understanding the current status of Zeneth's predictive capabilities and assessing gaps in predictivity. Using data from 27 small molecule drug substances from five pharmaceutical companies, the evolution of Zeneth predictions through knowledge base development since 2009 was evaluated. The experimentally observed degradation products from forced degradation, accelerated, and long-term stability studies were compared to Zeneth predictions. Steady progress in predictive performance was observed as the knowledge bases grew and were refined. Over the course of the development covered within this evaluation, the ability of Zeneth to predict experimentally observed degradants increased from 31% to 54%. In particular, gaps in predictivity were noted in the areas of epimerizations, N-dealkylation of N-alkylheteroaromatic compounds, photochemical decarboxylations, and electrocyclic reactions. The results of this study show that knowledge base development efforts have increased the ability of Zeneth to predict relevant degradation products and aid pharmaceutical research. This study has also provided valuable information to help guide further improvements to Zeneth and its knowledge base. PMID:25364862

  13. A Golgi-based KDELR-dependent signalling pathway controls extracellular matrix degradation

    PubMed Central

    Grossi, Mauro; Picciani, Benedetta; Di Martino, Rosaria; Capitani, Mirco; Buccione, Roberto; Luini, Alberto; Sallese, Michele

    2015-01-01

    We recently identified an endomembrane-based signalling cascade that is activated by the KDEL receptor (KDELR) on the Golgi complex. At the Golgi, the KDELR acts as a traffic sensor (presumably via binding to chaperones that leave the ER) and triggers signalling pathways that balance membrane fluxes between ER and Golgi. One such pathway relies on Gq and Src. Here, we examine if KDELR might control other cellular modules through this pathway. Given the central role of Src in extracellular matrix (ECM) degradation, we investigated the impact of the KDELR-Src pathway on the ability of cancer cells to degrade the ECM. We find that activation of the KDELR controls ECM degradation by increasing the number of the degradative structures known as invadopodia. The KDELR induces Src activation at the invadopodia and leads to phosphorylation of the Src substrates cortactin and ASAP1, which are required for basal and KDELR-stimulated ECM degradation. This study furthers our understanding of the regulatory circuitry underlying invadopodia-dependent ECM degradation, a key phase in metastases formation and invasive growth. PMID:25682866

  14. Evolution of efficient pathways for degradation of anthropogenic chemicals

    Microsoft Academic Search

    Shelley D Copley

    2009-01-01

    Anthropogenic compounds used as pesticides, solvents and explosives often persist in the environment and can cause toxicity to humans and wildlife. The persistence of anthropogenic compounds is due to their recent introduction into the environment; microbes in soil and water have had relatively little time to evolve efficient mechanisms for degradation of these new compounds. Some anthropogenic compounds are easily

  15. Bacterial Degradation of Chlorophenols: Pathways, Biochemica, and Genetic Aspects

    Microsoft Academic Search

    Inna P. Solyanikova; Ludmila A. Golovleva

    2004-01-01

    Chlorophenols belong to the group of toxic and persistent to microbial attack xenobiotics. Nevertheless, due to the adaptation microorganisms acquire the ability to use chlorophenols as the sole source of carbon and energy. The present review describes the diversity of aerobic pathways for the utilization of halogenated phenols by bacteria with the emphasis on the main reactions and intermediates formed,

  16. Investigating Bacterial Sources of Toxicity as an Environmental Contributor to Dopaminergic Neurodegeneration

    Microsoft Academic Search

    Kim A. Caldwell; Michelle L. Tucci; Jafa Armagost; Tyler W. Hodges; Jue Chen; Shermeen B. Memon; Jeana E. Blalock; Susan M. DeLeon; Robert H. Findlay; Qingmin Ruan; Philip J. Webber; David G. Standaert; Julie B. Olson; Guy A. Caldwell

    2009-01-01

    Parkinson disease (PD) involves progressive neurodegeneration, including loss of dopamine (DA) neurons from the substantia nigra. Select genes associated with rare familial forms of PD function in cellular pathways, such as the ubiquitin-proteasome system (UPS), involved in protein degradation. The misfolding and accumulation of proteins, such as ?-synuclein, into inclusions termed Lewy Bodies represents a clinical hallmark of PD. Given

  17. Annu. Rev. Cell Dev. Biol. 2003. 19:26186 doi: 10.1146/annurev.cellbio.19.111301.112449

    E-print Network

    Deng, Xing-Wang

    .deng@yale.edu Key Words CSN, Jab1, SCF, Nedd8/Rub1, protein degradation, ubiquitin-proteasome pathway s Abstract/Jab1 subunit, which removes the post-translational modification of a ubiquitin-likeprotein,Nedd8/Rub1 Characteristics of CSN1, CSN2, and CSN5/Jab1 . . . . . . . . 265 THE BIOCHEMICAL ACTIVITY AND CELLULAR FUNCTIONS

  18. EXPRESSION OF K6W-UBIQUITIN IN LENS EPITHELIAL CELLS LEADS TO UP-REGULATION OF A BROAD SPECTRUM OF MOLECULAR CHAPERONES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Purpose: Accumulation and precipitation of abnormal proteins are associated with many age-related diseases. The ubiquitin-proteasome pathway (UPP) is one of the protein quality control mechanisms that selectively degrade damaged or obsolete proteins. The other arm of the protein quality control me...

  19. Ubiquitin Domain Proteins in Disease

    Microsoft Academic Search

    Louise Madsen; Andrea Schulze; Michael Seeger; Rasmus Hartmann-Petersen

    2007-01-01

    The human genome encodes several ubiquitin-like (UBL) domain proteins (UDPs). Members of this protein family are involved in a variety of cellular functions and many are connected to the ubiquitin proteasome system, an essential pathway for protein degradation in eukaryotic cells. Despite their structural similarity, the UBL domains appear to have a range of different targets, resulting in a considerable

  20. Ubiquitin domain proteins in disease

    Microsoft Academic Search

    Louise Madsen; Andrea Schulze; Michael Seeger; Rasmus Hartmann-Petersen

    2007-01-01

    The human genome encodes several ubiquitin-like (UBL) domain proteins (UDPs). Members of this protein family are involved in a variety of cellular functions and many are connected to the ubiquitin proteasome system, an essential pathway for protein degradation in eukaryotic cells. Despite their structural similarity, the UBL domains appear to have a range of different targets, resulting in a considerable

  1. Ubiquitin control of S phase: a new role for the ubiquitin conjugating enzyme, UbcH7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Events within and transitions between the phases of the eukaryotic cell cycle are tightly controlled by transcriptional and post-translational processes. Prominent among them is a profound role for the ubiquitin proteasome proteolytic pathway. The timely degradation of proteins balances the increase...

  2. AEROBIC DEGRADATION OF DINITROTOLUENES AND PATHWAY FOR BACTERIAL DEGRADATION OF 2,6-DINITROTOLUENE

    EPA Science Inventory

    An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2,4-DNT) by Burkhoderia sp. strain DNT has been reported previously. We report here the isolation of additional strains with the ability to mineralize dinitrotoluene (2,6-DNT) by a different pathway. Burkhoderia ...

  3. Intracellular degradation of the HIV-1 envelope glycoprotein. Evidence for, and some characteristics of, an endoplasmic reticulum degradation pathway.

    PubMed

    Courageot, J; Fenouillet, E; Bastiani, P; Miquelis, R

    1999-03-01

    Analysis of the fate of HIV-1 envelope protein gp160 (Env) has shown that newly synthesized proteins may be degraded within the biosynthetic pathway and that this degradation may take place in compartments other than the lysosomes. The fate of newly synthesized Env was studied in living BHK-21 cells with the recombinant vaccinia virus expression system. We found that gp160 not only undergoes physiological endoproteolytic cleavage, producing gp120, but is also degraded, producing proteolytic fragments of 120 kDa to 26 kDa in size, as determined by SDS/PAGE in non reducing conditions. Analysis of the 120-kDa proteolytic fragment, and comparison with gp120, showed that it is composed of peptides linked by disulfides bonds and lacks the V3-loop epitope and the C-terminal domain of gp120 (amino acids 506-516). A permeabilized cell system, with impaired transport of labeled Env from the endoplasmic reticulum (ER) to Golgi compartments, was developed to determine the site of degradation and to define some biochemical characteristics of the intracellular degradation process. In the semipermeable BHK-21 cells, there was: (a) no gp120 production (b), a progressive decrease in the amount of newly synthesized gp160 and a concomitant increase in the amount of a 120-kDa proteolytic fragment. This fragment had the same biochemical characteristics as the 120-kDa proteolytic fragment found in living nonpermeabilized cells, and (c) susceptibility of the V3 loop. This degradation process occurred in the ER, as shown by both biochemical and indirect immunofluorescence analysis. Furthermore, there was evidence that changes in redox state are involved in the ER-dependent envelope degradation pathway because adding reducing agents to permeabilized cells caused dose-dependent degradation of the 120-kDa proteolytic fragment and of the remaining gp160 glycoprotein. Thus our results provide direct evidence that regulated degradation of the HIV-1 envelope glycoprotein may take place in the ER of infected cells. PMID:10095785

  4. Photolysis of chlorantraniliprole and cyantraniliprole in water and soil: verification of degradation pathways via kinetics modeling.

    PubMed

    Sharma, Ashok K; Zimmerman, William T; Singles, Suzanne K; Malekani, Kalumbu; Swain, Scott; Ryan, David; Mcquorcodale, Gordon; Wardrope, Laura

    2014-07-16

    Photodegradation of [(14)C]-chlorantraniliprole (CLAP) and [(14)C]-cyantraniliprole (CNAP) was investigated in sterile buffer solutions, in natural water, and on soil surfaces. Both compounds displayed rapid degradation in aqueous buffers when exposed to light at concentrations which could result from direct overspray to a shallow water body. While the main products observed had analogous structures, a substantial difference was noted in the rate of degradation of the two compounds despite minimal differences in their structures. Transformations observed were primarily intramolecular rearrangements and degradations resulting from addition of hydroxyl radicals leading to molecular cleavage. Some of the degradation products were transient, and several degradates had isomeric molecular compositions. The sequence of transformations was established definitively with the help of kinetics modeling. Utility of kinetics analysis in verification of the proposed pathways is illustrated. PMID:24971760

  5. Complete and Integrated Pyrene Degradation Pathway in Mycobacterium vanbaalenii PYR-1 Based on Systems Biology? †

    PubMed Central

    Kim, Seong-Jae; Kweon, Ohgew; Jones, Richard C.; Freeman, James P.; Edmondson, Ricky D.; Cerniglia, Carl E.

    2007-01-01

    Mycobacterium vanbaalenii PYR-1 was the first bacterium isolated by virtue of its ability to metabolize the high-molecular-weight polycyclic aromatic hydrocarbon (PAH) pyrene. We used metabolic, genomic, and proteomic approaches in this investigation to construct a complete and integrated pyrene degradation pathway for M. vanbaalenii PYR-1. Genome sequence analyses identified genes involved in the pyrene degradation pathway that we have proposed for this bacterium. To identify proteins involved in the degradation, we conducted a proteome analysis of cells exposed to pyrene using one-dimensional gel electrophoresis in combination with liquid chromatography-tandem mass spectrometry. Database searching performed with the M. vanbaalenii PYR-1 genome resulted in identification of 1,028 proteins with a protein false discovery rate of <1%. Based on both genomic and proteomic data, we identified 27 enzymes necessary for constructing a complete pathway for pyrene degradation. Our analyses indicate that this bacterium degrades pyrene to central intermediates through o-phthalate and the ?-ketoadipate pathway. Proteomic analysis also revealed that 18 enzymes in the pathway were upregulated more than twofold, as indicated by peptide counting when the organism was grown with pyrene; three copies of the terminal subunits of ring-hydroxylating oxygenase (NidAB2, MvanDraft_0817/0818, and PhtAaAb), dihydrodiol dehydrogenase (MvanDraft_0815), and ring cleavage dioxygenase (MvanDraft_3242) were detected only in pyrene-grown cells. The results presented here provide a comprehensive picture of pyrene metabolism in M. vanbaalenii PYR-1 and a useful framework for understanding cellular processes involved in PAH degradation. PMID:17085566

  6. Benzene degradation pathways investigated by a human-tissue-based biosensor

    Microsoft Academic Search

    Luigi Campanella; Patrizia Ercole; Rosella Grossi; Mario V. Russo; Giovanni Visco

    2004-01-01

    Benzene is determined by a biosensor based on human kidney tissue as biological component. Dioxygenase catalyse substrate oxidation. The useful analytical range results to be complementary to that of one or more traditional models based on Pseudomonas. The biosensors has got an other aim as its functioning is able to clear up the degradation pathways of benzene. The behavior of

  7. Unveiling New Degradation Intermediates/Pathways from the Photocatalytic Degradation of Microcystin-LR

    EPA Science Inventory

    This study focuses on the identification of reaction intermediates formed during the photocatalytic degradation of the cyanotoxin microcystin-LR with immobilized TiO2 Tphotocatalysts at neutral pH. To differentiate between impurities already existing in the MC-LR stand...

  8. Degradation pathway of dicyclanil in water in the presence of titanium dioxide. Comparison with photolysis.

    PubMed

    Goutailler, Gaëlle; Guillard, Chantal; Faure, René; Païssé, Olivier

    2002-08-28

    The solar photolytic behavior of the pesticide 4,6-diamino-2-cyclopropyl-pyrimidine-5-carbonitrile, currently known as dicyclanil, has been mimicked in a photoreactor operating with an artificial light flux. The rate and pathway of degradation were performed. An additional study, using TiO(2) photocatalysis, has been achieved in order to determine the efficiency of photocatalysis to degrade the molecule. The catalyst was titania Degussa P-25. The aim of this article was the identification of the intermediate products formed during the irradiation, to establish the degradation pathway of dicyclanil. The kinetics of the reactions were followed by liquid chromatography with a diode array detector (LC-DAD). Most of the organic compounds occurring during the photodegradation have been identified by means of liquid chromatography and mass spectrometry coupled techniques (HPLC-MS). Additional analyses were carried out to evaluate the mineralization rates into nitrate and ammonium ions. PMID:12188616

  9. New Hydrocarbon Degradation Pathways in the Microbial Metagenome from Brazilian Petroleum Reservoirs

    PubMed Central

    Sierra-García, Isabel Natalia; Correa Alvarez, Javier; Pantaroto de Vasconcellos, Suzan; Pereira de Souza, Anete; dos Santos Neto, Eugenio Vaz; de Oliveira, Valéria Maia

    2014-01-01

    Current knowledge of the microbial diversity and metabolic pathways involved in hydrocarbon degradation in petroleum reservoirs is still limited, mostly due to the difficulty in recovering the complex community from such an extreme environment. Metagenomics is a valuable tool to investigate the genetic and functional diversity of previously uncultured microorganisms in natural environments. Using a function-driven metagenomic approach, we investigated the metabolic abilities of microbial communities in oil reservoirs. Here, we describe novel functional metabolic pathways involved in the biodegradation of aromatic compounds in a metagenomic library obtained from an oil reservoir. Although many of the deduced proteins shared homology with known enzymes of different well-described aerobic and anaerobic catabolic pathways, the metagenomic fragments did not contain the complete clusters known to be involved in hydrocarbon degradation. Instead, the metagenomic fragments comprised genes belonging to different pathways, showing novel gene arrangements. These results reinforce the potential of the metagenomic approach for the identification and elucidation of new genes and pathways in poorly studied environments and contribute to a broader perspective on the hydrocarbon degradation processes in petroleum reservoirs. PMID:24587220

  10. Degradation of DIAP1 by the N-end rule pathway is essential for regulating apoptosis.

    PubMed

    Ditzel, Mark; Wilson, Rebecca; Tenev, Tencho; Zachariou, Anna; Paul, Angela; Deas, Emma; Meier, Pascal

    2003-05-01

    Some members of the inhibitor of apoptosis (IAP) protein family block apoptosis by binding to and neutralizing active caspases. We recently demonstrated that a physical association between IAP and caspases alone is insufficient to regulate caspases in vivo and that an additional level of control is provided by IAP-mediated ubiquitination of both itself and the associated caspases. Here we show that Drosophila IAP 1 (DIAP1) is degraded by the 'N-end rule' pathway and that this process is indispensable for regulating apoptosis. Caspase-mediated cleavage of DIAP1 at position 20 converts the more stable pro-N-degron of DIAP1 into the highly unstable, Asn-bearing, DIAP1 N-degron of the N-end rule degradation pathway. Thus, DIAP1 represents the first known metazoan substrate of the N-end rule pathway that is targeted for degradation through its amino-terminal Asn residue. We demonstrate that the N-end rule pathway is required for regulation of apoptosis induced by Reaper and Hid expression in the Drosophila melanogaster eye. Our data suggest that DIAP1 instability, mediated through caspase activity and subsequent exposure of the N-end rule pathway, is essential for suppression of apoptosis. We suggest that DIAP1 safeguards cell viability through the coordinated mutual destruction of itself and associated active caspases. PMID:12692559

  11. Kinetic models and pathways of ronidazole degradation by chlorination, UV irradiation and UV/chlorine processes.

    PubMed

    Qin, Lang; Lin, Yi-Li; Xu, Bin; Hu, Chen-Yan; Tian, Fu-Xiang; Zhang, Tian-Yang; Zhu, Wen-Qian; Huang, He; Gao, Nai-Yun

    2014-11-15

    Degradation kinetics and pathways of ronidazole (RNZ) by chlorination (Cl2), UV irradiation and combined UV/chlorine processes were investigated in this paper. The degradation kinetics of RNZ chlorination followed a second-order behavior with the rate constants calculated as (2.13 ± 0.15) × 10(2) M(-2) s(-1), (0.82 ± 0.52) × 10(-2) M(-1) s(-1) and (2.06 ± 0.09) × 10(-1) M(-1) s(-1) for the acid-catalyzed reaction, as well as the reactions of RNZ with HOCl and OCl(-), respectively. Although UV irradiation degraded RNZ more effectively than chlorination did, very low quantum yield of RNZ at 254 nm was obtained as 1.02 × 10(-3) mol E(-1). RNZ could be efficiently degraded and mineralized in the UV/chlorine process due to the generation of hydroxyl radicals. The second-order rate constant between RNZ and hydroxyl radical was determined as (2.92 ± 0.05) × 10(9) M(-1) s(-1). The degradation intermediates of RNZ during the three processes were identified with Ultra Performance Liquid Chromatography - Electrospray Ionization - mass spectrometry and the degradation pathways were then proposed. Moreover, the variation of chloropicrin (TCNM) and chloroform (CF) formation after the three processes were further evaluated. Enhanced formation of CF and TCNM precursors during UV/chlorine process deserves extensive attention in drinking water treatment. PMID:25141357

  12. Upregulation of macrophage-specific functions by oxidized LDL: lysosomal degradation-dependent and -independent pathways.

    PubMed

    Radhika, A; Sudhakaran, P R

    2013-01-01

    Formation of foam cells from macrophages, which are formed by the differentiation of blood-borne monocytes, is a critical early event in atherogenesis. To examine how pre-exposure of monocytes to modified proteins, such as oxLDL, influences their differentiation to macrophages, an in vitro model system using isolated PBMC maintained in culture in the presence of oxLDL was used. Pretreatment of monocytes with oxLDL caused a faster rate of expression of macrophage-specific functions and loss of monocyte-specific functions compared to unmodified LDL. The effect of oxidation of lipid component of LDL by CuSO(4) and its protein component by HOCl, on mo-m? differentiation was studied by monitoring the upregulation of macrophage-specific functions, particularly MMP-9. Chloroquine, a lysosomal degradation blocker, significantly reversed the effect mediated by CuSO(4) oxLDL, indicating the involvement of lysosomal degradation products, while no such effect was observed in HOCl oxLDL-treated cells, indicating the existence of a pathway independent of its lysosomal degradation products. Reversal of the effect of oxLDL by NAC and Calphostin C, an inhibitor of PKC, suggested the activation of RO-mediated signaling pathways. Use of inhibitors of signaling pathways showed that CuSO(4) oxLDL upregulated m?-specific MMP-9 through p38 MAPK and Akt-dependent pathways, while HOCl oxLDL utilized ERK ½ and Akt. Further analysis showed the activation of PPAR? and AP-1 in CuSO(4) oxLDL, while HOCl-oxLDL-mediated effect involved NF?B and AP-1. These results suggest that lipid oxLDL- and protein oxLDL-mediated upregulation of mo-m?-specific functions involve lysosomal degradation-dependent and -independent activation of intracellular signaling pathways. PMID:23054190

  13. Metagenomic Identification of Bacterioplankton Taxa and Pathways Involved in Microcystin Degradation in Lake Erie

    PubMed Central

    Mou, Xiaozhen; Lu, Xinxin; Jacob, Jisha; Sun, Shulei; Heath, Robert

    2013-01-01

    Cyanobacterial harmful blooms (CyanoHABs) that produce microcystins are appearing in an increasing number of freshwater ecosystems worldwide, damaging quality of water for use by human and aquatic life. Heterotrophic bacteria assemblages are thought to be important in transforming and detoxifying microcystins in natural environments. However, little is known about their taxonomic composition or pathways involved in the process. To address this knowledge gap, we compared the metagenomes of Lake Erie free-living bacterioplankton assemblages in laboratory microcosms amended with microcystins relative to unamended controls. A diverse array of bacterial phyla were responsive to elevated supply of microcystins, including Acidobacteria, Actinobacteria, Bacteroidetes, Planctomycetes, Proteobacteria of the alpha, beta, gamma, delta and epsilon subdivisions and Verrucomicrobia. At more detailed taxonomic levels, Methylophilales (mainly in genus Methylotenera) and Burkholderiales (mainly in genera Bordetella, Burkholderia, Cupriavidus, Polaromonas, Ralstonia, Polynucleobacter and Variovorax) of Betaproteobacteria were suggested to be more important in microcystin degradation than Sphingomonadales of Alphaproteobacteria. The latter taxa were previously thought to be major microcystin degraders. Homologs to known microcystin-degrading genes (mlr) were not overrepresented in microcystin-amended metagenomes, indicating that Lake Erie bacterioplankton might employ alternative genes and/or pathways in microcystin degradation. Genes for xenobiotic metabolism were overrepresented in microcystin-amended microcosms, suggesting they are important in bacterial degradation of microcystin, a phenomenon that has been identified previously only in eukaryotic systems. PMID:23637924

  14. Microbial degradation of chloroaromatics: use of the meta-cleavage pathway for mineralization of chlorobenzene.

    PubMed Central

    Mars, A E; Kasberg, T; Kaschabek, S R; van Agteren, M H; Janssen, D B; Reineke, W

    1997-01-01

    Pseudomonas putida GJ31 is able to simultaneously grow on toluene and chlorobenzene. When cultures of this strain were inhibited with 3-fluorocatechol while growing on toluene or chlorobenzene, 3-methylcatechol or 3-chlorocatechol, respectively, accumulated in the medium. To establish the catabolic routes for these catechols, activities of enzymes of the (modified) ortho- and meta-cleavage pathways were measured in crude extracts of cells of P. putida GJ31 grown on various aromatic substrates, including chlorobenzene. The enzymes of the modified ortho-cleavage pathway were never present, while the enzymes of the meta-cleavage pathway were detected in all cultures. This indicated that chloroaromatics and methylaromatics are both converted via the meta-cleavage pathway. Meta cleavage of 3-chlorocatechol usually leads to the formation of a reactive acylchloride, which inactivates the catechol 2,3-dioxygenase and blocks further degradation of catechols. However, partially purified catechol 2,3-dioxygenase of P. putida GJ31 converted 3-chlorocatechol to 2-hydroxy-cis,cis-muconic acid. Apparently, P. putida GJ31 has a meta-cleavage enzyme which is resistant to inactivation by the acylchloride, providing this strain with the exceptional ability to degrade both toluene and chlorobenzene via the meta-cleavage pathway. PMID:9226262

  15. New metabolic pathway for degradation of 2-nitrobenzoate by Arthrobacter sp. SPG

    PubMed Central

    Arora, Pankaj K.; Sharma, Ashutosh

    2015-01-01

    Arthrobacter sp. SPG utilized 2-nitrobenzoate as its sole source of carbon and energy and degraded it with accumulation of stoichiometric amounts of nitrite ions. Salicylate and catechol were detected as metabolites of the 2-nitrobenzoate degradation using high performance liquid chromatography and gas chromatography–mass spectrometry. Enzyme activities for 2-nitrobenzoate-2-monooxygenase, salicylate hydroxylase, and catechol-1,2-dioxygenase were detected in the crude extracts of the 2-nitrobenzoate-induced cells of strain SPG. The 2-nitrobenzoate-monooxygenase activity resulted in formation of salicylate and nitrite from 2-nitrobenzoate, whereas salicylate hydroxylase catalyzed the conversion of salicylate to catechol. The ring-cleaving enzyme, catechol-1,2-dioxygenase cleaved catechol to cis,cis-muconic acid. Cells of strain SPG were able to degrade 2-nitrobenzoate in sterile as well as non-sterile soil microcosms. The results of microcosm studies showed that strain SPG degraded more than 90% of 2-nitrobenzoate within 10–12 days. This study clearly shows that Arthrobacter sp. SPG degraded 2-nitrobenzoate via a new pathway with formation of salicylate and catechol as metabolites. Arthrobacter sp. SPG may be used for bioremediation of 2-nitrobenzoate-contaminated sites due to its ability to degrade 2-nitrobenzoate in soil.

  16. Degradation of 2,3-Dihydroxybenzoate by a Novel meta-Cleavage Pathway

    PubMed Central

    Marín, Macarena; Plumeier, Iris

    2012-01-01

    2,3-Dihydroxybenzoate is the precursor in the biosynthesis of several siderophores and an important plant secondary metabolite that, in bacteria, can be degraded via meta-cleavage of the aromatic ring. The dhb cluster of Pseudomonas reinekei MT1 encodes a chimeric meta-cleavage pathway involved in the catabolism of 2,3-dihydroxybenzoate. While the first two enzymes, DhbA and DhbB, are phylogenetically related to those involved in 2,3-dihydroxy-p-cumate degradation, the subsequent steps are catalyzed by enzymes related to those involved in catechol degradation (DhbCDEFGH). Characterization of kinetic properties of DhbA extradiol dioxygenase identified 2,3-dihydroxybenzoate as the preferred substrate. Deletion of the encoding gene impedes growth of P. reinekei MT1 on 2,3-dihydroxybenzoate. DhbA catalyzes 3,4-dioxygenation with 2-hydroxy-3-carboxymuconate as the product, which is then decarboxylated by DhbB to 2-hydroxymuconic semialdehyde. This compound is then subject to dehydrogenation and further degraded to citrate cycle intermediates. Transcriptional analysis revealed genes of the dhB gene cluster to be highly expressed during growth with 2,3-dihydroxybenzoate, whereas a downstream-localized gene encoding 2-hydroxymuconic semialdehyde hydrolase, dispensable for 2,3-dihydroxybenzoate metabolism but crucial for 2,3-dihydroxy-p-cumate degradation, was only marginally expressed. This is the first report describing a gene cluster encoding enzymes for the degradation of 2,3-dihydroxybenzoate. PMID:22609919

  17. Regulation of lysosomal and ubiquitin degradative pathways in differentiating human intestinal Caco-2 cells

    Microsoft Academic Search

    Yan Zhang; Debra A. Wick; Arthur L. Haas; Bellur Seetharam; Nancy M. Dahms

    1995-01-01

    The expression of various components of the lysosomal and ubiquitin-dependent degradative pathways was characterized in an in vitro model of differentiating enterocytes, the human colon adenocarcinoma Caco-2 cell line. The activities of the cell-associated lysosomal enzymes ?-d-mannosidase, ?-hexosaminidase, ?-glucuronidase, and ?-galactosidase increased ? 2- to 4-fold as differentiation proceeded. In contrast, the protein levels of the two mannose 6-phosphate receptors

  18. Evidence for the involvement of the anthranilate degradation pathway in Pseudomonas aeruginosa biofilm formation

    PubMed Central

    Costaglioli, Patricia; Barthe, Christophe; Claverol, Stephane; Brözel, Volker S; Perrot, Michel; Crouzet, Marc; Bonneu, Marc; Garbay, Bertrand; Vilain, Sebastien

    2012-01-01

    Bacterial biofilms are complex cell communities found attached to surfaces and surrounded by an extracellular matrix composed of exopolysaccharides, DNA, and proteins. We investigated the whole-genome expression profile of Pseudomonas aeruginosa sessile cells (SCs) present in biofilms developed on a glass wool substratum. The transcriptome and proteome of SCs were compared with those of planktonic cell cultures. Principal component analysis revealed a biofilm-specific gene expression profile. Our study highlighted the overexpression of genes controlling the anthranilate degradation pathway in the SCs grown on glass wool for 24 h. In this condition, the metabolic pathway that uses anthranilate for Pseudomonas quinolone signal production was not activated, which suggested that anthranilate was primarily being consumed for energy metabolism. Transposon mutants defective for anthranilate degradation were analyzed in a simple assay of biofilm formation. The phenotypic analyses confirmed that P. aeruginosa biofilm formation partially depended on the activity of the anthranilate degradation pathway. This work points to a new feature concerning anthranilate metabolism in P. aeruginosa SCs. PMID:23170231

  19. Thermally induced degradation pathways of three different antibody-based drug development candidates.

    PubMed

    Fincke, Anja; Winter, Jonas; Bunte, Thomas; Olbrich, Carsten

    2014-10-01

    Protein-based medicinal products are prone to undergo a variety of chemical and physical degradation pathways. One of the most important exogenous stress condition to consider during manufacturing, transport and storage processes is temperature, because antibody-based therapeutics are only stable in a limited temperature range. In this study, three different formats of antibody-based molecules (IgG1, a bispecific scFv and a fab fragment) were exposed to thermal stress conditions occurring during transport and storage. For evaluation, an analytical platform was developed for the detection and characterization of relevant degradation pathways of different antibody-based therapeutics. The effect of thermal stress conditions on the stability of the three antibody-based formats was therefore investigated using visual inspection, different spectroscopic measurements, dynamic light scattering (DLS), differential scanning calorimetry (DSC), electrophoresis, asymmetric flow field-flow fractionation (AF4) and surface plasmon resonance technology (SPR). In summary, thermal stress led to heterogeneous chemical and physical degradation pathways of all three antibody-based formats used. In addition, identical exogenous stress conditions resulted in different kinds and levels of aggregates and fragmentation products. This knowledge is fundamental for a systematic and successful stabilization of protein-based therapeutics by the use of formulation additives. PMID:24878389

  20. Degradation of benzotrifluoride via the dioxygenase pathway in Rhodococcus sp. 065240.

    PubMed

    Yano, Kenichi; Wachi, Masaaki; Tsuchida, Sakiko; Kitazume, Tomoya; Iwai, Noritaka

    2015-01-01

    We previously isolated Rhodococcus sp. 065240, which catalyzes the defluorination of benzotrifluoride (BTF). In order to investigate the mechanism of this degradation of BTF, we performed proteomic analysis of cells grown with or without BTF. Three proteins, which resemble dioxygenase pathway enzymes responsible for isopropylbenzene degradation from Rhodococcus erythropolis BD2, were induced by BTF. Genomic PCR and DNA sequence analysis revealed that the Rhodococcus sp. 065240 carries the gene cluster, btf, which is highly homologous to the ipb gene cluster from R. erythropolis BD2. A mutant strain, which could not catalyze BTF defluorination, was isolated from 065240 strain by UV mutagenesis. The mutant strain had one mutation in the btfT gene, which encodes a response regulator of the two component system. The defluorinating ability of the mutant strain was recovered by complementation of btfT. These results suggest that the btf gene cluster is responsible for degradation of BTF. PMID:25412819

  1. Secretion of intact proteins and peptide fragments by lysosomal pathways of protein degradation

    SciTech Connect

    Isenman, L.D.; Dice, J.F. (Tufts Univ. School of Medicine, Boston, MA (USA))

    1989-12-25

    We report that degradation of proteins microinjected into human fibroblasts is accompanied by release into the culture medium of peptide fragments and intact proteins as well as single amino acids. For the nine proteins and polypeptides microinjected, acid-precipitable radioactivity, i.e. peptide fragments and/or intact proteins, ranged from 10 to 67% of the total released radioactivity. Peptide fragments and/or intact protein accounted for 60% of the radioactivity released into the medium by cells microinjected with ribonuclease A. Two major radiolabeled peptide fragments were found, and one was of an appropriate size to function as an antigen in antigen-presenting cells. The peptides released from microinjected ribonuclease A were derived from lysosomal pathways of proteolysis based on several lines of evidence. Previous studies have shown that microinjected ribonuclease A is degraded to single amino acids entirely within lysosomes. We show that release of free amino acids and peptide fragments and/or intact protein was equivalently stimulated by serum deprivation and equivalently inhibited by NH4Cl. We also show that lysosomal degradation of endocytosed (3H)ribonuclease A was accompanied by the release of two peptide fragments similar in size and charge to those from microinjected ({sup 3}H)ribonuclease A. These findings demonstrate that degradation within lysosomes occurs in a manner that spares specific peptides; they also suggest a previously unsuspected pathway by which cells can secrete cytosol-derived polypeptides.

  2. p27Kip1 accumulation is associated with retinoic-induced neuroblastoma differentiation: evidence of a decreased proteasome-dependent degradation.

    PubMed

    Borriello, A; Pietra, V D; Criscuolo, M; Oliva, A; Tonini, G P; Iolascon, A; Zappia, V; Ragione, F D

    2000-01-01

    Development of human neuroblastoma is due to an arrest in the differentiation program of neural crest sympathoadrenal progenitor cells. However, neuroblastomas, as well as their derived cell lines, maintain the potentiality of terminal differentiation. We investigated the molecular mechanisms by which retinoic acid, a molecule introduced in clinical trials for chemotherapy, induces differentiation in neuroblastoma cell lines. Our findings demonstrate that the retinoic acid-dependent growth arrest of LAN-5 neuroblastoma cell line is associated to a very large accumulation (>tenfold) of p27Kip1 protein, a cyclin-dependent kinase inhibitor; the protein binds and inhibits cyclin-dependent kinase 2, 4 and 6 activities, thus hampering pRb and p107 phosphorylation. p27Kip1 build-up was observable as an early phenomenon (12 - 24 h) after retinoic exposure and resulted in a time-dependent accumulation of high quantities of a free p27Kip1 form. Furthermore, retinoic treatment causes an increase of cyclin-dependent kinase 5 level and activity; however, immunoprecipitation studies proved the absence of interaction with p27kip1. No noticeable variation of other components of G1 phase cell cycle engine was observed. Pulse-chase experiments showed a remarkable elongation of p27Kip1 half-life in retinoic-treated LAN-5, while no enhancement of p27Kip1 gene expression and of the translational efficiency of its messenger RNA were demonstrated. In vivo degradation of p27Kip1 was sensitive to two highly specific proteasome inhibitors, LLnL and lactacystin, while the calpain inhibitor II ALLM and the cysteine protease inhibitor E64 did not modify the level of the protein. LLnL treatment caused a very rapid (2 h) build-up of the Cdk inhibitor content and the accumulation of higher molecular weight anti-p27Kip1 immunoreactive bands, which probably represent ubiquitinated forms of the protein. Finally, in vitro experiments demonstrated that extracts prepared from retinoic-treated LAN-5 cells degraded recombinant p27Kip1 at a rate remarkably slower than the untreated cells. Our results indicate that retinoic acid strongly increases p27Kip1 levels by down-regulating the ubiquitin-proteasome p27Kip1 degrading pathway. PMID:10644979

  3. Metabolic pathway for degradation of 2-chloro-4-aminophenol by Arthrobacter sp. SPG.

    PubMed

    Arora, Pankaj Kumar; Mohanta, Tapan Kumar; Srivastava, Alok; Bae, Hanhong; Singh, Vijay Pal

    2014-01-01

    A degradation pathway of 2-chloro-4-aminophenol (2C4AP) was studied in an Arthrobacter sp. SPG that utilized 2C4AP as its sole source of carbon and energy. The 2C4AP degradation was initiated by a 2C4AP-deaminase that catalyzed the conversion of 2C4AP into chlorohydroquinone (CHQ) with removal of ammonium ion. In the next step, a CHQ-dehalogenase dehalogenated CHQ to hydroquinone (HQ) that cleaved into ?-hydroxymuconic semialdehyde by a HQ-dioxygenase. The 2C4AP degradation was also investigated in sterile and non-sterile soil microcosms using strain SPG. The results show that the SPG cells degraded 2C4AP more rapidly in sterile soil than non-sterile soil. Our studies showed that strain SPG may be used for bioremediation of 2C4AP-contaminated sites. This is the first report of the 2C4AP degradation by any bacteria. PMID:25427856

  4. Initial Steps in the Pathway for Bacterial Degradation of Two Tetrameric Lignin Model Compounds

    PubMed Central

    Jokela, Jouni; Pellinen, Jukka; Salkinoja-Salonen, Mirja

    1987-01-01

    We investigated the metabolic route by which a lignin tetramer-degrading mixed bacterial culture degraded two tetrameric lignin model compounds containing ?—O—4 and 5—5 biphenyl structures. The ?-hydroxyl groups in the propane chain of both phenolic and nonphenolic tetramers were first oxidized symmetrically in two successive steps to give monoketones and diketones. These ketone metabolites were decomposed through C?(=O)—C? cleavage, forming trimeric carboxyl acids which were further metabolized through another C?(=O)—C? cleavage. Dehydrodiveratric acid, which resulted from the cleavage of the carbon bonds of the nonphenol tetramer, was demethylated twice. Four metabolites of the phenolic tetramer were purified and identified. All of these were stable compounds in sterile mineral medium, but were readily degraded by lignin tetramer-degrading bacteria along the same pathway as the phenol tetramer. No monoaromatic metabolites accumulated. All metabolites were identified by mass and proton magnetic resonance spectrometry. The metabolic route by which the mixed bacterial culture degraded tetrameric lignin model compounds was different from the route of the main ligninase-catalyzed C?—C? cleavage by Phanerochaete chrysosporium. PMID:16347484

  5. Amyloid-beta protein clearance and degradation (ABCD) pathways and their role in Alzheimer's disease.

    PubMed

    Baranello, Robert J; Bharani, Krishna L; Padmaraju, Vasudevaraju; Chopra, Nipun; Lahiri, Debomoy K; Greig, Nigel H; Pappolla, Miguel A; Sambamurti, Kumar

    2015-01-01

    Amyloid-? proteins (A?) of 42 (A?42) and 40 aa (A?40) accumulate as senile plaques (SP) and cerebrovascular amyloid protein deposits that are defining diagnostic features of Alzheimer's disease (AD). A number of rare mutations linked to familial AD (FAD) on the A? precursor protein (APP), Presenilin-1 (PS1), Presenilin- 2 (PS2), Adamalysin10, and other genetic risk factors for sporadic AD such as the ?4 allele of Apolipoprotein E (ApoE-?4) foster the accumulation of A? and also induce the entire spectrum of pathology associated with the disease. A? accumulation is therefore a key pathological event and a prime target for the prevention and treatment of AD. APP is sequentially processed by ?-site APP cleaving enzyme (BACE1) and ?-secretase, a multisubunit PS1/PS2-containing integral membrane protease, to generate A?. Although A? accumulates in all forms of AD, the only pathways known to be affected in FAD increase A? production by APP gene duplication or via base substitutions on APP and ?-secretase subunits PS1 and PS2 that either specifically increase the yield of the longer A?42 or both A?40 and A?42. However, the vast majority of AD patients accumulate A? without these known mutations. This led to proposals that impairment of A? degradation or clearance may play a key role in AD pathogenesis. Several candidate enzymes, including Insulin-degrading enzyme (IDE), Neprilysin (NEP), Endothelin-converting enzyme (ECE), Angiotensin converting enzyme (ACE), Plasmin, and Matrix metalloproteinases (MMPs) have been identified and some have even been successfully evaluated in animal models. Several studies also have demonstrated the capacity of ?-secretase inhibitors to paradoxically increase the yield of A? and we have recently established that the mechanism is by skirting A? degradation. This review outlines major cellular pathways of A? degradation to provide a basis for future efforts to fully characterize the panel of pathways responsible for A? turnover. PMID:25523424

  6. Anoxic Androgen Degradation by the Denitrifying Bacterium Sterolibacterium denitrificans via the 2,3-seco Pathway

    PubMed Central

    Wang, Po-Hsiang; Yu, Chang-Ping; Lee, Tzong-Huei; Lin, Ching-Wen; Ismail, Wael; Wey, Shiaw-Pyng; Kuo, An-Ti

    2014-01-01

    The biodegradation of steroids is a crucial biochemical process mediated exclusively by bacteria. So far, information concerning the anoxic catabolic pathways of androgens is largely unknown, which has prevented many environmental investigations. In this work, we show that Sterolibacterium denitrificans DSMZ 13999 can anaerobically mineralize testosterone and some C19 androgens. By using a 13C-metabolomics approach and monitoring the sequential appearance of the intermediates, we demonstrated that S. denitrificans uses the 2,3-seco pathway to degrade testosterone under anoxic conditions. Furthermore, based on the identification of a C17 intermediate, we propose that the A-ring cleavage may be followed by the removal of a C2 side chain at C-5 of 17-hydroxy-1-oxo-2,3-seco-androstan-3-oic acid (the A-ring cleavage product) via retro-aldol reaction. The androgenic activities of the bacterial culture and the identified intermediates were assessed using the lacZ-based yeast androgen assay. The androgenic activity in the testosterone-grown S. denitrificans culture decreased significantly over time, indicating its ability to eliminate androgens. The A-ring cleavage intermediate (?500 ?M) did not exhibit androgenic activity, whereas the sterane-containing intermediates did. So far, only two androgen-degrading anaerobes (Sterolibacterium denitrificans DSMZ 13999 [a betaproteobacterium] and Steroidobacter denitrificans DSMZ 18526 [a gammaproteobacterium]) have been isolated and characterized, and both of them use the 2,3-seco pathway to anaerobically degrade androgens. The key intermediate 2,3-seco-androstan-3-oic acid can be used as a signature intermediate for culture-independent environmental investigations of anaerobic degradation of C19 androgens. PMID:24657867

  7. Autophagic Degradation of the 26S Proteasome Is Mediated by the Dual ATG8/Ubiquitin Receptor RPN10 in Arabidopsis.

    PubMed

    Marshall, Richard S; Li, Faqiang; Gemperline, David C; Book, Adam J; Vierstra, Richard D

    2015-06-18

    Autophagic turnover of intracellular constituents is critical for cellular housekeeping, nutrient recycling, and various aspects of growth and development in eukaryotes. Here 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 ensuing proteaphagy requiring the proteasome subunit RPN10, which can simultaneously bind both ATG8 and ubiquitin. 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. PMID:26004230

  8. Ubiquitin-fusion degradation pathway: A new strategy for inducing CD8 cells specific for mycobacterial HSP65

    SciTech Connect

    Shen Jianying; Hisaeda, Hajime; Chou Bin [Department of Parasitology, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Yu Qingsheng [Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Tu Liping [Department of Parasitology, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Himeno, Kunisuke [Department of Parasitology, Graduate School of Medical Sciences, Kyushu University, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)], E-mail: himeno@parasite.med.kyushu-u.ac.jp

    2008-01-25

    The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8{sup +} T cells. In this study, we exploited UPS to induce CD8{sup +} T cells specific for mycobacterial HSP65 (mHSP65), one of the leading vaccine candidates against infection with Mycobacterium tuberculosis. A chimeric DNA termed pU-HSP65 encoding a fusion protein between murine ubiquitin and mHSP65 was constructed, and C57BL/6 (B6) mice were immunized with the DNA using gene gun bombardment. Mice immunized with the chimeric DNA acquired potent resistance against challenge with the syngeneic B16F1 melanoma cells transfected with the mHSP65 gene (HSP65/B16F1), compared with those immunized with DNA encoding only mHSP65. Splenocytes from the former group of mice showed a higher grade of cytotoxic activity against HSP65/B16F1 cells and contained a larger number of granzyme B- or IFN-{gamma}-producing CD8{sup +} T cells compared with those from the latter group of mice.

  9. 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

    SciTech Connect

    Cho, Eun-Ah [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)] [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)] [Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

    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 DNA 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.

  10. Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation

    PubMed Central

    Deschênes-Simard, Xavier; Gaumont-Leclerc, Marie-France; Bourdeau, Véronique; Lessard, Frédéric; Moiseeva, Olga; Forest, Valérie; Igelmann, Sebastian; Mallette, Frédérick A.; Saba-El-Leil, Marc K.; Meloche, Sylvain; Saad, Fred; Mes-Masson, Anne-Marie; Ferbeyre, Gerardo

    2013-01-01

    Constitutive activation of growth factor signaling pathways paradoxically triggers a cell cycle arrest known as cellular senescence. In primary cells expressing oncogenic ras, this mechanism effectively prevents cell transformation. Surprisingly, attenuation of ERK/MAP kinase signaling by genetic inactivation of Erk2, RNAi-mediated knockdown of ERK1 or ERK2, or MEK inhibitors prevented the activation of the senescence mechanism, allowing oncogenic ras to transform primary cells. Mechanistically, ERK-mediated senescence involved the proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions, cell migration, RNA metabolism, and cell signaling. This senescence-associated protein degradation (SAPD) was observed not only in cells expressing ectopic ras, but also in cells that senesced due to short telomeres. Individual RNAi-mediated inactivation of SAPD targets was sufficient to restore senescence in cells transformed by oncogenic ras or trigger senescence in normal cells. Conversely, the anti-senescence viral oncoproteins E1A, E6, and E7 prevented SAPD. In human prostate neoplasms, high levels of phosphorylated ERK were found in benign lesions, correlating with other senescence markers and low levels of STAT3, one of the SAPD targets. We thus identified a mechanism that links aberrant activation of growth signaling pathways and short telomeres to protein degradation and cellular senescence. PMID:23599344

  11. Systematic unraveling of the unsolved pathway of nicotine degradation in Pseudomonas.

    PubMed

    Tang, Hongzhi; Wang, Lijuan; Wang, Weiwei; Yu, Hao; Zhang, Kunzhi; Yao, Yuxiang; Xu, Ping

    2013-10-01

    Microorganisms such as Pseudomonas putida play important roles in the mineralization of organic wastes and toxic compounds. To comprehensively and accurately elucidate key processes of nicotine degradation in Pseudomonas putida, we measured differential protein abundance levels with MS-based spectral counting in P. putida S16 grown on nicotine or glycerol, a non-repressive carbon source. In silico analyses highlighted significant clustering of proteins involved in a functional pathway in nicotine degradation. The transcriptional regulation of differentially expressed genes was analyzed by using quantitative reverse transcription-PCR. We observed the following key results: (i) The proteomes, containing 1,292 observed proteins, provide a detailed view of enzymes involved in nicotine metabolism. These proteins could be assigned to the functional groups of transport, detoxification, and amino acid metabolism. There were significant differences in the cytosolic protein patterns of cells growing in a nicotine medium and those in a glycerol medium. (ii) The key step in the conversion of 3-succinoylpyridine to 6-hydroxy-3-succinoylpyridine was catalyzed by a multi-enzyme reaction consisting of a molybdopeterin binding oxidase (spmA), molybdopterin dehydrogenase (spmB), and a (2Fe-2S)-binding ferredoxin (spmC) with molybdenum molybdopterin cytosine dinucleotide as a cofactor. (iii) The gene of a novel nicotine oxidoreductase (nicA2) was cloned, and the recombinant protein was characterized. The proteins and functional pathway identified in the current study represent attractive targets for degradation of environmental toxic compounds. PMID:24204321

  12. Systematic Unraveling of the Unsolved Pathway of Nicotine Degradation in Pseudomonas

    PubMed Central

    Tang, Hongzhi; Zhang, Kunzhi; Yao, Yuxiang; Xu, Ping

    2013-01-01

    Microorganisms such as Pseudomonas putida play important roles in the mineralization of organic wastes and toxic compounds. To comprehensively and accurately elucidate key processes of nicotine degradation in Pseudomonas putida, we measured differential protein abundance levels with MS-based spectral counting in P. putida S16 grown on nicotine or glycerol, a non-repressive carbon source. In silico analyses highlighted significant clustering of proteins involved in a functional pathway in nicotine degradation. The transcriptional regulation of differentially expressed genes was analyzed by using quantitative reverse transcription-PCR. We observed the following key results: (i) The proteomes, containing 1,292 observed proteins, provide a detailed view of enzymes involved in nicotine metabolism. These proteins could be assigned to the functional groups of transport, detoxification, and amino acid metabolism. There were significant differences in the cytosolic protein patterns of cells growing in a nicotine medium and those in a glycerol medium. (ii) The key step in the conversion of 3-succinoylpyridine to 6-hydroxy-3-succinoylpyridine was catalyzed by a multi-enzyme reaction consisting of a molybdopeterin binding oxidase (spmA), molybdopterin dehydrogenase (spmB), and a (2Fe-2S)-binding ferredoxin (spmC) with molybdenum molybdopterin cytosine dinucleotide as a cofactor. (iii) The gene of a novel nicotine oxidoreductase (nicA2) was cloned, and the recombinant protein was characterized. The proteins and functional pathway identified in the current study represent attractive targets for degradation of environmental toxic compounds. PMID:24204321

  13. A novel degradative pathway of 2-nitrobenzoate via 3-hydroxyanthranilate in Pseudomonas fluorescens strain KU-7.

    PubMed

    Hasegawa, Y; Muraki, T; Tokuyama, T; Iwaki, H; Tatsuno, M; Lau, P C

    2000-09-15

    A bacterial strain KU-7, identified as a Pseudomonas fluorescens by 16S rDNA sequencing, was one of the 12 new isolates that are able to grow on 2-nitrobenzoate as a sole source of carbon, nitrogen, and energy. Resting cells of KU-7 were found to accumulate ammonia in the medium indicating that degradation of 2-NBA proceeds through a reductive route. Metabolite analyses by thin layer chromatography and high pressure liquid chromatography indicated that 3-hydroxyanthranilate is an intermediate of 2-nitrobenzoate metabolism in KU-7 cells. This offers an alternative route to 2-nitrobenzoate metabolism since anthranilate (2-aminobenzoate) or catechol were detected as intermediates in other bacteria. Crude extracts of KU-7 cells converted 2-nitrobenzoate to 3-hydroxyanthranilate with oxidation of 2 mol of NADPH. Ring cleavage of 3-hydroxyanthranilate produced a transient yellow product, identified as 2-amino-3-carboxymuconic 6-semialdehyde, that has a maximum absorbance at 360 nm. The initial enzymes of the 2-nitrobenzoate degradation pathway were found to be inducible since succinate-grown cells produced very low enzyme activities. A pathway for 2-nitrobenzoate degradation in KU-7 was proposed. PMID:11034277

  14. Sequestration of a highly reactive intermediate in an evolving pathway for degradation of pentachlorophenol

    PubMed Central

    Yadid, Itamar; Rudolph, Johannes; Hlouchova, Klara; Copley, Shelley D.

    2013-01-01

    Microbes in contaminated environments often evolve new metabolic pathways for detoxification or degradation of pollutants. In some cases, intermediates in newly evolved pathways are more toxic than the initial compound. The initial step in the degradation of pentachlorophenol by Sphingobium chlorophenolicum generates a particularly reactive intermediate; tetrachlorobenzoquinone (TCBQ) is a potent alkylating agent that reacts with cellular thiols at a diffusion-controlled rate. TCBQ reductase (PcpD), an FMN- and NADH-dependent reductase, catalyzes the reduction of TCBQ to tetrachlorohydroquinone. In the presence of PcpD, TCBQ formed by pentachlorophenol hydroxylase (PcpB) is sequestered until it is reduced to the less toxic tetrachlorohydroquinone, protecting the bacterium from the toxic effects of TCBQ and maintaining flux through the pathway. The toxicity of TCBQ may have exerted selective pressure to maintain slow turnover of PcpB (0.02 s?1) so that a transient interaction between PcpB and PcpD can occur before TCBQ is released from the active site of PcpB. PMID:23676275

  15. Genetic immunization based on the ubiquitin-fusion degradation pathway against Trypanosoma cruzi

    SciTech Connect

    Chou, Bin [Department of Microbiology and Immunology, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180 (Japan) [Department of Microbiology and Immunology, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180 (Japan); Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 (Japan); Hiromatsu, Kenji, E-mail: khiromatsu@fukuoka-u.ac.jp [Department of Microbiology and Immunology, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180 (Japan)] [Department of Microbiology and Immunology, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180 (Japan); Hisaeda, Hajime; Duan, Xuefeng; Imai, Takashi [Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 (Japan)] [Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 (Japan); Murata, Shigeo; Tanaka, Keiji [Department of Molecular Oncology, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613 (Japan)] [Department of Molecular Oncology, The Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613 (Japan); Himeno, Kunisuke [Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 (Japan)] [Department of Parasitology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582 (Japan)

    2010-02-12

    Cytotoxic CD8{sup +} T cells are particularly important to the development of protective immunity against the intracellular protozoan parasite, Trypanosoma cruzi, the etiological agent of Chagas disease. We have developed a new effective strategy of genetic immunization by activating CD8{sup +} T cells through the ubiquitin-fusion degradation (UFD) pathway. We constructed expression plasmids encoding the amastigote surface protein-2 (ASP-2) of T. cruzi. To induce the UFD pathway, a chimeric gene encoding ubiquitin fused to ASP-2 (pUB-ASP-2) was constructed. Mice immunized with pUB-ASP-2 presented lower parasitemia and longer survival period, compared with mice immunized with pASP-2 alone. Depletion of CD8{sup +} T cells abolished protection against T. cruzi in mice immunized with pUB-ASP-2 while depletion of CD4{sup +} T cells did not influence the effective immunity. Mice deficient in LMP2 or LMP7, subunits of immunoproteasomes, were not able to develop protective immunity induced. These results suggest that ubiquitin-fused antigens expressed in antigen-presenting cells were effectively degraded via the UFD pathway, and subsequently activated CD8{sup +} T cells. Consequently, immunization with pUB-ASP-2 was able to induce potent protective immunity against infection of T. cruzi.

  16. Involvement of the Nrf2-proteasome pathway in the endoplasmic reticulum stress response in pancreatic ?-cells

    SciTech Connect

    Lee, Sanghwan; Hur, Eu-gene [Yeungnam University, College of Pharmacy, Gyeongsan-si, Gyeongsangbuk-do 712?749 (Korea, Republic of)] [Yeungnam University, College of Pharmacy, Gyeongsan-si, Gyeongsangbuk-do 712?749 (Korea, Republic of); Ryoo, In-geun; Jung, Kyeong-Ah [The Catholic University of Korea, College of Pharmacy, Wonmi-gu, Bucheon, Gyeonggi-do 420?743 (Korea, Republic of)] [The Catholic University of Korea, College of Pharmacy, Wonmi-gu, Bucheon, Gyeonggi-do 420?743 (Korea, Republic of); Kwak, Jiyeon [Inha University, College of Medicine, 253 Yonghyun-dong, Nam-gu, Incheon 402?751 (Korea, Republic of)] [Inha University, College of Medicine, 253 Yonghyun-dong, Nam-gu, Incheon 402?751 (Korea, Republic of); Kwak, Mi-Kyoung, E-mail: mkwak@catholic.ac.kr [The Catholic University of Korea, College of Pharmacy, Wonmi-gu, Bucheon, Gyeonggi-do 420?743 (Korea, Republic of)] [The Catholic University of Korea, College of Pharmacy, Wonmi-gu, Bucheon, Gyeonggi-do 420?743 (Korea, Republic of)

    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 ?-cell line, ?TC-6. shRNA-mediated silencing of Nrf2 expression in ?TC-6 cells significantly increased tunicamycin-induced cytotoxicity, elevated the expression of the pro-apoptotic ER 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 ?TC-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 ?-cells by enhancing proteasome-mediated ERAD. -- Highlights: ? Nrf2 silencing in pancreatic ?-cells enhanced tunicamycin-mediated ER stress. ? Expression of the proteasome was inducible by Nrf2 signaling. ? Nrf2 activator D3T protected ?-cells from tunicamycin-mediated ER stress. ? Protective effect of D3T was associated with Nrf2-dependent proteasome induction.

  17. Experimental design optimization for electrochemical removal of gentamicin: toxicity evaluation and degradation pathway.

    PubMed

    Dhuria, Radhey S; Bhatti, Rajbir; Bhatti, Manpreet S; Singh, Palwinder; Whitcomb, Patrick J; Thukral, Ashwani K

    2013-01-01

    Electrochemical degradation of gentamicin was achieved using a laboratory scale electrochemical reactor by optimizing pH, current density and treatment time. A two step statistical optimization was performed as per factorial design and center composite design (CCD). A Pareto chart was used for selecting statistically significant effects and an analysis of variance (ANOVA) table indicated significant curvature. Thus adding additional experimental runs improved the model fitting through a second order model. Maximum degradation was predicted at a pH of 6.7, 70 A m(-2) and 45 min. The experimental data fitted well through a reduced quadratic model with R(2) equal to 0.945. The toxicity of degradation products as determined by disc diffusion assay employing Pseudomonas aeruginosa strain was found to be reduced by 55%. The degradation pathway of gentamicin was studied using mass spectral (MS) analysis. Pure gentamicin showed a molecular ion peak at m/z 478 ([M + 1](+)), and after addition of NaCl as electrolyte, the mass peak was observed at m/z 523. After 15 min of electrochemical treatment, a new peak appeared at m/z 316 due to the loss of one pyran moiety. After 45 min of electrochemical treatment, another peak appeared at m/z of 478 due to loss of two Na(+) from gentamicin. PMID:23656945

  18. The exoribonuclease Dis3L2 defines a novel eukaryotic RNA degradation pathway

    PubMed Central

    Malecki, Michal; Viegas, Sandra C; Carneiro, Tiago; Golik, Pawel; Dressaire, Clémentine; Ferreira, Miguel G; Arraiano, Cecília M

    2013-01-01

    The final step of cytoplasmic mRNA degradation proceeds in either a 5?-3? direction catalysed by Xrn1 or in a 3?-5? direction catalysed by the exosome. Dis3/Rrp44, an RNase II family protein, is the catalytic subunit of the exosome. In humans, there are three paralogues of this enzyme: DIS3, DIS3L, and DIS3L2. In this work, we identified a novel Schizosaccharomyces pombe exonuclease belonging to the conserved family of human DIS3L2 and plant SOV. Dis3L2 does not interact with the exosome components and localizes in the cytoplasm and in cytoplasmic foci, which are docked to P-bodies. Deletion of dis3l2+ is synthetically lethal with xrn1?, while deletion of dis3l2+ in an lsm1? background results in the accumulation of transcripts and slower mRNA degradation rates. Accumulated transcripts show enhanced uridylation and in vitro Dis3L2 displays a preference for uridylated substrates. Altogether, our results suggest that in S. pombe, and possibly in most other eukaryotes, Dis3L2 is an important factor in mRNA degradation. Therefore, this novel 3?-5? RNA decay pathway represents an alternative to degradation by Xrn1 and the exosome. PMID:23503588

  19. The exoribonuclease Dis3L2 defines a novel eukaryotic RNA degradation pathway.

    PubMed

    Malecki, Michal; Viegas, Sandra C; Carneiro, Tiago; Golik, Pawel; Dressaire, Clémentine; Ferreira, Miguel G; Arraiano, Cecília M

    2013-07-01

    The final step of cytoplasmic mRNA degradation proceeds in either a 5'-3' direction catalysed by Xrn1 or in a 3'-5' direction catalysed by the exosome. Dis3/Rrp44, an RNase II family protein, is the catalytic subunit of the exosome. In humans, there are three paralogues of this enzyme: DIS3, DIS3L, and DIS3L2. In this work, we identified a novel Schizosaccharomyces pombe exonuclease belonging to the conserved family of human DIS3L2 and plant SOV. Dis3L2 does not interact with the exosome components and localizes in the cytoplasm and in cytoplasmic foci, which are docked to P-bodies. Deletion of dis3l2(+) is synthetically lethal with xrn1?, while deletion of dis3l2(+) in an lsm1? background results in the accumulation of transcripts and slower mRNA degradation rates. Accumulated transcripts show enhanced uridylation and in vitro Dis3L2 displays a preference for uridylated substrates. Altogether, our results suggest that in S. pombe, and possibly in most other eukaryotes, Dis3L2 is an important factor in mRNA degradation. Therefore, this novel 3'-5' RNA decay pathway represents an alternative to degradation by Xrn1 and the exosome. PMID:23503588

  20. Degradation of 2,4 Dichlorobiphenyl Via Meta-cleavage Pathway by Pseudomonas spp. Consortium.

    PubMed

    Jayanna, Shobha K; Gayathri, Devaraja

    2015-06-01

    Two bacterial isolates (Pseudomonas sp. GSa and Pseudomonas sp. GSb) were in close association able to assimilate 2,4 dichlorobiphenyl (2,4 CB), a PCB congener. GC-MS analysis of spent culture medium of the consortium with 2,4 CB as substrate showed 90 % degradation (according to Electron capture detection values) with catechol as one of the important intermediate compounds through meta-cleavage pathway. Further, ability of the consortium to utilise PCB congeners, Methoxychlor, Aroclor 1016, Chlorobenzoic acids and Monoaromatic compounds indicated that the consortium of GSa and GSb would be an ideal candidate for in situ bioremediation of PCB. PMID:25800378

  1. Benzene degradation pathways investigated by a human-tissue-based biosensor

    NASA Astrophysics Data System (ADS)

    Campanella, Luigi; Ercole, Patrizia; Grossi, Rosella; Russo, Mario V.; Visco, Giovanni

    2004-03-01

    Benzene is determined by a biosensor based on human kidney tissue as biological component. Dioxygenase catalyse substrate oxidation. The useful analytical range results to be complementary to that of one or more traditional models based on Pseudomonas. The biosensors has got an other aim as its functioning is able to clear up the degradation pathways of benzene. The behavior of a biosensor based on cancerous human kidney tissue compared to healthy one let to hypothize a poor activity of dioxygenase in cancerous kidney, possibly to be tested with prevention strategy.

  2. Lysine degradation through the saccharopine pathway in mammals: involvement of both bifunctional and monofunctional lysine-degrading enzymes in mouse.

    PubMed Central

    Papes, F; Kemper, E L; Cord-Neto, G; Langone, F; Arruda, P

    1999-01-01

    Lysine-oxoglutarate reductase and saccharopine dehydrogenase are enzymic activities that catalyse the first two steps of lysine degradation through the saccharopine pathway in upper eukaryotes. This paper describes the isolation and characterization of a cDNA clone encoding a bifunctional enzyme bearing domains corresponding to these two enzymic activities. We partly purified those activities from mouse liver and showed for the first time that both a bifunctional lysine-oxoglutarate reductase/saccharopine dehydrogenase and a monofunctional saccharopine dehydrogenase are likely to be present in this organ. Northern analyses indicate the existence of two mRNA species in liver and kidney. The longest molecule, 3.4 kb in size, corresponds to the isolated cDNA and encodes the bifunctional enzyme. The 2.4 kb short transcript probably codes for the monofunctional dehydrogenase. Sequence analyses show that the bifunctional enzyme is likely to be a mitochondrial protein. Furthermore, enzymic and expression analyses suggest that lysine-oxoglutarate reductase/saccharopine dehydrogenase levels increase in livers of mice under starvation. Lysine-injected mice also show an increase in lysine-oxoglutarate reductase and saccharopine dehydrogenase levels. PMID:10567240

  3. Molecular and biochemical characterization of the tetralin degradation pathway in Rhodococcus sp. strain TFB

    PubMed Central

    Tomás?Gallardo, Laura; Santero, Eduardo; Camafeita, Emilio; Calvo, Enrique; Schlömann, Michael; Floriano, Belén

    2009-01-01

    Summary The tetralin biodegradation pathway in Rhodococcus sp. strain TFB, a Gram?positive bacterium resistant to genetic manipulation, was characterized using a proteomic approach. Relative protein expression in cell free extracts from tetralin? and glucose?grown cells was compared using the 2D?DIGE technique. Identification of proteins specifically expressed in tetralin?grown cells was used to characterize a complete set of genes involved in tetralin degradation by reverse genetics. We propose a tetralin degradation pathway analogous to that described for Sphingomonas macrogolitabida strain TFA. TFB thn genes are organized into three operons; two contain all of the structural genes and are transcribed in the same direction, while the third operon, thnST, is transcribed in the opposite direction and encodes a two?component regulatory system, whose transcription is higher in tetralin?grown cells. In addition to tetralin induction, TFB thn structural genes are subject to glucose repression. Primer extension assays and translational thnA1::gfp and thnS::gfp fusions were used to characterize putative promoter regions. A mutational analysis of the thnA1 promoter region allowed us to define nucleotides within the cis regulatory elements that are important for the control of thn gene expression. PMID:21261920

  4. Characterization of a novel cometabolic degradation carbazole pathway by a phenol-cultivated Arthrobacter sp. W1.

    PubMed

    Shi, Shengnan; Qu, Yuanyuan; Zhou, Hao; Ma, Qiao; Ma, Fang

    2015-10-01

    Arthrobacter sp. W1 was used to characterize the pathways involved in cometabolic degradation of carbazole (CA) with phenol as the primary substrate. To clarify the upper pathway of cometabolic degradation CA, Escherichia coli strain BL21 expressing phenol hydroxylase from strain W1 (PHIND) was investigated to degrade CA. Firstly, CA was initially monohydroxylated at C-2 and C-4 positions to produce 2- and 4-hydroxycarbazole, followed by successively hydroxylated to the corresponding 1,2- and 3,4-dihydroxycarbazole, of which 3,4-dihydroxycarbazole was unequivocally identified for the first time. To characterize the downstream cometabolic degradation CA pathway, purified 3,4-dihydroxycarbazole was used as the substrate for phenol-grown W1, and a series of novel indole derivatives were identified. These results suggested that a novel pathway of CA catabolism was employed by strain W1 via a successive hydroxylation and meta-cleavage pathway. These findings provide new insights into the cometabolic degradation CA process and have potential applications in biotechnology and bioremediation. PMID:26142994

  5. Candida albicans Utilizes a Modified ?-Oxidation Pathway for the Degradation of Toxic Propionyl-CoA*

    PubMed Central

    Otzen, Christian; Bardl, Bettina; Jacobsen, Ilse D.; Nett, Markus; Brock, Matthias

    2014-01-01

    Propionyl-CoA arises as a metabolic intermediate from the degradation of propionate, odd-chain fatty acids, and some amino acids. Thus, pathways for catabolism of this intermediate have evolved in all kingdoms of life, preventing the accumulation of toxic propionyl-CoA concentrations. Previous studies have shown that fungi generally use the methyl citrate cycle for propionyl-CoA degradation. Here, we show that this is not the case for the pathogenic fungus Candida albicans despite its ability to use propionate and valerate as carbon sources. Comparative proteome analyses suggested the presence of a modified ?-oxidation pathway with the key intermediate 3-hydroxypropionate. Gene deletion analyses confirmed that the enoyl-CoA hydratase/dehydrogenase Fox2p, the putative 3-hydroxypropionyl-CoA hydrolase Ehd3p, the 3-hydroxypropionate dehydrogenase Hpd1p, and the putative malonate semialdehyde dehydrogenase Ald6p essentially contribute to propionyl-CoA degradation and its conversion to acetyl-CoA. The function of Hpd1p was further supported by the detection of accumulating 3-hydroxypropionate in the hpd1 mutant on propionyl-CoA-generating nutrients. Substrate specificity of Hpd1p was determined from recombinant purified enzyme, which revealed a preference for 3-hydroxypropionate, although serine and 3-hydroxyisobutyrate could also serve as substrates. Finally, virulence studies in a murine sepsis model revealed attenuated virulence of the hpd1 mutant, which indicates generation of propionyl-CoA from host-provided nutrients during infection. PMID:24497638

  6. The degradative pathway of the s-triazine melamine. The steps to ring cleavage.

    PubMed Central

    Jutzi, K; Cook, A M; Hütter, R

    1982-01-01

    1. The degradative pathway of melamine (1,3,5-triazine-2,4,6-triamine) was examined in Pseudomonas sp. strain A. 2. The bacterium grew with melamine, ammeline, ammelide, cyanuric acid or NH+4 as sole source of nitrogen, and each substrate was entirely metabolized. Utilization of ammeline, ammelide, cyanuric acid or NH+4 was concomitant with growth. But with melamine as substrate, a transient intermediate was detected, which was identified as ammeline by three methods. 3. Enzymes from strain A were separated by chromatography on DEAE-cellulose, and four activities were examined. 4. Melamine was converted stoichiometrically into equimolar amounts of ammeline and NH+4. 5. Ammeline was converted stoichiometrically into equimolar amounts of ammelide and NH+4; ammelide was identified by four methods. 6. Ammelide was converted stoichiometrically into equimolar amounts of cyanuric acid and NH+4; cyanuric acid was identified by four methods. 7. Cyanuric acid was converted by an enzyme preparation into an unidentified product with negligible release of NH+4. 8. The specific activities of the degradative enzymes (greater than or equal to 0.3 mkat/kg of protein) were high enough to explain the growth rate of the organism. 9. The bacterium converted 0.4 mM-melamine anaerobically into 2.3 mM-NH+4. 10. Two other pseudomonads and two strains of Klebsiella pneumoniae were also examined, with similar results. 11. The degradative pathway of melamine appears to be hydrolytic, and proceeds by three successive deaminations to cyanuric acid, which is further metabolized. PMID:6762212

  7. Stress-Induced Nuclear RNA Degradation Pathways Regulate Yeast Bromodomain Factor 2 to Promote Cell Survival

    PubMed Central

    Roy, Kevin; Chanfreau, Guillaume

    2014-01-01

    Bromodomain proteins are key regulators of gene expression. How the levels of these factors are regulated in specific environmental conditions is unknown. Previous work has established that expression of yeast Bromodomain factor 2 (BDF2) is limited by spliceosome-mediated decay (SMD). Here we show that BDF2 is subject to an additional layer of post-transcriptional control through RNase III-mediated decay (RMD). We found that the yeast RNase III Rnt1p cleaves a stem-loop structure within the BDF2 mRNA to down-regulate its expression. However, these two nuclear RNA degradation pathways play distinct roles in the regulation of BDF2 expression, as we show that the RMD and SMD pathways of the BDF2 mRNA are differentially activated or repressed in specific environmental conditions. RMD is hyper-activated by salt stress and repressed by hydroxyurea-induced DNA damage while SMD is inactivated by salt stress and predominates during DNA damage. Mutations of cis-acting signals that control SMD and RMD rescue numerous growth defects of cells lacking Bdf1p, and show that SMD plays an important role in the DNA damage response. These results demonstrate that specific environmental conditions modulate nuclear RNA degradation pathways to control BDF2 expression and Bdf2p-mediated gene regulation. Moreover, these results show that precise dosage of Bromodomain factors is essential for cell survival in specific environmental conditions, emphasizing their importance for controlling chromatin structure and gene expression in response to environmental stress. PMID:25232960

  8. Further characterization of o-nitrobenzaldehyde degrading bacterium Pseudomonas sp. ONBA-17 and deduction on its metabolic pathway

    PubMed Central

    Yu, Fang-Bo; Li, Xiao-Dan; Ali, Shinawar Waseem; Shan, Sheng-Dao; Luo, Lin-Ping; Guan, Li-Bo

    2014-01-01

    A previously reported o-nitrobenzaldehyde (ONBA) degrading bacterium Pseudomonas sp. ONBA-17 was further identified and characterized. Based on results of DNA base composition and DNA-DNA hybridization, the strain was identified as P. putida. Its degradation effect enhanced with increase of inoculum amount and no lag phase was observed. Higher removal rate was achieved under shaking conditions. All tested ONBA with different initial concentrations could be completely degraded within 5 d. In addition, degradative enzyme(s) involved was confirmed as intra-cellular distributed and constitutively expressed. Effects of different compounds on relative activity of degradative enzyme(s) within cell-free extract were also evaluated. Finally, 2-nitrobenzoic acid and 2, 3-dihydroxybenzoic acid were detected as metabolites of ONBA degradation by P. putida ONBA-17, and relevant metabolic pathway was preliminary proposed. This study might help with future research in better understanding of nitroaromatics biodegradation. PMID:25763034

  9. Apoprotein B100 has a Prolonged Interaction with the Translocon during which Its Lipidation and Translocation Change from Dependence on the Microsomal Triglyceride Transfer Protein to Independence

    Microsoft Academic Search

    Deborah M. Mitchell; Mingyue Zhou; Rajalakshmi Pariyarath; Hongxing Wang; John D. Aitchison; Henry N. Ginsberg; Edward A. Fisher

    1998-01-01

    When lipid synthesis is limited in HepG2 cells, apoprotein B100 (apoB100) is not secreted but rapidly degraded by the ubiquitin-proteasome pathway. To investigate apoB100 biosynthesis and secretion further, the physical and functional states of apoB100 destined for either degradation or lipoprotein assembly were studied under conditions in which lipid synthesis, proteasomal activity, and microsomal triglyceride transfer protein (MTP) lipid-transfer activity

  10. Analysis of CFTR Folding and Degradation in Transiently Transfected Cells

    PubMed Central

    Grove, Diane E.; Rosser, Meredith F.N.; Watkins, Richard L.; Cyr, Douglas M.

    2015-01-01

    Misfolding and premature degradation of F508del-CFTR is the major cause of cystic fibrosis. Components of the ubiquitin-proteasome system function on the surface of the endoplasmic reticulum to select misfolded proteins for degradation. The folding status of F508del-CFTR is monitored by at least two ER quality control checkpoints. The ER-associated Derlin-1/RMA1 E3 complex appears to recognize folding defects in CFTR that involve misassembly of NBD1 into a complex with the R-domain. In contrast, the cytosolic Hsp70/CHIP E3 complex appears to sense folding defects that occur after synthesis of NBD2. Herein we describe methods that allow for the study of how modulation of these ER quality control factors by siRNA impacts CFTR folding and degradation. The experimental system described employs transiently transfected HEK293 cells and is utilized to monitor the biogenesis of CFTR by both Western blot and pulse chase studies. Methods to detect complexes formed between CFTR folding intermediates and ER quality control factors will also be described. PMID:21594788

  11. Carbon and chlorine isotope analysis to identify abiotic degradation pathways of 1,1,1-trichloroethane.

    PubMed

    Palau, Jordi; Shouakar-Stash, Orfan; Hunkeler, Daniel

    2014-12-16

    This study investigates dual C-Cl isotope fractionation during 1,1,1-TCA transformation by heat-activated persulfate (PS), hydrolysis/dehydrohalogenation (HY/DH) and Fe(0). Compound-specific chlorine isotope analysis of 1,1,1-TCA was performed for the first time, and transformation-associated isotope fractionation ? bulk C and ? bulk Cl values were -4.0 ± 0.2‰ and no chlorine isotope fractionation with PS, -1.6 ± 0.2‰ and -4.7 ± 0.1‰ for HY/DH, -7.8 ± 0.4‰ and -5.2 ± 0.2‰ with Fe(0). Distinctly different dual isotope slopes (??13C/??37Cl): ? with PS, 0.33 ± 0.04 for HY/DH and 1.5 ± 0.1 with Fe(0) highlight the potential of this approach to identify abiotic degradation pathways of 1,1,1-TCA in the field. The trend observed with PS agreed with a C-H bond oxidation mechanism in the first reaction step. For HY/DH and Fe(0) pathways, different slopes were obtained although both pathways involve cleavage of a C-Cl bond in their initial reaction step. In contrast to the expected larger primary carbon isotope effects relative to chlorine for C-Cl bond cleavage, ? bulk C < ? bulk Cl was observed for HY/DH and in a similar range for reduction by Fe(0), suggesting the contribution of secondary chlorine isotope effects. Therefore, different magnitude of secondary chlorine isotope effects could at least be partly responsible for the distinct slopes between HY/DH and Fe(0) pathways. Following this dual isotope approach, abiotic transformation processes can unambiguously be identified and quantified. PMID:25379605

  12. Morpholine Degradation Pathway of Mycobacterium aurum MO1: Direct Evidence of Intermediates by In Situ 1H Nuclear Magnetic Resonance

    PubMed Central

    Combourieu, B.; Besse, P.; Sancelme, M.; Veschambre, H.; Delort, A. M.; Poupin, P.; Truffaut, N.

    1998-01-01

    Resting Mycobacterium aurum MO1 cells were incubated with morpholine, a waste from the chemical industry. The kinetics of biodegradation was monitored by using in situ nuclear magnetic resonance (NMR). The incubation medium was directly analyzed by 1H NMR. This technique allowed the unambiguous identification of two intermediates of the metabolic pathway involved in the biodegradation process, glycolate and 2-(2-aminoethoxy)acetate. The latter compound, which was not commercially available, was synthesized, in three steps, from 2-(2-aminoethoxy)ethanol. Quantitative analysis of the kinetics of degradation of morpholine was performed by integrating the signals of the different metabolites in 1H-NMR spectra. Morpholine was degraded within 10 h. The intermediates increased during the first 10 h and finally disappeared after 20 h incubation. Assays of degradation were also carried out with glycolate and ethanolamine, hypothetical intermediates of the morpholine degradation pathway. They were degraded within 4 and 8 h, respectively. Until now, no tool for direct detection of intermediates or even morpholine has been available, consequently, only hypothetical pathways have been proposed. The approach described here gives both qualitative and quantitative information about the metabolic routes used in morpholine degradation by M. aurum MO1. It could be used to investigate many biodegradative processes. PMID:9435073

  13. M2-like macrophages are responsible for collagen degradation through a mannose receptor–mediated pathway

    PubMed Central

    Madsen, Daniel H.; Leonard, Daniel; Masedunskas, Andrius; Moyer, Amanda; Jürgensen, Henrik Jessen; Peters, Diane E.; Amornphimoltham, Panomwat; Selvaraj, Arul; Yamada, Susan S.; Brenner, David A.; Burgdorf, Sven; Engelholm, Lars H.; Behrendt, Niels; Holmbeck, Kenn; Weigert, Roberto

    2013-01-01

    Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase–dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor–associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process. PMID:24019537

  14. Statin-induced depletion of geranylgeranyl pyrophosphate inhibits cell proliferation by a novel pathway of Skp2 degradation

    PubMed Central

    Vosper, Jonathan; Masuccio, Alessia; Kullmann, Michael; Ploner, Christian; Geley, Stephan; Hengst, Ludger

    2015-01-01

    Statins, such as lovastatin, can induce a cell cycle arrest in the G1 phase. This robust antiproliferative activity remains intact in many cancer cells that are deficient in cell cycle checkpoints and leads to an increased expression of CDK inhibitor proteins p27Kip1 and p21Cip1. The molecular details of this statin-induced growth arrest remains unclear. Here we present evidence that lovastatin can induce the degradation of Skp2, a subunit of the SCFSkp2 ubiquitin ligase that targets p27Kip1 and p21Cip1 for proteasomal destruction. The statin-induced degradation of Skp2 is cell cycle phase independent and does not require its well characterised degradation pathway mediated by APC/CCdh1- or Skp2 autoubiquitination. An N-terminal domain preceding the F-box of Skp2 is both necessary and sufficient for its statin mediated degradation. The degradation of Skp2 results from statin induced depletion of geranylgeranyl isoprenoid intermediates of cholesterol biosynthesis. Inhibition of geranylgeranyl-transferase-I also promotes APC/CCdh1-independent degradation of Skp2, indicating that de-modification of a geranylgeranylated protein triggers this novel pathway of Skp2 degradation. PMID:25605247

  15. Genetic Investigation of the Catabolic Pathway for Degradation of Abietane Diterpenoids by Pseudomonas abietaniphila BKME-9

    PubMed Central

    Martin, Vincent J. J.; Mohn, William W.

    2000-01-01

    We have cloned and sequenced the dit gene cluster encoding enzymes of the catabolic pathway for abietane diterpenoid degradation by Pseudomonas abietaniphila BKME-9. The dit gene cluster is located on a 16.7-kb DNA fragment containing 13 complete open reading frames (ORFs) and 1 partial ORF. The genes ditA1A2A3 encode the ? and ? subunits and the ferredoxin of the dioxygenase which hydroxylates 7-oxodehydroabietic acid to 7-oxo-11,12-dihydroxy-8,13-abietadien acid. The dioxygenase mutant strain BKME-941 (ditA1::Tn5) did not grow on nonaromatic abietanes, and transformed palustric and abietic acids to 7-oxodehydroabietic acid in cell suspension assays. Thus, nonaromatic abietanes are aromatized prior to further degradation. Catechol 2,3-dioxygenase activity of xylE transcriptional fusion strains showed induction of ditA1 and ditA3 by abietic, dehydroabietic, and 7-oxodehydroabietic acids, which support the growth of strain BKME-9, as well as by isopimaric and 12,14-dichlorodehydroabietic acids, which are diterpenoids that do not support the growth of strain BKME-9. In addition to the aromatic-ring-hydroxylating dioxygenase genes, the dit cluster includes ditC, encoding an extradiol ring cleavage dioxygenase, and ditR, encoding an IclR-type transcriptional regulator. Although ditR is not strictly required for the growth of strain BKME-9 on abietanes, a ditR::Kmr mutation in a ditA3::xylE reporter strain demonstrated that it encodes an inducer-dependent transcriptional activator of ditA3. An ORF with sequence similarity to genes encoding permeases (ditE) is linked with genes involved in abietane degradation. PMID:10850995

  16. Structural basis of lentiviral subversion of a cellular protein degradation pathway

    NASA Astrophysics Data System (ADS)

    Schwefel, David; Groom, Harriet C. T.; Boucherit, Virginie C.; Christodoulou, Evangelos; Walker, Philip A.; Stoye, Jonathan P.; Bishop, Kate N.; Taylor, Ian A.

    2014-01-01

    Lentiviruses contain accessory genes that have evolved to counteract the effects of host cellular defence proteins that inhibit productive infection. One such restriction factor, SAMHD1, inhibits human immunodeficiency virus (HIV)-1 infection of myeloid-lineage cells as well as resting CD4+ T cells by reducing the cellular deoxynucleoside 5'-triphosphate (dNTP) concentration to a level at which the viral reverse transcriptase cannot function. In other lentiviruses, including HIV-2 and related simian immunodeficiency viruses (SIVs), SAMHD1 restriction is overcome by the action of viral accessory protein x (Vpx) or the related viral protein r (Vpr) that target and recruit SAMHD1 for proteasomal degradation. The molecular mechanism by which these viral proteins are able to usurp the host cell's ubiquitination machinery to destroy the cell's protection against these viruses has not been defined. Here we present the crystal structure of a ternary complex of Vpx with the human E3 ligase substrate adaptor DCAF1 and the carboxy-terminal region of human SAMHD1. Vpx is made up of a three-helical bundle stabilized by a zinc finger motif, and wraps tightly around the disc-shaped DCAF1 molecule to present a new molecular surface. This adapted surface is then able to recruit SAMHD1 via its C terminus, making it a competent substrate for the E3 ligase to mark for proteasomal degradation. The structure reported here provides a molecular description of how a lentiviral accessory protein is able to subvert the cell's normal protein degradation pathway to inactivate the cellular viral defence system.

  17. Megaplasmid and Chromosomal Loci for the Phb Degradation Pathway in Rhizobium (Sinorhizobium) Meliloti

    PubMed Central

    Charles, T. C.; Cai, C.; Aneja, P.

    1997-01-01

    Chromosomal and megaplasmid loci that affect the poly-3-hydroxybutyrate (PHB) degradation pathway in Rhizobium meliloti were identified. A clone that restores the ability of certain R. meliloti mutants with defined deletions in megaplasmid pRmeSU47b to use 3-hydroxybutyrate or acetoacetate as the sole carbon source was isolated from a cosmid library of R. meliloti genomic DNA. Tn5 insertion mutagenesis, followed by merodiploid complementation analysis, demonstrated that the locus consists of at least four transcriptional units, bhbA-D. We also identified loci involved in 3-hydroxybutyrate and/or acetoacetate utilization by screening for mutants that had lost the ability to use 3-hydroxybutyrate as the sole carbon source while retaining the ability to use acetate (thus ensuring an intact glyoxylate cycle and gluconeogenic pathway). These mutants fell into four classes, as determined by replicon mobilization experiments and genetic linkage in phage transduction; one class corresponded to the bhb locus on pRmeSU47b, two classes mapped to different regions on the chromosome and the fourth, bdhA, represented by a single mutant, mapped to another pRmeSU47b locus, near bacA. The bdhA mutant is deficient in 3-hydroxybutrate dehydrogenase activity. PMID:9258668

  18. The Whole Genome Sequence of Sphingobium chlorophenolicum L-1: Insights into the Evolution of the Pentachlorophenol Degradation Pathway

    PubMed Central

    Copley, Shelley D.; Rokicki, Joseph; Turner, Pernilla; Daligault, Hajnalka; Nolan, Matt; Land, Miriam

    2012-01-01

    Sphingobium chlorophenolicum Strain L-1 can mineralize the toxic pesticide pentachlorophenol (PCP). We have sequenced the genome of S. chlorophenolicum Strain L-1. The genome consists of a primary chromosome that encodes most of the genes for core processes, a secondary chromosome that encodes primarily genes that appear to be involved in environmental adaptation, and a small plasmid. The genes responsible for degradation of PCP are found on chromosome 2. We have compared the genomes of S. chlorophenolicum Strain L-1 and Sphingobium japonicum, a closely related Sphingomonad that degrades lindane. Our analysis suggests that the genes encoding the first three enzymes in the PCP degradation pathway were acquired via two different horizontal gene transfer events, and the genes encoding the final two enzymes in the pathway were acquired from the most recent common ancestor of these two bacteria. PMID:22179583

  19. An iron-regulated and glycosylation-dependent proteasomal degradation pathway for the plasma membrane metal transporter ZIP14

    PubMed Central

    Zhao, Ningning; Zhang, An-Sheng; Worthen, Christal; Knutson, Mitchell D.; Enns, Caroline A.

    2014-01-01

    Protein degradation is instrumental in regulating cellular function. Plasma membrane proteins targeted for degradation are internalized and sorted to multivesicular bodies, which fuse with lysosomes, where they are degraded. ZIP14 is a newly identified iron transporter with multitransmembrane domains. In an attempt to dissect the molecular mechanisms by which iron regulates ZIP14 levels, we found that ZIP14 is endocytosed, extracted from membranes, deglycosylated, and degraded by proteasomes. This pathway did not depend on the retrograde trafficking to the endoplasmic reticulum and thus did not involve the well-defined endoplasmic reticulum-associated protein degradation pathway. Iron inhibited membrane extraction of internalized ZIP14, resulting in higher steady-state levels of ZIP14. Asparagine-linked (N-linked) glycosylation of ZIP14, particularly the glycosylation at N102, was required for efficient membrane extraction of ZIP14 and therefore is necessary for its iron sensitivity. These findings highlight the importance of proteasomes in the degradation of endocytosed plasma membrane proteins. PMID:24927598

  20. Reaction pathway of the degradation of the p-hydroxybenzoic acid by sulfate radical generated by ionizing radiations

    NASA Astrophysics Data System (ADS)

    Criquet, Justine; Leitner, Nathalie Karpel Vel

    2015-01-01

    The degradation of p-hydroxybenzoic acid (HBA) in aqueous solutions by ionizing radiation was studied. The phenolic pollutant was easily removed by the electron beam irradiation, as more than 80% of the initial 100 ?M introduced was degraded for a dose of 600 Gy. It was shown that the addition of persulfate, producing the sulfate radical as additional reactive species, induced a change in the reaction pathway. LC-MS analyses were performed in order to identify the different by-products formed. In the absence of persulfate, the main by-product formed was 3,4-dihydroxybenzoic acid, while in presence of persulfate, 1,4-benzoquinone was detected and the hydroxylated by-products were not present. A reaction pathway of HBA degradation by hydroxyl and sulfate radicals was proposed from the identification of the chemical structure of the different by-products detected. The influences of pH and dissolved oxygen were also studied. A high decline of HBA degradation was observed at pH 11 compared to pH 4.5, this decrease was minimized in the presence of persulfate. The dissolved oxygen concentration was found to be a limiting parameter of HBA degradation, however an excess of dissolved oxygen in solution did not improve the degradation to a large extent.

  1. Connecting lignin-degradation pathway with pre-treatment inhibitor sensitivity of Cupriavidus necator.

    PubMed

    Wang, Wei; Yang, Shihui; Hunsinger, Glendon B; Pienkos, Philip T; Johnson, David K

    2014-01-01

    To produce lignocellulosic biofuels economically, the complete release of monomers from the plant cell wall components, cellulose, hemicellulose, and lignin, through pre-treatment and hydrolysis (both enzymatic and chemical), and the efficient utilization of these monomers as carbon sources, is crucial. In addition, the identification and development of robust microbial biofuel production strains that can tolerate the toxic compounds generated during pre-treatment and hydrolysis is also essential. In this work, Cupriavidus necator was selected due to its capabilities for utilizing lignin monomers and producing polyhydroxylbutyrate (PHB), a bioplastic as well as an advanced biofuel intermediate. We characterized the growth kinetics of C. necator in pre-treated corn stover slurry as well as individually in the pre-sence of 11 potentially toxic compounds in the saccharified slurry. We found that C. necator was sensitive to the saccharified slurry produced from dilute acid pre-treated corn stover. Five out of 11 compounds within the slurry were characterized as toxic to C. necator, namely ammonium acetate, furfural, hydroxymethylfurfural (HMF), benzoic acid, and p-coumaric acid. Aldehydes (e.g., furfural and HMF) were more toxic than the acetate and the lignin degradation products benzoic acid and p-coumaric acid; furfural was identified as the most toxic compound. Although toxic to C. necator at high concentration, ammonium acetate, benzoic acid, and p-coumaric acid could be utilized by C. necator with a stimulating effect on C. necator growth. Consequently, the lignin degradation pathway of C. necator was reconstructed based on genomic information and literature. The efficient conversion of intermediate catechol to downstream products of cis,cis-muconate or 2-hydroxymuconate-6-semialdehyde may help improve the robustness of C. necator to benzoic acid and p-coumaric acid as well as improve PHB productivity. PMID:24904560

  2. Simultaneous degradation of chloro- and methyl-substituted aromatic compounds: competition between Pseudomonas strains using the ortho and meta pathway or the ortho pathway exclusively

    Microsoft Academic Search

    A. C. Franck-Mokroß; E. Schmidt

    1998-01-01

    Pseudomonas sp. D7-4 and Pseudomonas sp. B13 FR1(pFRC20P) degraded mixtures of chloro- and methyl-substituted benzoates exclusively via an extended ortho pathway, whereas in Pseudomonas putida WR201 both ortho and meta fission were induced by mixtures of 3-chloro- and 3-methylbenzoate or even by 3-chlorobenzoate alone. The competition behaviour\\u000a of these strains was compared in batch and in chemostat cultures. Despite misrouting

  3. Loss of autophagy in the central nervous system causes neurodegeneration in mice

    Microsoft Academic Search

    Masaaki Komatsu; Satoshi Waguri; Tomoki Chiba; Shigeo Murata; Jun-Ichi Iwata; Isei Tanida; Takashi Ueno; Masato Koike; Yasuo Uchiyama; Eiki Kominami; Keiji Tanaka

    2006-01-01

    Protein quality-control, especially the removal of proteins with aberrant structures, has an important role in maintaining the homeostasis of non-dividing neural cells. In addition to the ubiquitin-proteasome system, emerging evidence points to the importance of autophagy-the bulk protein degradation pathway involved in starvation-induced and constitutive protein turnover-in the protein quality-control process. However, little is known about the precise roles of

  4. Enhancement of radiosensitivity by proteasome inhibition: Implications for a role of NF-?B

    Microsoft Academic Search

    Suzanne M Russo; Joel E Tepper; Albert S Baldwin; Rong Liu; Julian Adams; Peter Elliott; James C Cusack

    2001-01-01

    Purpose: NF-?B is activated by tumor necrosis factor, certain chemotherapeutic agents, and ionizing radiation, leading to inhibition of apoptosis. NF-?B activation is regulated by phosphorylation of I?B inhibitor molecules that are subsequently targeted for degradation by the ubiquitin-proteasome pathway. PS-341 is a specific and selective inhibitor of the proteasome that inhibits NF-?B activation and enhances cytotoxic effects of chemotherapy in

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

    Microsoft Academic Search

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

    2005-01-01

    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-1?, is hydroxylated on specific proline residues to target HIF-1? 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

  6. The Physical Association of Multiple Molecular Chaperone Proteins with Mutant p53 Is Altered by Geldanamycin, an hsp90Binding Agent

    Microsoft Academic Search

    LUKE WHITESELL; PATRICK D. SUTPHIN; ELIZABETH J. PULCINI; JESSE D. MARTINEZ; PAUL H. COOK

    1998-01-01

    Wild-type p53 is a short-lived protein which turns over very rapidly via selective proteolysis in the ubiquitin- proteasome pathway. Most p53 mutations, however, encode for protein products which display markedly increased intracellular levels and are associated with positive tumor-promoting activity. The mechanism by which mutation leads to impairment of ubiquitination and proteasome-mediated degradation is unknown, but it has been noted

  7. Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway

    PubMed Central

    Hermkens, Dorien; Wlodarski, Marcin W.; Da Costa, Lydie; MacInnes, Alyson W.

    2015-01-01

    Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes. PMID:26132763

  8. Ribosomal Protein Mutations Result in Constitutive p53 Protein Degradation through Impairment of the AKT Pathway.

    PubMed

    Antunes, Ana T; Goos, Yvonne J; Pereboom, Tamara C; Hermkens, Dorien; Wlodarski, Marcin W; Da Costa, Lydie; MacInnes, Alyson W

    2015-07-01

    Mutations in ribosomal protein (RP) genes can result in the loss of erythrocyte progenitor cells and cause severe anemia. This is seen in patients with Diamond-Blackfan anemia (DBA), a pure red cell aplasia and bone marrow failure syndrome that is almost exclusively linked to RP gene haploinsufficiency. While the mechanisms underlying the cytopenia phenotype of patients with these mutations are not completely understood, it is believed that stabilization of the p53 tumor suppressor protein may induce apoptosis in the progenitor cells. In stark contrast, tumor cells from zebrafish with RP gene haploinsufficiency are unable to stabilize p53 even when exposed to acute DNA damage despite transcribing wild type p53 normally. In this work we demonstrate that p53 has a limited role in eliciting the anemia phenotype of zebrafish models of DBA. In fact, we find that RP-deficient embryos exhibit the same normal p53 transcription, absence of p53 protein, and impaired p53 response to DNA damage as RP haploinsufficient tumor cells. Recently we reported that RP mutations suppress activity of the AKT pathway, and we show here that this suppression results in proteasomal degradation of p53. By re-activating the AKT pathway or by inhibiting GSK-3, a downstream modifier that normally represses AKT signaling, we are able to restore the stabilization of p53. Our work indicates that the anemia phenotype of zebrafish models of DBA is dependent on factors other than p53, and may hold clinical significance for both DBA and the increasing number of cancers revealing spontaneous mutations in RP genes. PMID:26132763

  9. Degradation pathways of 4-methylmethcathinone in alkaline solution and stability of methcathinone analogs in various pH solutions.

    PubMed

    Tsujikawa, Kenji; Mikuma, Toshiyasu; Kuwayama, Kenji; Miyaguchi, Hajime; Kanamori, Tatsuyuki; Iwata, Yuko T; Inoue, Hiroyuki

    2012-07-10

    Analogs of methcathinone (MC), a psychoactive stimulant, are in circulation all over the world. These analogs have been assumed to be unstable in alkaline solutions, as is MC itself. The aims of this study were: (i) to identify the degradation products of 4-methylmethcathinone (4-MMC), a typical MC analog, in solution at pH 12 and to determine the degradation pathway, (ii) to investigate the effects of antioxidants such as l-ascorbic acid and sodium sulfite on the degradation of 4-MMC, and (iii) to investigate the stability of seven MC analogs (4-MMC, 4-, 3-, or 2-fluoromethcathinone, 4-methoxymethcathinone, N-ethylcathinone, and N,N-dimethylcathinone) in solutions at different pHs.1-(4-Methylphenyl)-1,2-propanedione (MPPD), 4-methylbenzoic acid (MBA), N,4-dimethylbenzamide (DMBA), and N-acetyl-4-MMC (N-Ac-4-MMC) were identified as the degradation products of 4-MMC in pH 12 solution by gas chromatography-mass spectrometry. There are two degradation pathways for 4-MMC as follows: (a) 4-MMC?MPPD?MBA?DMBA and (b) 4-MMC?N-Ac-4-MMC. Oxidants such as dissolved oxygen were presumed to be involved in this degradation based on the suppressive effects generated by the addition of antioxidants. All of the seven MC analogs tested were stable in acidic (pH 4) solution but degraded in neutral-to-basic solutions. Their degradation rates increased with increasing pH, and varied with their chemical structures. These findings will be very useful for not only forensic analysis but also future pharmacokinetic analysis. PMID:22402273

  10. Facilitated Tau Degradation by USP14 Aptamers via Enhanced Proteasome Activity

    PubMed Central

    Lee, Jung Hoon; Shin, Seung Kyun; Jiang, Yanxialei; Choi, Won Hoon; Hong, Chaesun; Kim, Dong-Eun; Lee, Min Jae

    2015-01-01

    The ubiquitin-proteasome system (UPS) is the primary mechanism by which intracellular proteins, transcription factors, and many proteotoxic proteins with aggregation-prone structures are degraded. The UPS is reportedly downregulated in various neurodegenerative disorders, with increased proteasome activity shown to be beneficial in many related disease models. Proteasomes function under tonic inhibitory conditions, possibly via the ubiquitin chain-trimming function of USP14, a proteasome-associated deubiquitinating enzyme (DUB). We identified three specific RNA aptamers of USP14 (USP14-1, USP14-2, and USP14-3) that inhibited its deubiquitinating activity. The nucleotide sequences of these non-cytotoxic USP14 aptamers contained conserved GGAGG motifs, with G-rich regions upstream, and similar secondary structures. They efficiently elevated proteasomal activity, as determined by the increased degradation of small fluorogenic peptide substrates and physiological polyubiquitinated Sic1 proteins. Additionally, proteasomal degradation of tau proteins was facilitated in the presence of the UPS14 aptamers in vitro. Our results indicate that these novel inhibitory UPS14 aptamers can be used to enhance proteasome activity, and to facilitate the degradation of proteotoxic proteins, thereby protecting cells from various neurodegenerative stressors. PMID:26041011

  11. The molecular components of the extracellular protein-degradation pathways of the ectomycorrhizal fungus Paxillus involutus

    PubMed Central

    Shah, Firoz; Rineau, Francois; Canbäck, Björn; Johansson, Tomas; Tunlid, Anders

    2013-01-01

    Proteins contribute to a major part of the organic nitrogen (N) in forest soils. This N is mobilized and becomes available to trees as a result of the depolymerizing activities of symbiotic ectomycorrhizal fungi. The mechanisms by which these fungi depolymerize proteins and assimilate the released N are poorly characterized. Biochemical analysis and transcriptome profiling were performed to examine the proteolytic machinery and the uptake system of the ectomycorrhizal basidiomycete Paxillus involutus during the assimilation of organic N from various protein sources and extracts of organic matter. All substrates induced secretion of peptidase activity with an acidic pH optimum, mostly contributed by aspartic peptidases. The peptidase activity was transiently repressed by ammonium. Transcriptional analysis revealed a large number of extracellular endo- and exopeptidases. The expression levels of these peptidases were regulated in parallel with transporters and enzymes involved in the assimilation and metabolism of the released peptides and amino acids. For the first time the molecular components of the protein degradation pathways of an ectomycorrhizal fungus are described. The data suggest that the transcripts encoding these components are regulated in response to the chemical properties and the availability of the protein substrates. PMID:23902518

  12. Analysis of hydroxycinnamic acid degradation in Agrobacterium fabrum reveals a coenzyme A-dependent, beta-oxidative deacetylation pathway.

    PubMed

    Campillo, Tony; Renoud, Sébastien; Kerzaon, Isabelle; Vial, Ludovic; Baude, Jessica; Gaillard, Vincent; Bellvert, Floriant; Chamignon, Cécile; Comte, Gilles; Nesme, Xavier; Lavire, Céline; Hommais, Florence

    2014-06-01

    The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-?-hydroxypropionyl-CoA, 4-hydroxy-3-methoxyphenyl-?-ketopropionyl-CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-?-ketopropionic acid (HMPKP)-CoA ?-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent ?-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials. PMID:24657856

  13. Analysis of Hydroxycinnamic Acid Degradation in Agrobacterium fabrum Reveals a Coenzyme A-Dependent, Beta-Oxidative Deacetylation Pathway

    PubMed Central

    Campillo, Tony; Renoud, Sébastien; Kerzaon, Isabelle; Vial, Ludovic; Baude, Jessica; Gaillard, Vincent; Bellvert, Floriant; Chamignon, Cécile; Comte, Gilles; Lavire, Céline; Hommais, Florence

    2014-01-01

    The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-?-hydroxypropionyl–CoA, 4-hydroxy-3-methoxyphenyl-?-ketopropionyl–CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-?-ketopropionic acid (HMPKP)–CoA ?-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent ?-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials. PMID:24657856

  14. Modulation of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway.

    PubMed Central

    Falnes, P O; Olsnes, S

    1998-01-01

    The enzymatically active A-fragment of diphtheria toxin enters the cytosol of sensitive cells where it inhibits protein synthesis by inactivating elongation factor 2 (EF-2). We have constructed a number of diphtheria toxin mutants that are degraded by the N-end rule pathway in Vero cells, and that display a wide range of intracellular stabilities. The degradation could be inhibited by the proteasome inhibitor lactacystin, indicating that the proteasome is responsible for N-end rule-mediated degradation in mammalian cells. Previously, the N-end rule has been investigated by studying the co-translational degradation of intracellularly expressed beta-galactosidase. Our work shows that a mature protein entering the cytosol from the exterior can also be degraded by the N-end rule pathway with a similar, but not identical specificity to that previously found. We found a correlation between the intracellular stability of the mutants and their toxic effect on cells, thus demonstrating a novel manner of modulating the toxicity of a protein toxin. The data also indicate that the inactivation of EF-2 is the rate-limiting step in the intoxication process. PMID:9430652

  15. Molecular Characterization of the 4-Hydroxyphenylacetate Catabolic Pathway ofEscherichia coliW: Engineering a Mobile Aromatic Degradative Cluster

    Microsoft Academic Search

    MARIA A. PRIETO; EDUARDO DIAZ

    1996-01-01

    Wehavedeterminedandanalyzedthenucleicacidsequenceofa14,855-bpregionthatcontainsthecomplete gene cluster encoding the 4-hydroxyphenylacetic acid (4-HPA) degradative pathway of Escherichia coli W (ATCC 11105). This catabolic pathway is composed by 11 genes, i.e., 8 enzyme-encoding genes distributed in two putative operons,hpaBC(4-HPA hydroxylase operon) andhpaGEDFHI(meta-cleavage operon); 2 regula- tory genes, hpaR and hpaA; and the gene, hpaX, that encodes a protein related to the superfamily of trans- membrane facilitators

  16. Coxsackievirus B5 induced apoptosis of HeLa cells: Effects on p53 and SUMO

    SciTech Connect

    Gomes, Rogerio, E-mail: roggomes@usp.b [Department of Cell Biology, University of Sao Paulo School of Medicine at Ribeirao Preto, Av. dos Bandeirantes, 3900, 14049-900, SP (Brazil); Guerra-Sa, Renata [Federal University of Ouro Preto, MG (Brazil); Arruda, Eurico [Department of Cell Biology, University of Sao Paulo School of Medicine at Ribeirao Preto, Av. dos Bandeirantes, 3900, 14049-900, SP (Brazil); Virology Research Center, University of Sao Paulo School of Medicine at Ribeirao Preto, SP (Brazil)

    2010-01-20

    Coxsackievirus B5 (CVB5), a human enterovirus of the family Picornaviridae, is a frequent cause of acute and chronic human diseases. The pathogenesis of enteroviral infections is not completely understood, and the fate of the CVB5-infected cell has a pivotal role in this process. We have investigated the CVB5-induced apoptosis of HeLa cells and found that it happens by the intrinsic pathway by a mechanism dependent on the ubiquitin-proteasome system, associated with nuclear aggregation of p53. Striking redistribution of both SUMO and UBC9 was noted at 4 h post-infection, simultaneously with a reduction in the levels of the ubiquitin-ligase HDM2. Taken together, these results suggest that CVB5 infection of HeLa cells elicit the intrinsic pathway of apoptosis by MDM2 degradation and p53 activation, destabilizing protein sumoylation, by a mechanism that is dependent on a functional ubiquitin-proteasome system.

  17. Androgens Upregulate Cdc25C Protein by Inhibiting Its Proteasomal and Lysosomal Degradation Pathways

    PubMed Central

    Muniyan, Sakthivel; Ahmad, Humera; Kumar, Satyendra; Alam, Syed Mahfuzul; Lin, Ming-Fong

    2013-01-01

    Cdc25C is a cell cycle protein of the dual specificity phosphatase family essential for activating the cdk1/Cyclin B1 complex in cells entering into mitosis. Since altered cell cycle is a hallmark of human cancers, we investigated androgen regulation of Cdc25C protein in human prostate cancer (PCa) cells, including androgen-sensitive (AS) LNCaP C-33 cells and androgen-independent (AI) LNCaP C-81 as well as PC-3 cells. In the regular culture condition containing fetal bovine serum (FBS), Cdc25C protein levels were similar in these PCa cells. In a steroid-reduced condition, Cdc25C protein was greatly decreased in AS C-33 cells but not AI C-81 or PC-3 cells. In androgen-treated C-33 cells, the Cdc25C protein level was greatly elevated, following a dose- and a time-dependent manner, correlating with increased cell proliferation. This androgen effect was blocked by Casodex, an androgen receptor blocker. Nevertheless, epidermal growth factor (EGF), a growth stimulator of PCa cells, could only increase Cdc25C protein level by about 1.5-fold. Altered expression of Cdc25C in C-33 cells and PC-3 cells by cDNA and/or shRNA transfection is associated with the corresponding changes of cell growth and Cyclin B1 protein level. Actinomycin D and cycloheximide could only partially block androgen-induced Cdc25C protein level. Treatments with both proteasomal and lysosomal inhibitors resulted in elevated Cdc25C protein levels. Immunoprecipitation revealed that androgens reduced the ubiquitination of Cdc25C proteins. These results show for the first time that Cdc25C protein plays a role in regulating PCa cell growth, and androgen treatments, but not EGF, greatly increase Cdc25C protein levels in AS PCa cells, which is in part by decreasing its degradation. These results can lead to advanced PCa therapy via up-regulating the degradation pathways of Cdc25C protein. PMID:23637932

  18. Chemical intervention in bacterial lignin degradation pathways: Development of selective inhibitors for intradiol and extradiol catechol dioxygenases.

    PubMed

    Sainsbury, Paul D; Mineyeva, Yelena; Mycroft, Zoe; Bugg, Timothy D H

    2015-06-01

    Bacterial lignin degradation could be used to generate aromatic chemicals from the renewable resource lignin, provided that the breakdown pathways can be manipulated. In this study, selective inhibitors of enzymatic steps in bacterial degradation pathways were developed and tested for their effects upon lignin degradation. Screening of a collection of hydroxamic acid metallo-oxygenase inhibitors against two catechol dioxygenase enzymes, protocatechuate 3,4-dioxygenase (3,4-PCD) and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB), resulted in the identification of selective inhibitors D13 for 3,4-PCD (IC50 15?M) and D3 for MhpB (IC50 110?M). Application of D13 to Rhodococcus jostii RHA1 in minimal media containing ferulic acid led to the appearance of metabolic precursor protocatechuic acid at low concentration. Application of 1mM disulfiram, an inhibitor of mammalian aldehyde dehydrogenase, to R. jostii RHA1, gave rise to 4-carboxymuconolactone on the ?-ketoadipate pathway, whereas in Pseudomonas fluorescens Pf-5 disulfiram treatment gave rise to a metabolite found to be glycine betaine aldehyde. PMID:25984987

  19. Porcine arterivirus activates the NF-{kappa}B pathway through I{kappa}B degradation

    SciTech Connect

    Lee, Sang-Myeong [Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri-Columbia, MO 65211 (United States); Kleiboeker, Steven B. [Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri-Columbia, MO 65211 (United States) and Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri-Columbia, MO 65211 (United States)]. E-mail: KleiboekerS@Missouri.edu

    2005-11-10

    Nuclear factor-kappaB (NF-{kappa}B) is a critical regulator of innate and adaptive immune function as well as cell proliferation and survival. The present study demonstrated for the first time that a virus belonging to the Arteriviridae family activates NF-{kappa}B in MARC-145 cells and alveolar macrophages. In porcine reproductive and respiratory syndrome virus (PRRSV)-infected cells, NF-{kappa}B activation was characterized by translocation of NF-{kappa}B from the cytoplasm to the nucleus, increased DNA binding activity, and NF-{kappa}B-regulated gene expression. NF-{kappa}B activation was increased as PRRSV infection progressed and in a viral dose-dependent manner. UV-inactivation of PRRSV significantly reduced the level of NF-{kappa}B activation. Degradation of I{kappa}B protein was detected late in PRRSV infection, and overexpression of the dominant negative form of I{kappa}B{alpha} (I{kappa}B{alpha}DN) significantly suppressed NF-{kappa}B activation induced by PRRSV. However, I{kappa}B{alpha}DN did not affect viral replication and viral cytopathic effect. PRRSV infection induced oxidative stress in cells by generating reactive oxygen species (ROS), and antioxidants inhibited NF-{kappa}B DNA binding activity in PRRSV-infected cells, suggesting ROS as a mechanism by which NF-{kappa}B was activated by PRRSV infection. Moreover, NF-{kappa}B-dependent expression of matrix metalloproteinase (MMP)-2 and MMP-9 was observed in PRRSV-infected cells, an observation which implies that NF-{kappa}B activation is a biologically significant aspect of PRRSV pathogenesis. The results presented here provide a basis for understanding molecular pathways of pathology and immune evasion associated with disease caused by PRRSV.

  20. Structure of 2,6-dihydroxypyridine 3-hydroxylase from a nicotine-degrading pathway.

    PubMed

    Treiber, Nora; Schulz, Georg E

    2008-05-23

    The enzyme 2,6-dihydroxypyridine-3-hydroxylase catalyzes the sixth step of the nicotine degradation pathway in Arthrobacter nicotinovorans. The enzyme was produced in Escherichia coli, purified and crystallized. The crystal structure was solved at 2.6 A resolution, revealing a significant structural relationship with the family of FAD-dependent aromatic hydroxylases, but essentially no sequence homology. The structure was aligned with those of the established family members, showing that the FAD molecules are bound at virtually identical locations. The reported enzyme is a dimer like most other family members, but its dimerization contact differs from the others. The binding position of NAD(P)H to this enzyme family is not clear. Since the reported enzyme accepts only NADH for flavin reduction in contrast to the other established members using NADPH, we searched through the structural alignment and found an indication for the position of the 2'-phosphate of NADPH that is in general agreement with mutational studies on a related enzyme, but contradicts a crystal soaking experiment. Using a bound glycerol molecule and the known substrate positions of three related enzymes as a guide, the substrate 2,6-dihydroxypyridine was placed into the active center. The access to the binding site is discussed. The new active center geometry introduces constraints that render some reaction scenarios more likely than others. It suggests that flavin is reduced at its out-position and then drawn into its in-position, where it binds molecular oxygen. The geometry is consistent with the proposal that peroxy-flavin is protonated by the solvent to yield the electrophilic hydroperoxy-flavin. The substrate is activated by two buried histidines but there is no appropriate base to store the surplus proton of the hydroxylated carbon atom. The implications of this problem are discussed. PMID:18440023

  1. Differential ubiquitination and degradation of huntingtin fragments modulated by ubiquitin-protein ligase E3A

    PubMed Central

    Bhat, Kavita P.; Yan, Sen; Wang, Chuan-En; Li, Shihua; Li, Xiao-Jiang

    2014-01-01

    Ubiquitination of misfolded proteins, a common feature of many neurodegenerative diseases, is mediated by different lysine (K) residues in ubiquitin and alters the levels of toxic proteins. In Huntington disease, polyglutamine expansion causes N-terminal huntingtin (Htt) to misfold, inducing neurodegeneration. Here we report that shorter N-terminal Htt fragments are more stable than longer fragments and find differential ubiquitination via K63 of ubiquitin. Aging decreases proteasome-mediated Htt degradation, at the same time increasing K63-mediated ubiquitination and subsequent Htt aggregation in HD knock-in mice. The association of Htt with the K48-specific E3 ligase, Ube3a, is decreased in aged mouse brain. Overexpression of Ube3a in HD mouse brain reduces K63-mediated ubiquitination and Htt aggregation, enhancing its degradation via the K48 ubiquitin–proteasome system. Our findings suggest that aging-dependent Ube3a levels result in differential ubiquitination and degradation of Htt fragments, thereby contributing to the age-related neurotoxicity of mutant Htt. PMID:24706802

  2. HUWE1 ubiquitinates MyoD and targets it for proteasomal degradation

    SciTech Connect

    Noy, Tahel; Suad, Oded [Cancer and Vascular Biology Research Center, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa 31096 (Israel)] [Cancer and Vascular Biology Research Center, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa 31096 (Israel); Taglicht, Daniel [Proteologics Ltd., Weizmann Science Park, Rehovot 76704 (Israel)] [Proteologics Ltd., Weizmann Science Park, Rehovot 76704 (Israel); Ciechanover, Aaron, E-mail: c_tzachy@netvision.net.il [Cancer and Vascular Biology Research Center, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa 31096 (Israel)] [Cancer and Vascular Biology Research Center, The Rappaport Faculty of Medicine and Research Institute, Technion-Israel Institute of Technology, Haifa 31096 (Israel)

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer HUWE1 ubiquitinates MyoD in vitro and in cells. Black-Right-Pointing-Pointer The ubiquitination by HUWE1 targets MyoD for proteasomal degradation. Black-Right-Pointing-Pointer HUWE1 can modify MyoD on its N-terminal residue. -- Abstract: MyoD is a tissue-specific transcriptional activator that acts as a master switch for muscle development. It activates a broad array of muscle-specific genes, which leads to conversion of proliferating myoblasts into mature myotubes. The ubiquitin proteasome system (UPS) plays an important role in controlling MyoD. Both its N-terminal residue and internal lysines can be targeted by ubiquitin, and both modifications appear to direct it for proteasomal degradation. The protein is short-lived and has a half-life of {approx}45 min in different cells. It was reported that MyoD can be ubiquitinated by MAFbx/AT-1, but accumulating lines of experimental evidence showed that other ligase(s) may also participate in its targeting. Here we describe the involvement of HUWE1 in the ubiquitination and proteasomal degradation of MyoD. Furthermore, we show that the ligase can ubiquitinate the protein in its N-terminal residue.

  3. The poly(A)-dependent degradation pathway of rpsO mRNA is primarily mediated by RNase R

    PubMed Central

    Andrade, José M.; Hajnsdorf, Eliane; Régnier, Philippe; Arraiano, Cecília M.

    2009-01-01

    Polyadenylation is an important factor controlling RNA degradation and RNA quality control mechanisms. In this report we demonstrate for the first time that RNase R has in vivo affinity for polyadenylated RNA and can be a key enzyme involved in poly(A) metabolism. RNase II and PNPase, two major RNA exonucleases present in Escherichia coli, could not account for all the poly(A)-dependent degradation of the rpsO mRNA. RNase II can remove the poly(A) tails but fails to degrade the mRNA as it cannot overcome the RNA termination hairpin, while PNPase plays only a modest role in this degradation. We now demonstrate that in the absence of RNase E, RNase R is the relevant factor in the poly(A)-dependent degradation of the rpsO mRNA. Moreover, we have found that the RNase R inactivation counteracts the extended degradation of this transcript observed in RNase II-deficient cells. Elongated rpsO transcripts harboring increasing poly(A) tails are specifically recognized by RNase R and strongly accumulate in the absence of this exonuclease. The 3? oligo(A) extension may stimulate the binding of RNase R, allowing the complete degradation of the mRNA, as RNase R is not susceptible to RNA secondary structures. Moreover, this regulation is shown to occur despite the presence of PNPase. Similar results were observed with the rpsT mRNA. This report shows that polyadenylation favors in vivo the RNase R-mediated pathways of RNA degradation. PMID:19103951

  4. Pathways of reductive degradation of crystal violet in wastewater using free-strain Burkholderia vietnamiensis C09V.

    PubMed

    Gan, Li; Cheng, Ying; Palanisami, Thavamani; Chen, Zuliang; Megharaj, Mallavarapu; Naidu, Ravendra

    2014-09-01

    A new strain isolated from activated sludge and identified as Burkholderia vietnamiensis C09V was used to biodegrade crystal violet (CV) from aqueous solution. To understand the degradation pathways of CV, batch experiments showed that the degradation using B. vietnamiensis C09V significantly depended on conditions such as pH, initial dye concentration and media components, carbon and nitrogen sources. Acceleration in the biodegradation of CV was observed in presence of metal ions such as Cd and Mn. More than 98.86C of CV (30 mg l(-1)) was degraded within 42 h at pH 5 and 30 °C. The biodegradation kinetics of CV corresponded to the pseudo first-order rate model with a rate constant of 0.046 h(-1). UV-visible and Fourier transform infrared spectroscopy (FTIR) were used to identify degradation metabolites. Which further confirmed by LC-MS analysis, indicating that CV was biodegraded to N,N-dimethylaminophenol and Michler's ketone prior to these intermediates being further degraded. Finally, the ability of B. vietnamiensis C09V to remove CV in wastewater was demonstrated. PMID:24862483

  5. Phagocytic Receptor CED1 Initiates a Signaling Pathway for Degrading Engulfed Apoptotic Cells

    Microsoft Academic Search

    Xiaomeng Yu; Nan Lu; Zheng Zhou

    2008-01-01

    Apoptotic cells in animals are engulfed by phagocytic cells and subsequently degraded inside phagosomes. To study the mechanisms controlling the degradation of apoptotic cells, we developed time-lapse imaging protocols in developing Caenorhabditis elegans embryos and established the temporal order of multiple events during engulfment and phagosome maturation. These include sequential enrichment on phagocytic membranes of phagocytic receptor cell death abnormal

  6. REACTION PATHWAY OF THE DIKETONITRILE DEGRADATE OF ISOXAFLUTOLE (BALANCE(TM)) WITH HYPOCHLORITE IN WATER

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Isoxaflutole (IXF; Balance(TM)) belongs to the new class of isoxazole herbicides. Isoxaflutole has a very short half-life in soil and rapidly degrades to a stable and phytotoxic degradate, diketonitrile (DKN). DKN was previously discovered to rapidly react with hypochlorite (OCl-) in tap water, yie...

  7. Prediction of CL-20 chemical degradation pathways, theoretical and experimental evidence for dependence on competing modes of reaction.

    PubMed

    Qasim, M; Fredrickson, H; Honea, P; Furey, J; Leszczynski, J; Okovytyy, S; Szecsody, J; Kholod, Y

    2005-10-01

    Highest occupied and lowest unoccupied molecular orbital energies, formation energies, bond lengths and FTIR spectra all suggest competing CL-20 degradation mechanisms. This second of two studies investigates recalcitrant, toxic, aromatic CL-20 intermediates that absorb from 370 to 430 nm. Our earlier study (Struct. Chem., 15, 2004) revealed that these intermediates were formed at high OH(-) concentrations via the chemically preferred pathway of breaking the C-C bond between the two cyclopentanes, thereby eliminating nitro groups, forming conjugated pi bonds, and resulting in a pyrazine three-ring aromatic intermediate. In attempting to find and make dominant a more benign CL-20 transformation pathway, this current research validates hydroxylation results from both studies and examines CL-20 transformations via photo-induced free radical reactions. This article discusses CL-20 competing modes of degradation revealed through: computational calculation; UV/VIS and SF spectroscopy following alkaline hydrolysis; and photochemical irradiation to degrade CL-20 and its byproducts at their respective wavelengths of maximum absorption. PMID:16272046

  8. Degradation pathway and kinetics of 1-alkyl-3-methylimidazolium bromides oxidation in an ultrasonic nanoscale zero-valent iron/hydrogen peroxide system.

    PubMed

    Zhou, Haimei; Shen, Yuanyuan; Lv, Ping; Wang, Jianji; Li, Pu

    2015-03-01

    Fenton and Fenton-like oxidation has been already demonstrated to be efficient for the degradation of imidazolium ionic liquids (ILs), but little is known for their degradation pathway and kinetics in such systems. In this work, degradation pathway and kinetics of 1-alkyl-3-methylimidazolium bromides ([Cnmim]Br, n=2, 4, 6, 8, and 10) were investigated in an ultrasound nanoscale zero-valent iron/hydrogen peroxide (US-nZVI/H2O2) system. For this purpose, 1-butyl-3-methylimidazolium bromide ([C4mim]Br) was used as a representative ionic liquid to optimize pH value, nZVI dose, and H2O2 concentration for the degradation reaction. Then, the degradation kinetics of [Cnmim]Br was investigated under optimal conditions, and their degradation intermediates were monitored by gas chromatography-mass spectrometry (GC-MS). It was shown that the degradation of [Cnmim]Br in such a heterogeneous Fenton-like system could be described by a second order kinetic model, and a number of intermediate products were detected. Based on these intermediate products, detailed pathways were proposed for the degradation of [Cnmim]Br in the ultrasound-assisted nZVI/H2O2 system. These findings may be useful for the better understanding of degradation mechanism of the imidazolium ILs in aqueous solutions. PMID:25463239

  9. Ubiquitin-specific protease 14 modulates degradation of cellular prion protein.

    PubMed

    Homma, Takujiro; Ishibashi, Daisuke; Nakagaki, Takehiro; Fuse, Takayuki; Mori, Tsuyoshi; Satoh, Katsuya; Atarashi, Ryuichiro; Nishida, Noriyuki

    2015-01-01

    Prion diseases are fatal neurodegenerative disorders characterized by the accumulation of prion protein (PrP(C)). To date, there is no effective treatment for the disease. The accumulated PrP, termed PrP(Sc), forms amyloid fibrils and could be infectious. It has been suggested that PrP(Sc) is abnormally folded and resistant to proteolytic degradation, and also inhibits proteasomal functions in infected cells, thereby inducing neuronal death. Recent work indicates that the ubiquitin-proteasome system is involved in quality control of PrP(C). To reveal the significance of prion protein ubiqitination, we focused on ubiquitin-specific protease 14 (USP14), a deubiqutinating enzyme that catalyzes trimming of polyubiquitin chains and plays a role in regulation of proteasomal processes. Results from the present study showed that treatment with a selective inhibitor of USP14 reduced PrP(C), as well as PrP(Sc), levels in prion-infected neuronal cells. Overexpression of the dominant negative mutant form of USP14 reduced PrP(Sc), whereas wildtype USP14 increased PrP(Sc) in prion-infected cells. These results suggest that USP14 prevents degradation of both normal and abnormal PrP. Collectively, a better understanding about the regulation of PrP(Sc) clearance caused by USP14 might contribute greatly to the development of therapeutic strategies for prion diseases. PMID:26061634

  10. Ubiquitin-specific protease 14 modulates degradation of cellular prion protein

    PubMed Central

    Homma, Takujiro; Ishibashi, Daisuke; Nakagaki, Takehiro; Fuse, Takayuki; Mori, Tsuyoshi; Satoh, Katsuya; Atarashi, Ryuichiro; Nishida, Noriyuki

    2015-01-01

    Prion diseases are fatal neurodegenerative disorders characterized by the accumulation of prion protein (PrPC). To date, there is no effective treatment for the disease. The accumulated PrP, termed PrPSc, forms amyloid fibrils and could be infectious. It has been suggested that PrPSc is abnormally folded and resistant to proteolytic degradation, and also inhibits proteasomal functions in infected cells, thereby inducing neuronal death. Recent work indicates that the ubiquitin-proteasome system is involved in quality control of PrPC. To reveal the significance of prion protein ubiqitination, we focused on ubiquitin-specific protease 14 (USP14), a deubiqutinating enzyme that catalyzes trimming of polyubiquitin chains and plays a role in regulation of proteasomal processes. Results from the present study showed that treatment with a selective inhibitor of USP14 reduced PrPC, as well as PrPSc, levels in prion-infected neuronal cells. Overexpression of the dominant negative mutant form of USP14 reduced PrPSc, whereas wildtype USP14 increased PrPSc in prion-infected cells. These results suggest that USP14 prevents degradation of both normal and abnormal PrP. Collectively, a better understanding about the regulation of PrPSc clearance caused by USP14 might contribute greatly to the development of therapeutic strategies for prion diseases. PMID:26061634

  11. [Study on the degradation of AO7 by UV/K2S2O8, system: kinetics and pathways].

    PubMed

    Chen, Xiao-yang; Wang, Wei-ping; Zhu, Feng-xiang; Hong, Chun-lai; Xue, Zhi-yong

    2010-07-01

    The UV photolysis of S2O8(2-) is a novel advanced oxidation technologies (AOTs), which leads to the formation of strong oxidizing radicals, sulfate radicals (SO4(*-)). The effect of oxidant K2S2O8 concentration, the initial pH of solution and various inorganic anions (H2PO4-, HCO3-, NO3- and Cl-) were investigated using AO7, a kind of azo dye, as a model pollutant. The degradation kinetics of AO7 followed pseudo first-order kinetics and reaction rates related to PMS concentrations. When the initial concentration of AO7 was 0.14 mmol/L, the optimal molar ratio of oxidant K2S2O8 to pollutants AO7 was 20. The effect of initial pH had great effect on the AO7 degradation rate during UV/K2S2O8 system. Increasing system pH results in the formation of *OH from SO4(*-). The effects of four inorganic anions (H2PO4-, HCO3-, NO3- and Cl-) all had some negative effect on the degradation of AO7. Based on the results of GC/MS, three main intermediates (2-naphthalenol, coumarin and 1,2-benzenedicarboxylic acid) were identified, thus the degradation pathway for SO4(*-) induced by UV/K2S2O8 was proposed accordingly. PMID:20825022

  12. Novel Pathway for the Degradation of 2-Chloro-4-Nitrobenzoic Acid by Acinetobacter sp. Strain RKJ12?†

    PubMed Central

    Prakash, Dhan; Kumar, Ravi; Jain, R. K.; Tiwary, B. N.

    2011-01-01

    The organism Acinetobacter sp. RKJ12 is capable of utilizing 2-chloro-4-nitrobenzoic acid (2C4NBA) as a sole source of carbon, nitrogen, and energy. In the degradation of 2C4NBA by strain RKJ12, various metabolites were isolated and identified by a combination of chromatographic, spectroscopic, and enzymatic activities, revealing a novel assimilation pathway involving both oxidative and reductive catabolic mechanisms. The metabolism of 2C4NBA was initiated by oxidative ortho dehalogenation, leading to the formation of 2-hydroxy-4-nitrobenzoic acid (2H4NBA), which subsequently was metabolized into 2,4-dihydroxybenzoic acid (2,4-DHBA) by a mono-oxygenase with the concomitant release of chloride and nitrite ions. Stoichiometric analysis indicated the consumption of 1 mol O2 per conversion of 2C4NBA to 2,4-DHBA, ruling out the possibility of two oxidative reactions. Experiments with labeled H218O and 18O2 indicated the involvement of mono-oxygenase-catalyzed initial hydrolytic dechlorination and oxidative denitration mechanisms. The further degradation of 2,4-DHBA then proceeds via reductive dehydroxylation involving the formation of salicylic acid. In the lower pathway, the organism transformed salicylic acid into catechol, which was mineralized by the ortho ring cleavage catechol-1,2-dioxygenase to cis, cis-muconic acid, ultimately forming tricarboxylic acid cycle intermediates. Furthermore, the studies carried out on a 2C4NBA? derivative and a 2C4NBA+ transconjugant demonstrated that the catabolic genes for the 2C4NBA degradation pathway possibly reside on the ?55-kb transmissible plasmid present in RKJ12. PMID:21803909

  13. Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway

    PubMed Central

    2014-01-01

    Background Due to an increasing demand of transportation fuels, a lower availability of cheap crude oil and a lack of sustainability of fossil fuels, a gradual shift from petroleum based fuels towards alternative and renewable fuel resources will be required in the near future. Fatty acid ethyl esters (FAEEs) have properties similar to current crude diesel and could therefore form an important contribution to the development of sustainable transportation fuels in future. It is important to develop novel cell factories for efficient production of FAEEs and their precursors. Results Here, a Saccharomyces cerevisiae cell factory expressing a heterologous wax ester synthase (ws2) from Marinobacter hydrocarbonoclasticus was used to produce FAEEs from ethanol and acyl-coenzyme A (acyl-CoA). The production of acyl-CoA requires large amounts of NADPH and acetyl-CoA. Therefore, two metabolic engineering strategies for improved provision of NADPH and acetyl-CoA were evaluated. First, the ethanol degradation pathway was employed to re-channel carbon flow towards the synthesis of acetyl-CoA. Therefore, ADH2 and ALD6 encoding, respectively, alcohol dehydrogenase and acetaldehyde dehydrogenase were overexpressed together with the heterologous gene acsSEL641P encoding acetyl-CoA synthetase. The co-overexpression of ADH2, ALD6 and acsSEL641P with ws2 resulted in 408?±?270 ?g FAEE gCDW?1, a 3-fold improvement. Secondly, for the expression of the PHK pathway two genes, xpkA and ack, both descending from Aspergillus nidulans, were co-expressed together with ws2 to catalyze, respectively, the conversion of xylulose-5-phosphate to acetyl phosphate and glyceraldehyde-3-phosphate and acetyl phosphate to acetate. Alternatively, ack was substituted with pta from Bacillus subtilis, encoding phosphotransacetylase for the conversion of acetyl phosphate to acetyl-CoA. Both PHK pathways were additionally expressed in a strain with multiple chromosomally integrated ws2 gene, which resulted in respectively 5100?±?509 and 4670?±?379 ?g FAEE gCDW?1, an up to 1.7-fold improvement. Conclusion Two different strategies for engineering of the central carbon metabolism for efficient provision of acetyl-CoA and NADPH required for fatty acid biosynthesis and hence FAEE production were evaluated and it was found that both the ethanol degradation pathway as well as the phosphoketolase pathway improve the yield of FAEEs. PMID:24618091

  14. Isolation of a novel beta-cypermethrin degrading strain Bacillus subtilis BSF01 and its biodegradation pathway.

    PubMed

    Xiao, Ying; Chen, Shaohua; Gao, Yuanqi; Hu, Wei; Hu, Meiying; Zhong, Guohua

    2015-03-01

    Continuous use of the pyrethroid insecticide beta-cypermethrin (beta-cp) has resulted in serious environmental contamination problems. We report here that a novel bacterial strain BSF01, which was isolated from activated sludge and identified as Bacillus subtilis (collection number: CCTCC AB 2014103), showed high efficiency in degrading beta-cp. Strain BSF01 was able to utilize beta-cp as the sole carbon source for growth and degraded 89.4 % of 50 mg L(-1) beta-cp within 7 days. The optimal conditions for beta-cp degradation were determined to be 34.5 °C, pH 6.7, and inocula amount 0.11 g dry wt L(-1) using response surface methodology. The kinetic parameters q max, K s, and K i were established to be 2.19 day(-1), 76.37 mg L(-1), and 54.14 mg L(-1), respectively. The critical inhibitor concentration was determined to be 64.30 mg L(-1). Seven metabolites were identified by gas chromatography-mass spectrometry. Furthermore, a novel biodegradation pathway for beta-cp was proposed on the basis of analysis of the metabolites. This strain was also capable of degrading a wide range of pyrethroid insecticides including cypermethrin, deltamethrin, cyhalothrin, and beta-cyfluthrin, which similar to beta-cp are hazardous chemicals. Taken together, our results depict the biodegradation pathway of beta-cp and highlight the promising potentials of strain BSF01 in bioremediation of pyrethroid-contaminated environments. PMID:25398281

  15. Ascorbic acid regulates osterix expression in osteoblasts by activation of prolyl hydroxylase and ubiquitination-mediated proteosomal degradation pathway

    PubMed Central

    Xing, Weirong; Pourteymoor, Sheila

    2011-01-01

    Mouse genetic studies reveal that ascorbic acid (AA) is essential for osteoblast (OB) differentiation and that osterix (Osx) was a key downstream target of AA action in OBs. To determine the molecular pathways for AA regulation of Osx expression, we evaluated if AA regulates Osx expression by regulating production and/or actions of local growth factors and extracellular matrix (ECM) proteins. Inhibition of actions of IGFs by inhibitory IGFBP-4, BMPs by noggin, and ECM-mediated integrin signaling by RGD did not block AA effects on Osx expression in OBs. Furthermore, blockade of components of MAPK signaling pathway had no effect on AA-induced Osx expression. Because AA is required for prolyl hydroxylase domain (PHD) activity and because PHD-induced prolyl-hydroxylation targets proteins to proteosomal degradation, we next tested if AA effect on Osx expression involves activation of PHD to hydroxylate and induce ubiquitin-proteosome-mediated degradation of transcriptional repressor(s) of Osx gene. Treatment of OBs with dimethyloxallyl glycine and ethyl 3, 4-dihydroxybenzoate, known inhibitors of PHD, completely blocked AA effect on Osx expression and OB differentiation. Knockdown of PHD2 expression by Lentivirus-mediated shRNA abolished AA-induced Osx induction and alkaline phosphatase activity. Furthermore, treatment of OBs with MG115, inhibitor of proteosomal degradation, completely blocked AA effects on Osx expression. Based on these data, we conclude that AA effect on Osx expression is mediated via a novel mechanism that involves PHD2 and proteosomal degradation of a yet to be identified transcriptional repressor that is independent of BMP, IGF-I, or integrin-mediated signaling in mouse OBs. PMID:21467157

  16. Unraveling the metabolic pathway in Leucosceptrum canum by isolation of new defensive leucosceptroid degradation products and biomimetic model synthesis.

    PubMed

    Luo, Shi-Hong; Hugelshofer, Cedric L; Hua, Juan; Jing, Shu-Xi; Li, Chun-Huan; Liu, Yan; Li, Xiao-Nian; Zhao, Xu; Magauer, Thomas; Li, Sheng-Hong

    2014-12-19

    Seven new leucosceptroid degradation products possessing a C20, C21, or C25 framework, norleucosceptroids D-H (1-5), leucosceptroids P (6), and Q (7), have been isolated from Leucosceptrum canum. Their structures were determined by comprehensive NMR, MS, and single-crystal X-ray diffraction analyses. Discovery of these key intermediates, together with the biomimetic oxidation of a model system, supports the hypothesis that two biosynthetic pathways are operative. Antifeedant activity was observed for compounds 1-3. PMID:25474304

  17. 3-Hydroxyphenylacetic Acid Induces the Burkholderia cenocepacia Phenylacetic Acid Degradation Pathway – Toward Understanding the Contribution of Aromatic Catabolism to Pathogenesis

    PubMed Central

    Imolorhe, Ijeme A.; Cardona, Silvia T.

    2011-01-01

    The phenylacetic acid (PA) degradative pathway is the central pathway by which various aromatic compounds (e.g., styrene) are degraded. Upper pathways for different aromatic compounds converge at common intermediate phenylacetyl-CoA (PA-CoA), which is then metabolized to succinyl-CoA and acetyl-CoA. We previously made a link in Burkholderia cenocepacia between PA degradation and virulence by showing that insertional mutagenesis of paaA and paaE genes, that encode part of a multicomponent oxidase of PA-CoA, results in PA-conditional growth and an attenuated killing phenotype in the Caenorhabditis elegans model of infection. However, insertional mutagenesis of paaK1, which encodes a phenylacetate-CoA ligase, did not result in a PA-conditional growth probably due to the presence of a putative paralog gene paaK2. Recently published crystallographic and enzyme kinetics data comparing the two PaaK ligases showed that PaaK1 is more active than PaaK2 and that the larger binding pocket of PaaK1 can accommodate hydroxylated PA derived molecules such as 3-hydroxyphenylacetic (3-OHPA) acid and 4-hydroxyphenylacetic acid (4-OHPA). The higher activity and broader substrate specificity suggested a more active role in pathogenesis. In this work, we aimed to determine the relevance of PaaK1 activity to the killing ability of B. cenocepacia to C. elegans. Using reporter activity assays, we demonstrate that 3-OHPA activated PA degradation gene promoters of Burkholderia cenocepacia K56-2 in a paaK1-dependent manner, while 4-OHPA had no effect. We compared the pathogenicity of a paaK1 deletion mutant with that of the wild type in C. elegans and observed no differences in the killing ability of the strains. Taken together, these studies suggest that 3-OHPA, but not 4-OHPA, can induce the PA pathway and that this induction is dependent on the paaK1 gene. However, the more active PaaK1 does not play a distinct role in pathogenesis of B. cenocepacia as previously suggested. PMID:22919580

  18. Identification and characterization of the furfural and 5-(hydroxymethyl)furfural degradation pathways of Cupriavidus basilensis HMF14

    PubMed Central

    Koopman, Frank; Wierckx, Nick; de Winde, Johannes H.; Ruijssenaars, Harald J.

    2010-01-01

    The toxic fermentation inhibitors in lignocellulosic hydrolysates pose significant problems for the production of second-generation biofuels and biochemicals. Among these inhibitors, 5-(hydroxymethyl)furfural (HMF) and furfural are specifically notorious. In this study, we describe the complete molecular identification and characterization of the pathway by which Cupriavidus basilensis HMF14 metabolizes HMF and furfural. The identification of this pathway enabled the construction of an HMF and furfural-metabolizing Pseudomonas putida. The genetic information obtained furthermore enabled us to predict the HMF and furfural degrading capabilities of sequenced bacterial species that had not previously been connected to furanic aldehyde metabolism. These results pave the way for in situ detoxification of lignocellulosic hydrolysates, which is a major step toward improved efficiency of utilization of lignocellulosic feedstock. PMID:20194784

  19. Identification and characterization of the furfural and 5-(hydroxymethyl)furfural degradation pathways of Cupriavidus basilensis HMF14.

    PubMed

    Koopman, Frank; Wierckx, Nick; de Winde, Johannes H; Ruijssenaars, Harald J

    2010-03-16

    The toxic fermentation inhibitors in lignocellulosic hydrolysates pose significant problems for the production of second-generation biofuels and biochemicals. Among these inhibitors, 5-(hydroxymethyl)furfural (HMF) and furfural are specifically notorious. In this study, we describe the complete molecular identification and characterization of the pathway by which Cupriavidus basilensis HMF14 metabolizes HMF and furfural. The identification of this pathway enabled the construction of an HMF and furfural-metabolizing Pseudomonas putida. The genetic information obtained furthermore enabled us to predict the HMF and furfural degrading capabilities of sequenced bacterial species that had not previously been connected to furanic aldehyde metabolism. These results pave the way for in situ detoxification of lignocellulosic hydrolysates, which is a major step toward improved efficiency of utilization of lignocellulosic feedstock. PMID:20194784

  20. Ozonation of chlorophenylurea pesticides in water: reaction monitoring and degradation pathways

    Microsoft Academic Search

    L Amir Tahmasseb; S Nélieu; L Kerhoas; J Einhorn

    2002-01-01

    The degradation of mono- and dichlorophenylureas under ozone\\/hydrogen peroxide conditions was investigated in order to establish the effect of the structural parameters. The N-dimethyl phenylureas (mono- and dichloro) appear to differ strongly from the corresponding N-methyl N-methoxy analogues in terms of disappearance of the parent pesticide and evolution of the by-products identified by MS and MS-MS. The degradation rate of

  1. Photo-induced degradation of diuron in aqueous solution by nitrites and nitrates: Kinetics and pathways

    Microsoft Academic Search

    M. V. Shankar; S. Nélieu; L. Kerhoas; J. Einhorn

    2007-01-01

    The photo-induced degradation of diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) in aqueous solution under simulated solar irradiation has been investigated in the presence of NO3-\\/NO2- ions. The degradation rates were compared by varying environmental parameters including substrate and inducer concentrations, oxygen content and pH. The photoproducts were identified by extensive LC–ESI–MS and LC–ESI–MS–MS studies after SPE preconcentration on prepacked cartridges. In both NO3- and

  2. The Sjögren-Larsson syndrome gene encodes a hexadecenal dehydrogenase of the sphingosine 1-phosphate degradation pathway.

    PubMed

    Nakahara, Kanae; Ohkuni, Aya; Kitamura, Takuya; Abe, Kensuke; Naganuma, Tatsuro; Ohno, Yusuke; Zoeller, Raphael A; Kihara, Akio

    2012-05-25

    Sphingosine 1-phosphate (S1P) functions not only as a bioactive lipid molecule, but also as an important intermediate of the sole sphingolipid-to-glycerolipid metabolic pathway. However, the precise reactions and the enzymes involved in this pathway remain unresolved. We report here that yeast HFD1 and the Sjögren-Larsson syndrome (SLS)-causative mammalian gene ALDH3A2 are responsible for conversion of the S1P degradation product hexadecenal to hexadecenoic acid. The absence of ALDH3A2 in CHO-K1 mutant cells caused abnormal metabolism of S1P/hexadecenal to ether-linked glycerolipids. Moreover, we demonstrate that yeast Faa1 and Faa4 and mammalian ACSL family members are acyl-CoA synthetases involved in the sphingolipid-to-glycerolipid metabolic pathway and that hexadecenoic acid accumulates in ?faa1 ?faa4 mutant cells. These results unveil the entire S1P metabolic pathway: S1P is metabolized to glycerolipids via hexadecenal, hexadecenoic acid, hexadecenoyl-CoA, and palmitoyl-CoA. From our results we propose a possibility that accumulation of the S1P metabolite hexadecenal contributes to the pathogenesis of SLS. PMID:22633490

  3. Age-related dysfunctions of the autophagy lysosomal pathway in hippocampal pyramidal neurons under proteasome stress.

    PubMed

    Gavilán, Elena; Pintado, Cristina; Gavilan, Maria P; Daza, Paula; Sánchez-Aguayo, Inmaculada; Castaño, Angélica; Ruano, Diego

    2015-05-01

    Autophagy plays a key role in the maintenance of cellular homeostasis, and autophagy deregulation gives rise to severe disorders. Many of the signaling pathways regulating autophagy under stress conditions are still poorly understood. Using a model of proteasome stress in rat hippocampus, we have characterized the functional crosstalk between the ubiquitin proteasome system and the autophagy-lysosome pathway, identifying also age-related modifications in the crosstalk between both proteolytic systems. Under proteasome inhibition, both autophagy activation and resolution were efficiently induced in young but not in aged rats, leading to restoration of protein homeostasis only in young pyramidal neurons. Importantly, proteasome stress inhibited glycogen synthase kinase-3? in young but activated in aged rats. This age-related difference could be because of a dysfunction in the signaling pathway of the insulin growth factor-1 under stress situations. Present data highlight the potential role of glycogen synthase kinase-3? in the coordination of both proteolytic systems under stress situation, representing a key molecular target to sort out this deleterious effect. PMID:25817083

  4. Degradation of 3-chlorobenzoate and phenol singly and in mixture by a mixed culture of two ortho-pathway-following Pseudomonas strains

    Microsoft Academic Search

    V. P. Jayachandran; A. A. M. Kunhi

    2009-01-01

    The compatibility and efficiency of two ortho-cleavage pathway-following pseudomonads viz. the 3-chlorobenzoate (3-CBA)-degrader, Pseudomonas aeruginosa 3mT (3mT) and the phenol-degrader, P. stutzeri SPC-2 (SPC-2) in a mixed culture for the degradation of these substrates singly and simultaneously in mixtures was studied.\\u000a Another phenol-degrading strain, Pseudomonas sp. SoPC-5 (SoPC-5) that utilizes a meta-cleavage mode also was tried in co-culture with 3mT.

  5. Relationship between the proteasomal system and autophagy

    PubMed Central

    Lilienbaum, Alain

    2013-01-01

    Two major pathways degrade most cellular proteins in eukaryotic cells: the ubiquitin–proteasome system (UPS), which usually degrades the majority of proteins, and autophagy, primarily responsible for the degradation of most long-lived or aggregated proteins and cellular organelles. Disruption of these processes can contribute to pathology of a variety of diseases. Further, both pathways are critical for the maintenance of several aspects of cellular homeostasis, but, until recently, were thought to be largely distinct. Recent advances in this field, however, now strongly suggest that their activities are carefully orchestrated through several interfacing elements that are presented and discussed in this review. PMID:23638318

  6. Targeting C4-demethylating genes in the cholesterol pathway sensitizes cancer cells to EGFR inhibitors via increased EGFR degradation

    PubMed Central

    Sukhanova, Anna; Gorin, Andrey; Serebriiskii, Ilya G.; Gabitova, Linara; Zheng, Hui; Restifo, Diana; Egleston, Brian L.; Cunningham, David; Bagnyukova, Tetyana; Liu, Hanqing; Nikonova, Anna; Adams, Gregory P.; Zhou, Yan; Yang, Dong-Hua; Mehra, Ranee; Burtness, Barbara; Cai, Kathy Q.; Klein-Szanto, Andres; Kratz, Lisa E.; Kelley, Richard I.; Weiner, Louis M.; Herman, Gail E.; Golemis, Erica A.; Astsaturov, Igor

    2012-01-01

    Persistent signaling by the oncogenic epidermal growth factor receptor (EGFR) is a major source of cancer resistance to EGFR targeting. We established that inactivation of two sterol biosynthesis pathway genes, SC4MOL (sterol C4-methyl oxidase-like) and its partner NSDHL (NADP-dependent steroid dehydrogenase-like), sensitized tumor cells to EGFR inhibitors. Bioinformatics modeling of interactions for the sterol pathway genes in eukaryotes allowed us to hypothesize, and then extensively validate an unexpected role for SC4MOL and NSDHL in controlling the signaling, vesicular trafficking and degradation of EGFR and its dimerization partners, ERBB2 and ERBB3. Metabolic block upstream of SC4MOL with ketoconazole or CYP51A1 siRNA rescued cancer cell viability and EGFR degradation. Inactivation of SC4MOL markedly sensitized A431 xenografts to cetuximab, a therapeutic anti-EGFR antibody. Analysis of Nsdhl-deficient Bpa1H/+ mice confirmed dramatic and selective loss of internalized PDGFR in fibroblasts, and reduced activation of EGFR and its effectors in regions of skin lacking NSDHL. PMID:23125191

  7. Toxoplasma gondii Virulence Factor ROP18 Inhibits the Host NF-?B Pathway by Promoting p65 Degradation*

    PubMed Central

    Du, Jian; An, Ran; Chen, Lijian; Shen, Yuxian; Chen, Ying; Cheng, Li; Jiang, Zhongru; Zhang, Aimei; Yu, Li; Chu, Deyong; Shen, Yujun; Luo, Qingli; Chen, He; Wan, Lijuan; Li, Min; Xu, Xiucai; Shen, Jilong

    2014-01-01

    The obligate intracellular parasite Toxoplasma gondii secretes effector molecules into the host cell to modulate host immunity. Previous studies have shown that T. gondii could interfere with host NF-?B signaling to promote their survival, but the effectors of type I strains remain unclear. The polymorphic rhoptry protein ROP18 is a key serine/threonine kinase that phosphorylates host proteins to modulate acute virulence. Our data demonstrated that the N-terminal portion of ROP18 is associated with the dimerization domain of p65. ROP18 phosphorylates p65 at Ser-468 and targets this protein to the ubiquitin-dependent degradation pathway. The kinase activity of ROP18 is required for p65 degradation and suppresses NF-?B activation. Consistently, compared with wild-type ROP18 strain, ROP18 kinase-deficient type I parasites displayed a severe inability to inhibit NF-?B, culminating in the enhanced production of IL-6, IL-12, and TNF-? in infected macrophages. In addition, studies have shown that transgenic parasites carrying kinase-deficient ROP18 induce M1-biased activation. These results demonstrate for the first time that the virulence factor ROP18 in T. gondii type I strains is responsible for inhibiting the host NF-?B pathway and for suppressing proinflammatory cytokine expression, thus providing a survival advantage to the infectious agent. PMID:24648522

  8. Novel Cell Death Signaling Pathways in Neurotoxicity Models of Dopaminergic Degeneration: Relevance to Oxidative Stress and Neuroinflammation in Parkinson’s Disease

    PubMed Central

    Kanthasamy, Anumantha; Jin, Huajun; Mehrotra, Suneet; Mishra, Rajakishore; Kanthasamy, Arthi; Rana, Ajay

    2009-01-01

    Parkinson’s disease (PD) is a common neurodegenerative movement disorder characterized by extensive degeneration of dopaminergic neurons in the nigrostriatal system. Neurochemical and neuropathological analyses clearly indicate that oxidative stress, mitochondrial dysfunction, neuroinflammation and impairment of the ubiquitin-proteasome system (UPS) are major mechanisms of dopaminergic degeneration. Evidence from experimental models and postmortem PD brain tissues demonstrates that apoptotic cell death is the common final pathway responsible for selective and irreversible loss of nigral dopaminergic neurons. Epidemiological studies imply both environmental neurotoxicants and genetic predisposition are risk factors for PD, though the cellular mechanisms underlying selective dopaminergic degeneration remain unclear. Recent progress in signal transduction research is beginning to unravel the complex mechanisms governing dopaminergic degeneration. During 12th International Neurotoxicology meeting, discussion at one symposium focused on several key signaling pathways of dopaminergic degeneration. This review summarizes two novel signaling pathways of nigral dopaminergic degeneration that have been elucidated using neurotoxicity models of PD. Dr. Anumantha Kanthasamy described a cell death pathway involving the novel protein kinase C delta isoform (PKC?) in oxidative stress-induced apoptotic cell death in experimental models of PD. Dr. Ajay Rana presented his recent work on the role of mixed lineage kinase-3 (MLK3) in neuroinflammatory processes in neurotoxic cell death. Collectively, PKC? and MLK3 signaling pathways provide new understanding of neurodegenerative processes in PD, and further exploration of these pathways may translate into effective neuroprotective drugs for the treatment of PD. PMID:20005250

  9. c-myc RNA degradation in growing and differentiating cells: Possible alternate pathways

    SciTech Connect

    Swartwout, S.G. (Roswell Park Memorial Inst., Buffalo, NY (USA)); Kinniburgh, A.J. (Roswell Park Memorial Inst., Buffalo, NY (USA). Dept. of Hematology Research)

    1989-01-01

    Transcripts of the proto-oncogene c-myc are composed of a rapidly degraded polyadenylated RNA species and an apparently much more stable, nonadenylated RNA species. In this report, the extended kinetics of c-myc RNA turnover have been examined in rapidly growing cells and in cells induced to differentiate. When transcription was blocked with actinomycin D in rapidly growing cells, poly(A)/sup +/ c-myc was rapidly degraded (t/sub 1/2/ = 12 min). c-myc RNA lacking poly (A) initially remained at or near control levels; however, after 80 to 90 min it was degraded with kinetics similar to those of poly (A)/sup +/ c-myc RNA. These bizarre kinetics are due to the deadenylation of poly (A)/sup +/ c-myc RNA to form poly (A)/sup -/ c-myc, thereby initially maintaining the poly (A)/sup -/ c-myc RNA pool when transcription is blocked. In contrast to growing cells, cells induced to differentiate degraded both poly (A)/sup +/ and poly (A)/sup -/ c-myc RNA rapidly. The rapid disappearance of both RNA species in differentiating cells suggests that a large proportion of the poly (A)/sup +/ c-myc RNA was directly degraded without first being converted to poly (A)/sup -/ c-myc RNA. Others have shown that transcriptional elongation of the c-myc gene is rapidly blocked in differentiating cells. The authors therefore hypothesize that in differentiating cells a direct, rapid degradation of poly (A)/sup +/ c-myc RNA may act as a backup or fail-safe system to ensure that c-myc protein is not synthesized.

  10. The Alkyl tert-Butyl Ether Intermediate 2-Hydroxyisobutyrate Is Degraded via a Novel Cobalamin-Dependent Mutase Pathway

    PubMed Central

    Rohwerder, Thore; Breuer, Uta; Benndorf, Dirk; Lechner, Ute; Müller, Roland H.

    2006-01-01

    Fuel oxygenates such as methyl and ethyl tert-butyl ether (MTBE and ETBE, respectively) are degraded only by a limited number of bacterial strains. The aerobic pathway is generally thought to run via tert-butyl alcohol (TBA) and 2-hydroxyisobutyrate (2-HIBA), whereas further steps are unclear. We have now demonstrated for the newly isolated ?-proteobacterial strains L108 and L10, as well as for the closely related strain CIP I-2052, that 2-HIBA was degraded by a cobalamin-dependent enzymatic step. In these strains, growth on substrates containing the tert-butyl moiety, such as MTBE, TBA, and 2-HIBA, was strictly dependent on cobalt, which could be replaced by cobalamin. Tandem mass spectrometry identified a 2-HIBA-induced protein with high similarity to a peptide whose gene sequence was found in the finished genome of the MTBE-degrading strain Methylibium petroleiphilum PM1. Alignment analysis identified it as the small subunit of isobutyryl-coenzyme A (CoA) mutase (ICM; EC 5.4.99.13), which is a cobalamin-containing carbon skeleton-rearranging enzyme, originally described only in Streptomyces spp. Sequencing of the genes of both ICM subunits from strain L108 revealed nearly 100% identity with the corresponding peptide sequences from M. petroleiphilum PM1, suggesting a horizontal gene transfer event to have occurred between these strains. Enzyme activity was demonstrated in crude extracts of induced cells of strains L108 and L10, transforming 2-HIBA into 3-hydroxybutyrate in the presence of CoA and ATP. The physiological and evolutionary aspects of this novel pathway involved in MTBE and ETBE metabolism are discussed. PMID:16751524

  11. Degradation of o-nitrobenzoate via anthranilic acid (o-aminobenzoate) by Arthrobacter protophormiae: a plasmid-encoded new pathway.

    PubMed

    Chauhan, A; Jain, R K

    2000-01-01

    An Arthrobacter protophormiae strain RKJ100, isolated by selective enrichment, was capable of utilizing o-nitrobenzoate (ONB(+)) as the sole carbon, nitrogen, and energy source. The degradation of ONB proceeds through an oxygen insensitive reductive route as shown by the release of ammonia in the culture medium aerobically rather than nitrite ions. Thin-layer chromatography, gas chromatography, and gas chromatography-mass spectrometry of the intermediates have shown that ONB is degraded by a two-electron reduction of the nitro moiety, yielding o-hydroxylaminobenzoate and anthranilic acid. Quantitation of the intermediates, inhibition studies, and simultaneous induction studies have shown that anthranilic acid is produced as the terminal aromatic intermediate of a catabolic energy-yielding pathway and not as a side reaction taking place concurrently which is the first such report. A plasmid of approximately 65 kb was found to be responsible for harboring genes for ONB degradation in this strain. The same plasmid also encoded resistance to cobalt ions. PMID:10623604

  12. Comparison of EJC-enhanced and EJC-independent NMD in human cells reveals two partially redundant degradation pathways

    PubMed Central

    Metze, Stefanie; Herzog, Veronika A.; Ruepp, Marc-David; Mühlemann, Oliver

    2013-01-01

    Nonsense-mediated mRNA decay (NMD) is a eukaryotic post-transcriptional gene regulation mechanism that eliminates mRNAs with the termination codon (TC) located in an unfavorable environment for efficient translation termination. The best-studied NMD-targeted mRNAs contain premature termination codons (PTCs); however, NMD regulates even many physiological mRNAs. An exon-junction complex (EJC) located downstream from a TC acts as an NMD-enhancing signal, but is not generally required for NMD. Here, we compared these “EJC-enhanced” and “EJC-independent” modes of NMD with regard to their requirement for seven known NMD factors in human cells using two well-characterized NMD reporter genes (immunoglobulin ? and ?-Globin) with or without an intron downstream from the PTC. We show that both NMD modes depend on UPF1 and SMG1, but detected transcript-specific differences with respect to the requirement for UPF2 and UPF3b, consistent with previously reported UPF2- and UPF3-independent branches of NMD. In addition and contrary to expectation, a higher sensitivity of EJC-independent NMD to reduced UPF2 and UPF3b concentrations was observed. Our data further revealed a redundancy of the endo- and exonucleolytic mRNA degradation pathways in both modes of NMD. Moreover, the relative contributions of both decay pathways differed between the reporters, with PTC-containing immunoglobulin ? transcripts being preferentially subjected to SMG6-mediated endonucleolytic cleavage, whereas ?-Globin transcripts were predominantly degraded by the SMG5/SMG7-dependent pathway. Overall, the surprising heterogeneity observed with only two NMD reporter pairs suggests the existence of several mechanistically distinct branches of NMD in human cells. PMID:23962664

  13. Regulation of protein degradation pathways by amino acids and insulin in skeletal muscle of neonatal pigs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rapid gain in lean mass in neonates requires greater rates of protein synthesis than degradation. We previously delineated the molecular mechanisms by which insulin and amino acids, especially leucine, modulate skeletal muscle protein synthesis and how this changes with development. In the curre...

  14. Windpipe Controls Drosophila Intestinal Homeostasis by Regulating JAK/STAT Pathway via Promoting Receptor Endocytosis and Lysosomal Degradation.

    PubMed

    Ren, Wenyan; Zhang, Yan; Li, Min; Wu, Longfei; Wang, Guolun; Baeg, Gyeong-Hun; You, Jia; Li, Zhouhua; Lin, Xinhua

    2015-04-01

    The adult intestinal homeostasis is tightly controlled by proper proliferation and differentiation of intestinal stem cells. The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway is essential for the regulation of adult stem cell activities and maintenance of intestinal homeostasis. Currently, it remains largely unknown how JAK/STAT signaling activities are regulated in these processes. Here we have identified windpipe (wdp) as a novel component of the JAK/STAT pathway. We demonstrate that Wdp is positively regulated by JAK/STAT signaling in Drosophila adult intestines. Loss of wdp activity results in the disruption of midgut homeostasis under normal and regenerative conditions. Conversely, ectopic expression of Wdp inhibits JAK/STAT signaling activity. Importantly, we show that Wdp interacts with the receptor Domeless (Dome), and promotes its internalization for subsequent lysosomal degradation. Together, these data led us to propose that Wdp acts as a novel negative feedback regulator of the JAK/STAT pathway in regulating intestinal homeostasis. PMID:25923769

  15. Toll-Like Receptor Signaling Induces Nrf2 Pathway Activation through p62-Triggered Keap1 Degradation.

    PubMed

    Yin, Shasha; Cao, Wangsen

    2015-08-01

    Toll-like receptors (TLRs) induce inflammation and tissue repair through multiple signaling pathways. The Nrf2 pathway plays a key role in defending against the tissue damage incurred by microbial infection or inflammation-associated diseases. The critical event that mediates TLR-induced Nrf2 activation is still poorly understood. In this study, we found that lipopolysaccharide (LPS) and other Toll-like receptor (TLR) agonists activate Nrf2 signaling and the activation is due to the reduction of Keap1, the key Nrf2 inhibitor. TLR signaling-induced Keap1 reduction promoted Nrf2 translocation from the cytoplasm to the nucleus, where it activated transcription of its target genes. TLR agonists modulated Keap1 at the protein posttranslation level through autophagy. TLR signaling increased the expression of autophagy protein p62 and LC3-II and induced their association with Keap1 in the autophagosome-like structures. We also characterized the interaction between p62 and Keap1 and found that p62 is indispensable for TLR-mediated Keap1 reduction: TLR signaling had no effect on Keap1 if cells lacked p62 or if cells expressed a mutant Keap1 that could not interact with p62. Our study indicates that p62-mediated Keap1 degradation through autophagy represents a critical linkage for TLR signaling regulation of the major defense network, the Nrf2 signaling pathway. PMID:26012548

  16. Windpipe Controls Drosophila Intestinal Homeostasis by Regulating JAK/STAT Pathway via Promoting Receptor Endocytosis and Lysosomal Degradation

    PubMed Central

    Li, Min; Wu, Longfei; Wang, Guolun; Baeg, Gyeong-Hun; You, Jia; Li, Zhouhua; Lin, Xinhua

    2015-01-01

    The adult intestinal homeostasis is tightly controlled by proper proliferation and differentiation of intestinal stem cells. The JAK/STAT (Janus Kinase/Signal Transducer and Activator of Transcription) signaling pathway is essential for the regulation of adult stem cell activities and maintenance of intestinal homeostasis. Currently, it remains largely unknown how JAK/STAT signaling activities are regulated in these processes. Here we have identified windpipe (wdp) as a novel component of the JAK/STAT pathway. We demonstrate that Wdp is positively regulated by JAK/STAT signaling in Drosophila adult intestines. Loss of wdp activity results in the disruption of midgut homeostasis under normal and regenerative conditions. Conversely, ectopic expression of Wdp inhibits JAK/STAT signaling activity. Importantly, we show that Wdp interacts with the receptor Domeless (Dome), and promotes its internalization for subsequent lysosomal degradation. Together, these data led us to propose that Wdp acts as a novel negative feedback regulator of the JAK/STAT pathway in regulating intestinal homeostasis. PMID:25923769

  17. Aggresomes protect cells by enhancing the degradation of toxic polyglutamine-containing protein.

    PubMed

    Taylor, J Paul; Tanaka, Fumiaki; Robitschek, Jon; Sandoval, C Miguel; Taye, Addis; Markovic-Plese, Silva; Fischbeck, Kenneth H

    2003-04-01

    Expression of misfolded protein in cultured cells frequently leads to the formation of juxtanuclear inclusions that have been termed 'aggresomes'. Aggresome formation is an active cellular response that involves trafficking of the offending protein along microtubules, reorganization of intermediate filaments and recruitment of components of the ubiquitin proteasome system. Whether aggresomes are benevolent or noxious is unknown, but they are of particular interest because of the appearance of similar inclusions in protein deposition diseases. Here we present evidence that aggresomes serve a cytoprotective function and are associated with accelerated turnover of mutant proteins. We show that mutant androgen receptor (AR), the protein responsible for X-linked spinobulbar muscular atrophy, forms insoluble aggregates and is toxic to cultured cells. Mutant AR was also found to form aggresomes in a process distinct from aggregation. Molecular and pharmacological interventions were used to disrupt aggresome formation, revealing their cytoprotective function. Aggresome-forming proteins were found to have an accelerated rate of turnover, and this turnover was slowed by inhibition of aggresome formation. Finally, we show that aggresome-forming proteins become membrane-bound and associate with lysosomal structures. Together, these findings suggest that aggresomes are cytoprotective, serving as cytoplasmic recruitment centers to facilitate degradation of toxic proteins. PMID:12651870

  18. Betulinic Acid Selectively Increases Protein Degradation and Enhances Prostate Cancer-Specific Apoptosis: Possible Role for Inhibition of Deubiquitinase Activity

    PubMed Central

    Reiner, Teresita; Parrondo, Ricardo; de las Pozas, Alicia; Palenzuela, Deanna; Perez-Stable, Carlos

    2013-01-01

    Inhibition of the ubiquitin-proteasome system (UPS) of protein degradation is a valid anti-cancer strategy and has led to the approval of bortezomib for the treatment of multiple myeloma. However, the alternative approach of enhancing the degradation of oncoproteins that are frequently overexpressed in cancers is less developed. Betulinic acid (BA) is a plant-derived small molecule that can increase apoptosis specifically in cancer but not in normal cells, making it an attractive anti-cancer agent. Our results in prostate cancer suggested that BA inhibited multiple deubiquitinases (DUBs), which resulted in the accumulation of poly-ubiquitinated proteins, decreased levels of oncoproteins, and increased apoptotic cell death. In normal fibroblasts, however, BA did not inhibit DUB activity nor increased total poly-ubiquitinated proteins, which was associated with a lack of effect on cell death. In the TRAMP transgenic mouse model of prostate cancer, treatment with BA (10 mg/kg) inhibited primary tumors, increased apoptosis, decreased angiogenesis and proliferation, and lowered androgen receptor and cyclin D1 protein. BA treatment also inhibited DUB activity and increased ubiquitinated proteins in TRAMP prostate cancer but had no effect on apoptosis or ubiquitination in normal mouse tissues. Overall, our data suggests that BA-mediated inhibition of DUBs and induction of apoptotic cell death specifically in prostate cancer but not in normal cells and tissues may provide an effective non-toxic and clinically selective agent for chemotherapy. PMID:23424652

  19. Role of the glutamine transaminase- -amidase pathway and glutaminase in glutamine degradation in Rhizobium etli

    Microsoft Academic Search

    S. Duran; J. Calderon

    1995-01-01

    Evidence for the participation of the glutamine transaminase-cu-amidase pathway and a glutaminase in the utilization of glutamine in Rhizobium etli has been obtained. The glutamine transaminase preferentially transaminates glyoxylate and pyruvate. Glutamine transaminase activity was similar under all growth conditions tested except on PY (rich medium) where it was low. Glutaminase activity was positively regulated by glutamine and negatively regulated

  20. Production of Pyomelanin, a Second Type of Melanin, via the Tyrosine Degradation Pathway in Aspergillus fumigatus

    Microsoft Academic Search

    Jeannette Schmaler-Ripcke; Venelina Sugareva; Peter Gebhardt; Robert Winkler; Olaf Kniemeyer; Thorsten Heinekamp; Axel A. Brakhage

    2009-01-01

    Aspergillus fumigatus is the most important airborne fungal pathogen of immunosuppressed humans. A. fumigatus is able to produce dihydroxynaphthalene melanin, which is predominantly present in the conidia. Its biosynthesis is an important virulence determinant. Here, we show that A. fumigatus is able to produce an alternative melanin, i.e., pyomelanin, by a different pathway, starting from L-tyrosine. Proteome analysis indicated that

  1. Catabolite-mediated mutations in alternate toluene degradative pathways in Pseudomonas putida.

    PubMed Central

    Leddy, M B; Phipps, D W; Ridgway, H F

    1995-01-01

    Pseudomonas putida 54g grew on mineral salts with toluene and exhibited catechol-2,3-dioxygenase (C23O) activity, indicating a meta pathway. After 10 to 15 days on toluene, nondegrading (Tol-) variants approached nearly 10% of total CFU. Auxotrophs were not detected among variants, suggesting selective loss of catabolic function(s). Variant formation was substrate dependent, since Tol- cells were observed on neither ethylbenzene, glucose, nor peptone-based media nor when toluene catabolism was suppressed by glucose. Unlike wild-type cells, variants did not grow on gasoline, toluene, benzene, ethylbenzene, benzoate, or catechol, suggesting loss of meta pathway function. Catabolic and C23O activities were restored to variants via transfer of a 78-mDa TOL-like plasmid from a wild-type Tol+ donor. Tests for reversion of variants to Tol+ were uniformly negative, suggesting possible delection or excision of catabolic genes. Deletions were confirmed in some variants by failure to hybridize with a DNA probe specific for the xylE gene encoding C23O. Cells grown on benzoate remained Tol+ but were C23O- and contained a plasmid of reduced size or were plasmid free, suggesting an alternate chromosomal catabolic pathway, also defective in variants. Cells exposed to benzyl alcohol, the initial oxidation product of toluene, accumulated > 13% variants in 5 days, even when cell division was repressed by nitrogen deprivation to abrogate selection processes. No variants formed in identical ethylbenzene-exposed controls. The results suggest that benzyl alcohol mediates irreversible defects in both a plasmid-associated meta pathway and an alternate chromosomal pathway. PMID:7642499

  2. A novel degradative pathway of 2-nitrobenzoate via 3-hydroxyanthranilate in Pseudomonas fluorescens strain KU-7

    Microsoft Academic Search

    Yoshie Hasegawa; Takamichi Muraki; Tai Tokuyama; Hiroaki Iwaki; Michiaki Tatsuno; Peter C. K. Lau

    2000-01-01

    A bacterial strain KU-7, identified as a Pseudomonas fluorescens by 16S rDNA sequencing, was one of the 12 new isolates that are able to grow on 2-nitrobenzoate as a sole source of carbon, nitrogen, and energy. Resting cells of KU-7 were found to accumulate ammonia in the medium indicating that degradation of 2-NBA proceeds through a reductive route. Metabolite analyses

  3. Ubiquitination and degradation of the zebrafish paired-like homeobox protein VSX-1.

    PubMed

    Kurtzman, A L; Gregori, L; Haas, A L; Schechter, N

    2000-07-01

    Vsx-1 is a paired-like : CVC homeobox protein dynamically expressed during zebrafish development. Previous results indicate that Vsx-1 influences bipolar cell differentiation and maintenance of these cells in the adult retina. To understand the developmental regulation of this transcription factor, we investigated ubiquitination as a possible posttranslational mechanism. In vitro, Vsx-1 was conjugated with multiple ubiquitin moieties. Proteasome inhibitors and added ubiquitin increased the accumulation of Vsx-1-ubiquitin(n) complexes and stabilized unmodified Vsx-1. Also, in transiently transfected COS-7 cells, Vsx-1 is ubiquitinated, and pulse-chase experiments show that Vsx-1 proteolysis occurs. Vsx-1 proteins with C-terminal deletions retained the capacity for initial modification by ubiquitin but lost the capacity for efficient chain elongation. These results show that Vsx-1 is a substrate of the ubiquitin/proteasome pathway and suggest that C-terminal sequences of Vsx-1 are critical for ubiquitin chain elongation. In addition, our findings suggest that ubiquitin-dependent proteolysis regulates Vsx-1 during zebrafish retinal development. PMID:10854246

  4. Degradation Pathways for Geogenic Volatile Organic Compounds (VOCs) in Soil Gases from the Solfatara Crater (Campi Flegrei, Southern Italy).

    NASA Astrophysics Data System (ADS)

    Tassi, F.; Venturi, S.; Cabassi, J.; Capecchiacci, F.; Nisi, B., Sr.; Vaselli, O.

    2014-12-01

    The chemical composition of volatile organic compounds (VOCs) in soil gases from the Solfatara crater (Campi Flegrei, Southern Italy) was analyzed to investigate the effects of biogeochemical processes occurring within the crater soil on gases discharged from the hydrothermal reservoir and released into the atmosphere through diffuse degassing. In this system, two fumarolic vents (namely Bocca Grande and Bocca Nuova) are the preferential pathways for hydrothermal fluid uprising. For our goal, the chemistry of VOCs discharged from these sites were compared to that of soil gases. Our results highlighted that C4-C9 alkanes, alkenes, S-bearing compounds and alkylated aromatics produced at depth were the most prone to degradation processes, such as oxidation-reduction and hydration-dehydration reactions, as well as to microbial activity. Secondary products, which were enriched in sites characterized by low soil gas fluxes, mostly consisted of aldheydes, ketons, esters, ethers, organic acids and, subordinately, alcohols. Benzene, phenol and hydrofluorocarbons (HCFCs) produced at depth were able to transit through the soil almost undisturbed, independently on the emission rate of diffuse degassing. The presence of cyclics was possibly related to an independent low-temperature VOC source, likely within sedimentary formations overlying the hydrothermal reservoir. Chlorofluorocarbons (CFCs) were possibly due to air contamination. This study demonstrated the strict control of biogeochemical processes on the behaviour of hydrothermal VOCs that, at least at a local scale, may have a significant impact on air quality. Laboratory experiments conducted at specific chemical-physical conditions and in presence of different microbial populations may provide useful information for the reconstruction of the degradation pathways controlling fate and behaviour of VOCs in the soil.

  5. A Shared Endoplasmic Reticulum-associated Degradation Pathway Involving the EDEM1 Protein for Glycosylated and Nonglycosylated Proteins*

    PubMed Central

    Shenkman, Marina; Groisman, Bella; Ron, Efrat; Avezov, Edward; Hendershot, Linda M.; Lederkremer, Gerardo Z.

    2013-01-01

    Studies of misfolded protein targeting to endoplasmic reticulum-associated degradation (ERAD) have largely focused on glycoproteins, which include the bulk of the secretory proteins. Mechanisms of targeting of nonglycosylated proteins are less clear. Here, we studied three nonglycosylated proteins and analyzed their use of known glycoprotein quality control and ERAD components. Similar to an established glycosylated ERAD substrate, the uncleaved precursor of asialoglycoprotein receptor H2a, its nonglycosylated mutant, makes use of calnexin, EDEM1, and HRD1, but only glycosylated H2a is a substrate for the cytosolic SCFFbs2 E3 ubiquitin ligase with lectin activity. Two nonglycosylated BiP substrates, NS-1? light chain and truncated Ig? heavy chain, interact with the ERAD complex lectins OS-9 and XTP3-B and require EDEM1 for degradation. EDEM1 associates through a region outside of its mannosidase-like domain with the nonglycosylated proteins. Similar to glycosylated substrates, proteasomal inhibition induced accumulation of the nonglycosylated proteins and ERAD machinery in the endoplasmic reticulum-derived quality control compartment. Our results suggest a shared ERAD pathway for glycosylated and nonglycosylated proteins composed of luminal lectin machinery components also capable of protein-protein interactions. PMID:23233672

  6. Molecular and Biochemical Characterization of the 5-Nitroanthranilic Acid Degradation Pathway in Bradyrhizobium sp. Strain JS329 ? †

    PubMed Central

    Qu, Yi; Spain, Jim C.

    2011-01-01

    Biodegradation pathways of synthetic nitroaromatic compounds and anilines are well documented, but little is known about those of nitroanilines. We previously reported that the initial step in 5-nitroanthranilic acid (5NAA) degradation by Bradyrhizobium sp. strain JS329 is a hydrolytic deamination to form 5-nitrosalicylic acid (5NSA), followed by ring fission catalyzed by 5NSA dioxygenase. The mechanism of release of the nitro group was unknown. In this study, we subcloned, sequenced, and expressed the genes encoding 5NAA deaminase (5NAA aminohydrolase, NaaA), 5NSA dioxygenase (NaaB) and lactonase (NaaC), the key genes responsible for 5NAA degradation. Sequence analysis and enzyme characterization revealed that NaaA is a hydrolytic metalloenzyme with a narrow substrate range. The nitro group is spontaneously eliminated as nitrite concomitant with the formation of a lactone from the ring fission product of 5NSA dioxygenation. The elimination of the nitro group during lactone formation is a previously unreported mechanism for denitration of nitro aliphatic compounds. PMID:21498645

  7. Electrochemical degradation of sulfonamides at BDD electrode: kinetics, reaction pathway and eco-toxicity evaluation.

    PubMed

    Fabia?ska, Aleksandra; Bia?k-Bieli?ska, Anna; Stepnowski, Piotr; Stolte, Stefan; Siedlecka, Ewa Maria

    2014-09-15

    The investigation dealt with electrochemical oxidation of five sulfonamides (SNs): sulfadiazine (SDZ), sulfathiazole (STZ), sulfamerazine (SMR), sulfamethazine (SMN) and sulfadimethoxine (SDM) in aqueous solution at boron-doped diamond (BDD) electrode. All studied sulfonamides were degraded according to a pseudo first order kinetics. The structure of SNs had no significant effect on the values of pseudo first order rate constants. Increased degradation efficiency was observed in higher temperature and in acidic pH. Due to the presence of chlorine and nitrate SNs were more effectively oxidized from municipal wastewater treatment plant (WWTP) effluents than from pure supporting electrolyte Na2SO4. The intermediates identified by LC-MS and GC-MS analysis suggested that the hydroxyl radicals attack mainly the SN bond, but also the aromatic ring systems (aniline, pyrimidine or triazole) of SNs. Finally, the toxicity of the SNs solutions and effluents after electrochemical treatment was assessed through the measurement of growth inhibition of green algae (Scenedesmus vacualatus) and duckweed (Lemna minor). Toxicity of SMR, STZ, SMN solutions before and after electrochemical oxidation and SDM solution after the process in L. minor test was observed. No significant toxicity of studied SNs was observed in algae test. PMID:25215656

  8. Sulfoacetate Is Degraded via a Novel Pathway Involving Sulfoacetyl-CoA and Sulfoacetaldehyde in Cupriavidus necator H16

    PubMed Central

    Weinitschke, Sonja; Hollemeyer, Klaus; Kusian, Bernhard; Bowien, Botho; Smits, Theo H. M.; Cook, Alasdair M.

    2010-01-01

    Bacterial degradation of sulfoacetate, a widespread natural product, proceeds via sulfoacetaldehyde and requires a considerable initial energy input. Whereas the fate of sulfoacetaldehyde in Cupriavidus necator (Ralstonia eutropha) H16 is known, the pathway from sulfoacetate to sulfoacetaldehyde is not. The genome sequence of the organism enabled us to hypothesize that the inducible pathway, which initiates sau (sulfoacetate utilization), involved a four-gene cluster (sauRSTU; H16_A2746 to H16_A2749). The sauR gene, divergently orientated to the other three genes, probably encodes the transcriptional regulator of the presumed sauSTU operon, which is subject to inducible transcription. SauU was tentatively identified as a transporter of the major facilitator superfamily, and SauT was deduced to be a sulfoacetate-CoA ligase. SauT was a labile protein, but it could be separated and shown to generate AMP and an unknown, labile CoA-derivative from sulfoacetate, CoA, and ATP. This unknown compound, analyzed by MALDI-TOF-MS, had a relative molecular mass of 889.7, which identified it as protonated sulfoacetyl-CoA (calculated 889.6). SauS was deduced to be sulfoacetaldehyde dehydrogenase (acylating). The enzyme was purified 175-fold to homogeneity and characterized. Peptide mass fingerprinting confirmed the sauS locus (H16_A2747). SauS converted sulfoacetyl-CoA and NADPH to sulfoacetaldehyde, CoA, and NADP+, thus confirming the hypothesis. PMID:20693281

  9. Sulfoacetate is degraded via a novel pathway involving sulfoacetyl-CoA and sulfoacetaldehyde in Cupriavidus necator H16.

    PubMed

    Weinitschke, Sonja; Hollemeyer, Klaus; Kusian, Bernhard; Bowien, Botho; Smits, Theo H M; Cook, Alasdair M

    2010-11-12

    Bacterial degradation of sulfoacetate, a widespread natural product, proceeds via sulfoacetaldehyde and requires a considerable initial energy input. Whereas the fate of sulfoacetaldehyde in Cupriavidus necator (Ralstonia eutropha) H16 is known, the pathway from sulfoacetate to sulfoacetaldehyde is not. The genome sequence of the organism enabled us to hypothesize that the inducible pathway, which initiates sau (sulfoacetate utilization), involved a four-gene cluster (sauRSTU; H16_A2746 to H16_A2749). The sauR gene, divergently orientated to the other three genes, probably encodes the transcriptional regulator of the presumed sauSTU operon, which is subject to inducible transcription. SauU was tentatively identified as a transporter of the major facilitator superfamily, and SauT was deduced to be a sulfoacetate-CoA ligase. SauT was a labile protein, but it could be separated and shown to generate AMP and an unknown, labile CoA-derivative from sulfoacetate, CoA, and ATP. This unknown compound, analyzed by MALDI-TOF-MS, had a relative molecular mass of 889.7, which identified it as protonated sulfoacetyl-CoA (calculated 889.6). SauS was deduced to be sulfoacetaldehyde dehydrogenase (acylating). The enzyme was purified 175-fold to homogeneity and characterized. Peptide mass fingerprinting confirmed the sauS locus (H16_A2747). SauS converted sulfoacetyl-CoA and NADPH to sulfoacetaldehyde, CoA, and NADP(+), thus confirming the hypothesis. PMID:20693281

  10. Molecular characterization of the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli W: engineering a mobile aromatic degradative cluster.

    PubMed Central

    Prieto, M A; Díaz, E; García, J L

    1996-01-01

    We have determined and analyzed the nucleic acid sequence of a 14,855-bp region that contains the complete gene cluster encoding the 4-hydroxyphenylacetic acid (4-HPA) degradative pathway of Escherichia coli W (ATCC 11105). This catabolic pathway is composed by 11 genes, i.e., 8 enzyme-encoding genes distributed in two putative operons, hpaBC (4-HPA hydroxylase operon) and hpaGEDFHI (meta-cleavage operon); 2 regulatory genes, hpaR and hpaA; and the gene, hpaX, that encodes a protein related to the superfamily of transmembrane facilitators and appears to be cotranscribed with hpaA. Although comparisons with other aromatic catabolic pathways revealed interesting similarities, some of the genes did not present any similarity to their corresponding counterparts in other pathways, suggesting different evolutionary origins. The cluster is flanked by two genes homologous to the estA (carbon starvation protein) and tsr (serine chemoreceptor) genes of E. coli K-12. A detailed genetic analysis of this region has provided a singular example of how E. coli becomes adapted to novel nutritional sources by the recruitment of a catabolic cassette. Furthermore, the presence of the pac gene in the proximity of the 4-HPA cluster suggests that the penicillin G acylase was a recent acquisition to improve the ability of E. coli W to metabolize a wider range of substrates, enhancing its catabolic versatility. Five repetitive extragenic palindromic sequences that might be involved in transcriptional regulation were found within the cluster. The complete 4-HPA cluster was cloned in plasmid and transposon cloning vectors that were used to engineer E. coli K-12 strains able to grow on 4-HPA. We report here also the in vitro design of new biodegradative capabilities through the construction of a transposable cassette containing the wide substrate range 4-HPA hydroxylase, in order to expand the ortho-cleavage pathway of Pseudomonas putida KT2442 and allow the new recombinant strain to use phenol as the only carbon source. PMID:8550403

  11. Draft Genome Sequence of Comamonas sp. Strain E6 (NBRC 107749), a Degrader of Phthalate Isomers through the Protocatechuate 4,5-Cleavage Pathway.

    PubMed

    Shimodaira, Jun; Kamimura, Naofumi; Hosoyama, Akira; Yamazoe, Atsushi; Fujita, Nobuyuki; Masai, Eiji

    2015-01-01

    Comamonas sp. strain E6 can degrade o-phthalate, terephthalate, and isophthalate via the protocatechuate 4,5-cleavage pathway. Here, we report the draft genome sequence of E6 in order to provide insights into its mechanisms in o-phthalate catabolism and its potential use for biotechnological applications. PMID:26089421

  12. Draft Genome Sequence of Comamonas sp. Strain E6 (NBRC 107749), a Degrader of Phthalate Isomers through the Protocatechuate 4,5-Cleavage Pathway

    PubMed Central

    Shimodaira, Jun; Kamimura, Naofumi; Hosoyama, Akira; Yamazoe, Atsushi; Fujita, Nobuyuki

    2015-01-01

    Comamonas sp. strain E6 can degrade o-phthalate, terephthalate, and isophthalate via the protocatechuate 4,5-cleavage pathway. Here, we report the draft genome sequence of E6 in order to provide insights into its mechanisms in o-phthalate catabolism and its potential use for biotechnological applications.

  13. Isotopic analysis of oxidative pollutant degradation pathways exhibiting large H isotope fractionation.

    PubMed

    Wijker, Reto S; Adamczyk, Pawel; Bolotin, Jakov; Paneth, Piotr; Hofstetter, Thomas B

    2013-12-01

    Oxidation of aromatic rings and its alkyl substituents are often competing initial steps of organic pollutant transformation. The use of compound-specific isotope analysis (CSIA) to distinguish between these two pathways quantitatively, however, can be hampered by large H isotope fractionation that precludes calculation of apparent (2)H-kinetic isotope effects (KIE) as well as the process identification in multi-element isotope fractionation analysis. Here, we investigated the C and H isotope fractionation associated with the transformation of toluene, nitrobenzene, and four substituted nitrotoluenes by permanganate, MnO4(-), to propose a refined evaluation procedure for the quantitative distinction of CH3-group oxidation and dioxygenation. On the basis of batch experiments, an isotopomer-specific kinetic model, and density functional theory (DFT) calculations, we successfully derived the large apparent (2)H-KIE of 4.033 ± 0.20 for the CH3-group oxidation of toluene from H isotope fractionation exceeding >1300‰ as well as the corresponding (13)C-KIE (1.0324 ± 0.0011). Experiment and theory also agreed well for the dioxygenation of nitrobenzene, which was associated with (2)H- and (13)C-KIEs of 0.9410 ± 0.0030 (0.9228 obtained by DFT) and 1.0289 ± 0.0003 (1.025). Consistent branching ratios for the competing CH3-group oxidation and dioxygenation of nitrotoluenes by MnO4(-) were obtained from the combined modeling of concentration as well as C and H isotope signature trends. Our approach offers improved estimates for the identification of contaminant microbial and abiotic oxidation pathways by CSIA. PMID:24175739

  14. Sources and Input Pathways of Glyphosate and its Degradation Product AMPA

    NASA Astrophysics Data System (ADS)

    Bischofberger, S.; Hanke, I.; Wittmer, I.; Singer, H.; Stamm, C.

    2009-04-01

    Despite being the pesticide used in the largest quantities worldwide, the environmental relevance of glyphosate has been considered low for many years. Reasons for this assessment were the observations that glyphosate degrades quickly into its degradation product AMPA and that it sorbs strongly to soil particles. Hence, little losses to water bodies had been expected. Research during the last few years however contradicts this expectation. Although glyphosate is a dominant pesticide used in agriculture, recent studies on other pesticides revealed that urban sources may play a significant role for water quality. Therefore this study compares glyphosate input into streams from agricultural and urban sources. For that purpose, a catchment of an area of 25 km2 was selected. It has by about 12'000 inhabitants and about 15 % of the area is used as arable land. Four sampling sites were selected in the river system in order to reflect different urban and agricultural sources. Additionally, we sampled a combined sewer overflow, a rain sewer and the outflow of a waste water treatment plant. At each site discharge was measured continuously from March to November 2007. During 16 rain events samples were taken by automatic devices at a high temporal resolution. To analyze the concentration of glyphosate and its degradation product AMPA, the samples were derivatized with FMOC-Cl at low pH conditions and then filtrated. The solid phase extraction was conducted with Strata-X sorbent cartridge. Glyphosate and AMPA were detected with API 4000 after the chromatography with X bridge column C18. To assure the data quality, interne standards of Glyphosate and AMPA were added to every sample. The limit of detection and quantification for glyphosate and AMPA are bellow 1ng/l. We analyzed two rain events at a high resolution for all stations and several events at the outlet of the catchment. We measured high glyphosate concentration in urban and agriculture dominated catchments with up to 3'600ng/l in the rainwater sewer and 2'500ng/l from agricultural origin. The highest glyphosate concentrations were detected during peak flow. The input of the waste water treatment plant (WWTP) was up to 500ng/l. We detected glyphosate above the detection limit in all samples throughout the sampling period. Even after the vegetation period in November, glyphosate peak flow concentration in the outlet of the entire catchment was 137 ng/l exceeding the Swiss water quality criteria for single pesticides. The AMPA concentrations were generally lower than those of Glyphosate except for the WWTP. Generally, AMPA concentrations varied less during a rain event than glyphosate concentrations. Despite the strong sorption to soil particles and short half-life, glyphosate occurs in high concentrations in surface waters affected by urban and agricultural sources. Concentrations were even higher than those of other widely used herbicides like atrazine and mecoprop.

  15. Amyloid domains in the cell nucleus controlled by nucleoskeletal protein lamin B1 reveal a new pathway of mercury neurotoxicity

    PubMed Central

    Arnhold, Florian; Gührs, Karl-Heinz

    2015-01-01

    Mercury (Hg) is a bioaccumulating trace metal that globally circulates the atmosphere and waters in its elemental, inorganic and organic chemical forms. While Hg represents a notorious neurotoxicant, the underlying cellular pathways are insufficiently understood. We identify amyloid protein aggregation in the cell nucleus as a novel pathway of Hg-bio-interactions. By mass spectrometry of purified protein aggregates, a subset of spliceosomal components and nucleoskeletal protein lamin B1 were detected as constituent parts of an Hg-induced nuclear aggregome network. The aggregome network was located by confocal imaging of amyloid-specific antibodies and dyes to amyloid cores within splicing-speckles that additionally recruit components of the ubiquitin-proteasome system. Hg significantly enhances global proteasomal activity in the nucleus, suggesting that formation of amyloid speckles plays a role in maintenance of protein homeostasis. RNAi knock down showed that lamin B1 for its part regulates amyloid speckle formation and thus likewise participates in nuclear protein homeostasis. As the Hg-induced cascade of interactions between the nucleoskeleton and protein homeostasis reduces neuronal signalling, amyloid fibrillation in the cell nucleus is introduced as a feature of Hg-neurotoxicity that opens new avenues of future research. Similar to protein aggregation events in the cytoplasm that are controlled by the cytoskeleton, amyloid fibrillation of nuclear proteins may be driven by the nucleoskeleton. PMID:25699204

  16. The Schizophrenia-Related Protein Dysbindin-1A Is Degraded and Facilitates NF-Kappa B Activity in the Nucleus

    PubMed Central

    Fu, Cheng; Chen, Dong; Chen, Ruijie; Hu, Qingsong; Wang, Guanghui

    2015-01-01

    Dystrobrevin-binding protein 1 (DTNBP1), a gene encoding dysbindin-1, has been identified as a susceptibility gene for schizophrenia. Functioning with partners in synapses or the cytoplasm, this gene regulates neurite outgrowth and neurotransmitter release. Loss of dysbindin-1 affects schizophrenia pathology. Dysbindin-1 is also found in the nucleus, however, the characteristics of dysbindin in the nucleus are not fully understood. Here, we found that dysbindin-1A is degraded in the nucleus via the ubiquitin-proteasome system and that amino acids 2-41 at the N-terminus are required for this process. By interacting with p65, dysbindin-1A promotes the transcriptional activity of NF-kappa B in the nucleus and positively regulates MMP-9 expression. Taken together, the data obtained in this study demonstrate that dysbindin-1A protein levels are highly regulated in the nucleus and that dysbindin-1A regulates transcription factor NF-kappa B activity to promote the expression of MMP-9 and TNF-?. PMID:26171858

  17. Activation of lysosomal degradative pathway in spinal cord tissues of carbon disulfide-treated rats.

    PubMed

    Gao, Yuan; Wang, Shasha; Yi, Anji; Kou, Ruirui; Xie, Keqin; Song, Fuyong

    2014-08-01

    Chronic exposure to carbon disulfide (CS?) can induce polyneuropathy in occupational worker and experimental animals, but underlying mechanism for CS? neuropathy is currently unknown. In the present study, male Wistar rats were randomly divided into three experimental groups and one control group. The rats in experimental groups were treated with CS? by gavage at dosages of 200, 400 and 600 mg/kg/day respectively, six times per week for 6 weeks. The formation of autophagosomes and lysosomes in motor neurons of rat spinal cord was observed by transmission electron microscopy, the level of autophagy-related proteins, lysosome-associated membrane protein 1 (LAMP-1), and cathepsin B in spinal cord tissues was determined by Western blot analysis, and the activity of cathepsin B was measured by fluorescence assay. The results demonstrated that the number of lysosomes in motor neurons was markedly increased in CS?-treated rats. In the meantime, the administration of CS? significantly increased the level of microtubule-associated protein light chain 3-II (LC3-II), Atg1, UVRAG and LAMP-1 in rat spinal cord. Furthermore, the content and activity of cathepsin B in rat spinal cord also showed a significant elevation. Taken together, this study suggested that CS? intoxication was associated with the activation of lysosomal degradative machinery, which might play a protective role against CS?-induced neuronal damage. PMID:24887698

  18. Abatement and degradation pathways of toluene in indoor air by positive corona discharge.

    PubMed

    Van Durme, J; Dewulf, J; Sysmans, W; Leys, C; Van Langenhove, H

    2007-08-01

    Indoor air concentrations of volatile organic compounds often exceed outdoor levels by a factor of 5. There is much interest in developing new technologies in order to improve indoor air quality. In this work non-thermal plasma (DC positive corona discharge) is explored as an innovative technology for indoor air purification. An inlet gas stream of 10 l min(-1) containing 0.50+/-0.02 ppm toluene was treated by the plasma reactor in atmospheric conditions. Toluene removal proved to be achievable with a characteristic energy density epsilon(0) of 50 J l(-1). Removal efficiencies were higher for 26% relative humidity (epsilon(0)=35 J l(-1)), compared with those at increased humidities (50% relative humidity, epsilon(0)=49 J l(-1)). Reaction products such as formic acid, benzaldehyde, benzyl alcohol, 3-methyl-4-nitrophenol, 4-methyl-2-nitrophenol, 4-methyl-2-propyl furan, 5-methyl-2-nitrophenol, 4-nitrophenol, 2-methyl-4,6-dinitrophenol are identified by means of mass spectrometry. Based on these by-products a toluene degradation mechanism is proposed. PMID:17490711

  19. Icariin reduces ?-synuclein over-expression by promoting ?-synuclein degradation.

    PubMed

    Zhang, Lan; Shen, Cong; Chu, Jin; Liu, Ying; Li, Yali; Zhang, Li; Li, Lin

    2015-08-01

    The objectives of this study are to investigate the effects of icariin (a main component extracted from Epimedium) on over-expression of ?-synuclein and to explore the underlying mechanisms. APPV717I transgenic (Tg) mice and A53T ?-synuclein-transfected PC12 cells were used in this study. The content of ?-synuclein mRNA was determined by reverse-transcription PCR (RT-PCR). Western blotting and immunohistochemistry were used to detect the protein expression of ?-synuclein, parkin, ubiquitin carboxy-terminal hydrolase L1 (UCH-L1), and heat shock protein 70 (HSP70). In 10-month-old APP Tg mice, ?-synuclein expression was increased, and the expression of Parkin, UCH-L1, and HSP70 was decreased in the hippocampus. Intragastrical administration of icariin (30 and 100 ?mol/kg) for 6 months (from 4 to 10 months old) decreased ?-synuclein expression and increased the expression of Parkin, UCH-L1, and HSP70 in the hippocampus of APP Tg mice. Incubation of icariin (40 and 80 ?M) with A53T ?-synuclein-transfected PC12 cells for 24 h showed no difference in the expressions of ?-synuclein mRNA among model group and icariin-treated groups, but decreased ?-synuclein protein expression in both monomer and tetramer. Along with the downregulation of ?-synuclein, icariin (40 and 80 ?M) elevated the expression of Parkin, UCH-L1, and HSP70 in A53T ?-synuclein-transfected cells. Icariin inhibited the over-expression of ?-synuclein both in vivo and in vitro. The mechanism of icariin may be related to upregulate Parkin and UCH-L1 expression in ubiquitin-proteasome system and HSP70 in molecular chaperone, thus enhancing the degradation of ?-synuclein. It is suggested that icariin may have the potential to treat Alzheimer's disease (AD) and other synucleinopathies. PMID:26169945

  20. Regulation of the pathway for the degradation of anthranilate in Aspergillus niger.

    PubMed

    Rao, P V; Sreeleela, N S; Premakumar, R; Vaidyanathan, C S

    1971-07-01

    Studies were carried out to determine the factors governing the induction of anthranilate hydroxylase and other enzymes in the pathway for the dissimilation of anthranilate by Aspergillus niger (UBC 814). The enzyme was induced by growth in the presence of tryptophan, kynurenine, anthranilate, and, surprisingly, by 3-hydroxyanthranilate, which was not an intermediate in the conversion of anthranilate to 2,3-dihydroxybenzoate. There was an initial lag in the synthesis of anthranilate hydroxylase when induced by tryptophan, anthranilate, and 3-hydroxyanthranilate. Cycloheximide inhibited the enzyme induction. Comparative studies on anthranilate hydroxylase, 2,3-dihydroxybenzoate carboxy-lyase, and catechol 1:2-oxygenase revealed that these enzymes were not coordinately induced by either anthranilate or 3-hydroxyanthranilate. Structural requirements for the induction of anthranilate hydroxylase were determined by using various analogues of anthranilate. The activity of the constitutive catechol oxygenase was increased threefold by exposure to anthranilate, 2,3-dihydroxybenzoate, or catechol. 3-Hydroxyanthranilate did not enhance the levels of catechol oxygenase activity. PMID:5563863

  1. Identification of Common Biological Pathways and Drug Targets Across Multiple Respiratory Viruses Based on Human Host Gene Expression Analysis

    PubMed Central

    Smith, Steven B.; Dampier, William; Tozeren, Aydin; Brown, James R.; Magid-Slav, Michal

    2012-01-01

    Background Pandemic and seasonal respiratory viruses are a major global health concern. Given the genetic diversity of respiratory viruses and the emergence of drug resistant strains, the targeted disruption of human host-virus interactions is a potential therapeutic strategy for treating multi-viral infections. The availability of large-scale genomic datasets focused on host-pathogen interactions can be used to discover novel drug targets as well as potential opportunities for drug repositioning. Methods/Results In this study, we performed a large-scale analysis of microarray datasets involving host response to infections by influenza A virus, respiratory syncytial virus, rhinovirus, SARS-coronavirus, metapneumonia virus, coxsackievirus and cytomegalovirus. Common genes and pathways were found through a rigorous, iterative analysis pipeline where relevant host mRNA expression datasets were identified, analyzed for quality and gene differential expression, then mapped to pathways for enrichment analysis. Possible repurposed drugs targets were found through database and literature searches. A total of 67 common biological pathways were identified among the seven different respiratory viruses analyzed, representing fifteen laboratories, nine different cell types, and seven different array platforms. A large overlap in the general immune response was observed among the top twenty of these 67 pathways, adding validation to our analysis strategy. Of the top five pathways, we found 53 differentially expressed genes affected by at least five of the seven viruses. We suggest five new therapeutic indications for existing small molecules or biological agents targeting proteins encoded by the genes F3, IL1B, TNF, CASP1 and MMP9. Pathway enrichment analysis also identified a potential novel host response, the Parkin-Ubiquitin Proteasomal System (Parkin-UPS) pathway, which is known to be involved in the progression of neurodegenerative Parkinson's disease. Conclusions Our study suggests that multiple and diverse respiratory viruses invoke several common host response pathways. Further analysis of these pathways suggests potential opportunities for therapeutic intervention. PMID:22432004

  2. Targeting of the ?6 gene to suppress degradation of ECM via inactivation of the MAPK pathway in breast adenocarcinoma cells

    PubMed Central

    ZHANG, YUHUA; WEI, LIJING; YU, JIN; LI, GUANG; ZHANG, XIURU; WANG, ANLIU; HE, YANJIAO; LI, HONGLI; YIN, DELING

    2014-01-01

    Integrin ???6 has emerged as a potential novel target for anticancer and plays a major role in promoting malignant tumor progression. Recent studies indicate that integrin ???6 occurs in many cancers. However, whether and how ???6 is regulated by genetic and epigenetic mechanisms in breast cancer remain unknown. In the present study, two different short hairpin RNAs (shRNAs) targeting the ?6 gene were designed and constructed into pSUPER, respectively, which were transfected into the MCF-7 human breast adenocarcinoma cell line. The ?6-shRNA stably transfected cells were successfully established, and significant lower levels of ???6 mRNA and protein expression were confirmed. Furthermore, inhibition of integrin ???6 markedly downregulated the expression of matrix metalloproteinase-9 (MMP-9), matrix metalloproteinase-3 (MMP-3) and urokinase plasminogen activator (uPA) in tumor conditioned medium. Furthermore, ?6-shRNA-mediated silencing of the ???6 gene obviously decreased the expression of ERK1/2. In particular, supression of integrin ???6 caused significant downregulation of the degradation of basement membrane type IV collagen secretion via modulation of the plasminogen activation cascade. Our results thus indicate that ???6 plays a fundamental role in promoting invasion and growth of breast adenocarcinoma cells. Taken together, this study revealed that targeting of the ?6 gene by RNA interference (RNAi) could efficiently downregulate ???6 expression and suppress the ERK1/2-dependent extracellular matrix degradation in vitro, which is dependent upon inactivation of the mitogen-activated protein kinase (MAPK) pathway. These findings may offer a useful therapeutic approach to block invasion and migration of breast cancer cells. PMID:25176506

  3. (Per)Chlorate-Reducing Bacteria Can Utilize Aerobic and Anaerobic Pathways of Aromatic Degradation with (Per)Chlorate as an Electron Acceptor

    PubMed Central

    Carlström, Charlotte I.; Loutey, Dana; Bauer, Stefan; Clark, Iain C.; Rohde, Robert A.; Iavarone, Anthony T.; Lucas, Lauren

    2015-01-01

    ABSTRACT The pathways involved in aromatic compound oxidation under perchlorate and chlorate [collectively known as (per)chlorate]-reducing conditions are poorly understood. Previous studies suggest that these are oxygenase-dependent pathways involving O2 biogenically produced during (per)chlorate respiration. Recently, we described Sedimenticola selenatireducens CUZ and Dechloromarinus chlorophilus NSS, which oxidized phenylacetate and benzoate, two key intermediates in aromatic compound catabolism, coupled to the reduction of perchlorate or chlorate, respectively, and nitrate. While strain CUZ also oxidized benzoate and phenylacetate with oxygen as an electron acceptor, strain NSS oxidized only the latter, even at a very low oxygen concentration (1%, vol/vol). Strains CUZ and NSS contain similar genes for both the anaerobic and aerobic-hybrid pathways of benzoate and phenylacetate degradation; however, the key genes (paaABCD) encoding the epoxidase of the aerobic-hybrid phenylacetate pathway were not found in either genome. By using transcriptomics and proteomics, as well as by monitoring metabolic intermediates, we investigated the utilization of the anaerobic and aerobic-hybrid pathways on different electron acceptors. For strain CUZ, the results indicated utilization of the anaerobic pathways with perchlorate and nitrate as electron acceptors and of the aerobic-hybrid pathways in the presence of oxygen. In contrast, proteomic results suggest that strain NSS may use a combination of the anaerobic and aerobic-hybrid pathways when growing on phenylacetate with chlorate. Though microbial (per)chlorate reduction produces molecular oxygen through the dismutation of chlorite (ClO2?), this study demonstrates that anaerobic pathways for the degradation of aromatics can still be utilized by these novel organisms. PMID:25805732

  4. Nitrate-induced photodegradation of atenolol in aqueous solution: kinetics, toxicity and degradation pathways.

    PubMed

    Ji, Yuefei; Zeng, Chao; Ferronato, Corinne; Chovelon, Jean-Marc; Yang, Xi

    2012-07-01

    The extensive utilization of ?-blockers worldwide led to frequent detection in natural water. In this study the photolysis behavior of atenolol (ATL) and toxicity of its photodegradation products were investigated in the presence of nitrate ions. The results showed that ATL photodegradation followed pseudo-first-order kinetics upon simulated solar irradiation. The photodegradation was found to be dependent on nitrate concentration and increasing the nitrate from 0.5 mML(-1) to 10 mML(-1) led to the enhancement of rate constant from 0.00101 min(-1) to 0.00716 min(-1). Hydroxyl radical was determined to play a key role in the photolysis process by using isopropanol as molecular probe. Increasing the solution pH from 4.8 to 10.4, the photodegradation rate slightly decreased from 0.00246 min(-1) to 0.00195 min(-1), probably due to pH-dependent effect of nitrate-induced .OH formation. Bicarbonate decreased the photodegradation of ATL in the presence of nitrate ions mainly through pH effect, while humic substance inhibited the photodegradation via both attenuating light and competing radicals. Upon irradiation for 240 min, only 10% reduction of total organic carbon (TOC) can be achieved in spite of 72% transformation rate of ATL, implying a majority of ATL transformed into intermediate products rather than complete mineralization. The main photoproducts of ATL were identified by using solid phase extraction-liquid chromatography-mass spectrometry (SPE-LC-MS) techniques and possible nitrate-induced photodegradation pathways were proposed. The toxicity of the phototransformation products was evaluated using aquatic species Daphnia magna, and the results revealed that photodegradation was an effective mechanism for ATL toxicity reduction in natural waters. PMID:22497785

  5. The Rut pathway for pyrimidine degradation: novel chemistry and toxicity problems.

    PubMed

    Kim, Kwang-Seo; Pelton, Jeffrey G; Inwood, William B; Andersen, Ulla; Kustu, Sydney; Wemmer, David E

    2010-08-01

    The Rut pathway is composed of seven proteins, all of which are required by Escherichia coli K-12 to grow on uracil as the sole nitrogen source. The RutA and RutB proteins are central: no spontaneous suppressors arise in strains lacking them. RutA works in conjunction with a flavin reductase (RutF or a substitute) to catalyze a novel reaction. It directly cleaves the uracil ring between N-3 and C-4 to yield ureidoacrylate, as established by both nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Although ureidoacrylate appears to arise by hydrolysis, the requirements for the reaction and the incorporation of (18)O at C-4 from molecular oxygen indicate otherwise. Mass spectrometry revealed the presence of a small amount of product with the mass of ureidoacrylate peracid in reaction mixtures, and we infer that this is the direct product of RutA. In vitro RutB cleaves ureidoacrylate hydrolytically to release 2 mol of ammonium, malonic semialdehyde, and carbon dioxide. Presumably the direct products are aminoacrylate and carbamate, both of which hydrolyze spontaneously. Together with bioinformatic predictions and published crystal structures, genetic and physiological studies allow us to predict functions for RutC, -D, and -E. In vivo we postulate that RutB hydrolyzes the peracid of ureidoacrylate to yield the peracid of aminoacrylate. We speculate that RutC reduces aminoacrylate peracid to aminoacrylate and RutD increases the rate of spontaneous hydrolysis of aminoacrylate. The function of RutE appears to be the same as that of YdfG, which reduces malonic semialdehyde to 3-hydroxypropionic acid. RutG appears to be a uracil transporter. PMID:20400551

  6. Dysregulation of protein degradation pathways may mediate the liver injury and phospholipidosis associated with a cationic amphiphilic antibiotic drug.

    PubMed

    Mosedale, Merrie; Wu, Hong; Kurtz, C Lisa; Schmidt, Stephen P; Adkins, Karissa; Harrill, Alison H

    2014-10-01

    A large number of antibiotics are known to cause drug-induced liver injury in the clinic; however, interpreting clinical risk is not straightforward owing to a lack of predictivity of the toxicity by standard preclinical species and a poor understanding of the mechanisms of toxicity. An example is PF-04287881, a novel ketolide antibiotic that caused elevations in liver function tests in Phase I clinical studies. In this study, a mouse diversity panel (MDP), comprised of 34 genetically diverse, inbred mouse strains, was utilized to model the toxicity observed with PF-04287881 treatment and investigate potential mechanisms that may mediate the liver response. Significant elevations in serum alanine aminotransferase (ALT) levels in PF-04287881-treated animals relative to vehicle-treated controls were observed in the majority (88%) of strains tested following a seven day exposure. The average fold elevation in ALT varied by genetic background and correlated with microscopic findings of hepatocellular hypertrophy, hepatocellular single cell necrosis, and Kupffer cell vacuolation (confirmed as phospholipidosis) in the liver. Global liver mRNA expression was evaluated in a subset of four strains to identify transcript and pathway differences that distinguish susceptible mice from resistant mice in the context of PF-04287881 treatment. The protein ubiquitination pathway was highly enriched among genes associated with PF-04287881-induced hepatocellular necrosis. Expression changes associated with PF-04287881-induced phospholipidosis included genes involved in drug transport, phospholipid metabolism, and lysosomal function. The findings suggest that perturbations in genes involved in protein degradation leading to accumulation of oxidized proteins may mediate the liver injury induced by this drug. PMID:24967691

  7. Distribution of the Coenzyme M Pathway of Epoxide Metabolism among Ethene- and Vinyl Chloride-Degrading Mycobacterium Strains

    PubMed Central

    Coleman , Nicholas V.; Spain, Jim C.

    2003-01-01

    An epoxyalkane:coenzyme M (CoM) transferase (EaCoMT) enzyme was recently found to be active in the aerobic vinyl chloride (VC) and ethene assimilation pathways of Mycobacterium strain JS60. In the present study, EaCoMT activity and genes were investigated in 10 different mycobacteria isolated on VC or ethene from diverse environmental samples. In all cases, epoxyethane metabolism in cell extracts was dependent on CoM, with average specific activities of EaCoMT between 380 and 2,910 nmol/min/mg of protein. PCR with primers based on conserved regions of EaCoMT genes from Mycobacterium strain JS60 and the propene oxidizers Xanthobacter strain Py2 and Rhodococcus strain B-276 yielded fragments (834 bp) of EaCoMT genes from all of the VC- and ethene-assimilating isolates. The Mycobacterium EaCoMT genes form a distinct cluster and are more closely related to the EaCoMT of Rhodococcus strain B-276 than that of Xanthobacter strain Py2. The incongruence of the EaCoMT and 16S rRNA gene trees and the fact that isolates from geographically distant locations possessed almost identical EaCoMT genes suggest that lateral transfer of EaCoMT among the Mycobacterium strains has occurred. Pulsed-field gel electrophoresis revealed large linear plasmids (110 to 330 kb) in all of the VC-degrading strains. In Southern blotting experiments, the strain JS60 EaCoMT gene hybridized to many of the plasmids. The CoM-mediated pathway of epoxide metabolism appears to be universal in alkene-assimilating mycobacteria, possibly because of plasmid-mediated lateral gene transfer. PMID:14532060

  8. An endogenous calcium-dependent, caspase-independent intranuclear degradation pathway in thymocyte nuclei: Antagonism by physiological concentrations of K{sup +} ions

    SciTech Connect

    Ajiro, Kozo; Bortner, Carl D.; Westmoreland, Jim [Laboratory of Signal Transduction, National Institute of Environmental Health Science, National Institute of Health, Research Triangle Park, NC 27709 (United States); Laboratory of Molecular Genetics, National Institute of Environmental Health Science, National Institute of Health, Research Triangle Park, NC 27709 (United States); Cidlowski, John A. [Laboratory of Signal Transduction, National Institute of Environmental Health Science, National Institute of Health, Research Triangle Park, NC 27709 (United States); Laboratory of Molecular Genetics, National Institute of Environmental Health Science, National Institute of Health, Research Triangle Park, NC 27709 (United States)], E-mail: cidlows1@niehs.nih.gov

    2008-04-01

    Calcium ions have been implicated in apoptosis for many years, however the precise role of this ion in the cell death process remains incomplete. We have extensively examined the role of Ca{sup 2+} on nuclear degradation in vitro using highly purified nuclei isolated from non-apoptotic rat thymocytes. We show that these nuclei are devoid of CAD (caspase-activated DNase), and DNA degradation occurs independent of caspase activity. Serine proteases rather than caspase-3 appear necessary for this Ca{sup 2+}-dependent DNA degradation in nuclei. We analyzed nuclei treated with various concentrations of Ca{sup 2+} in the presence of both a physiological (140 mM) and apoptotic (40 mM) concentration of KCl. Our results show that a 5-fold increase in Ca{sup 2+} is required to induce DNA degradation at the physiological KCl concentration compared to the lower, apoptotic concentration of the cation. Ca{sup 2+}-induced internucleosomal DNA degradation was also accompanied by the release of histones, however the apoptotic-specific phosphorylation of histone H2B does not occur in these isolated nuclei. Interestingly, physiological concentrations of K{sup +} inhibit both Ca{sup 2+}-dependent DNA degradation and histone release suggesting that a reduction of intracellular K{sup +} is necessary for this apoptosis-associated nuclear degradation in cells. Together, these data define an inherent caspase-independent catabolic pathway in thymocyte nuclei that is sensitive to physiological concentrations of intracellular cations.

  9. A JNK-mediated autophagy pathway that triggers c-IAP degradation and necroptosis for anticancer chemotherapy

    PubMed Central

    He, Weiyang; Wang, Qiong; Srinivasan, Balasubramanian; Xu, Jennings; Padilla, Mabel T.; Li, Zi; Wang, Xia; Liu, Yushi; Gou, Xin; Shen, Han-Ming; Xing, Chengguo; Lin, Yong

    2014-01-01

    Killing cancer cells through the induction of apoptosis is one of the main mechanisms of chemotherapy. However, numerous cancer cells have primary or acquired apoptosis resistance, resulting in chemoresistance. In this study, using a novel chalcone derivative chalcone-24 (Chal-24), we identified a novel anticancer mechanism through autophagy-mediated necroptosis (RIP1- and RIP3-dependent necrosis). Chal-24 potently killed different cancer cells with induction of necrotic cellular morphology while causing no detectable caspase activation. Blocking the necroptosis pathway with either necrostatin-1 or by knockdown of RIP1 and RIP3 effectively blocked the cytotoxicity of Chal-24, suggesting that Chal-24-induced cell death is associated with necroptosis. Chal-24 robustly activated JNK and ERK and blockage of which effectively suppressed Chal-24-induced cytotoxicity. In addition, Chal-24 strongly induced autophagy that is dependent on JNK-mediated phosphorylation of Bcl-2 and Bcl-xL and dissociation of Bcl-2 or Bcl-xL from Beclin1. Importantly, suppression of autophagy, with either pharmacological inhibitors or siRNAs targeting the essential autophagy components ATG7 and Beclin1, effectively attenuated Chal-24-induced cell death. Furthermore, we found that autophagy activation resulted in c-IAP1 and c-IAP2 degradation and formation of the Ripoptosome that contributes to necroptosis. These results thus establish a novel mechanism for killing cancer cells that involves autophagy-mediated necroptosis, which may be employed for overcoming chemoresistance. PMID:23831571

  10. Cathepsin?L Ameliorates Cardiac Hypertrophy Through Activation of the Autophagy–Lysosomal Dependent Protein Processing Pathways

    PubMed Central

    Sun, Mei; Ouzounian, Maral; de Couto, Geoffrey; Chen, Manyin; Yan, Ran; Fukuoka, Masahiro; Li, Guohua; Moon, Mark; Liu, Youan; Gramolini, Anthony; Wells, George J.; Liu, Peter P.

    2013-01-01

    Background Autophagy is critical in the maintenance of cellular protein quality control, the final step of which involves the fusion of autophagosomes with lysosomes. Cathepsin?L (CTSL) is a key member of the lysosomal protease family that is expressed in the murine and human heart, and it may play an important role in protein turnover. We hypothesized that CTSL is important in regulating protein processing in the heart, particularly under pathological stress. Methods and Results Phenylephrine?induced cardiac hypertrophy in vitro was more pronounced in CTSL?deficient neonatal cardiomyocytes than in in controls. This was accompanied by a significant accumulation of autophagosomes, increased levels of ubiquitin?conjugated protein, as well as impaired protein degradation and decreased cell viability. These effects were partially rescued with CTSL1 replacement via adeno?associated virus–mediated gene transfer. In the in vivo murine model of aortic banding (AB), a deficiency in CTSL markedly exacerbated cardiac hypertrophy, worsened cardiac function, and increased mortality. Ctsl?/? AB mice demonstrated significantly decreased lysosomal activity and increased sarcomere?associated protein aggregation. Homeostasis of the endoplasmic reticulum was also altered by CTSL deficiency, with increases in Bip and GRP94 proteins, accompanied by increased ubiquitin–proteasome system activity and higher levels of ubiquitinated proteins in response to AB. These changes ultimately led to a decrease in cellular ATP production, enhanced oxidative stress, and increased cellular apoptosis. Conclusions Lysosomal CTSL attenuates cardiac hypertrophy and preserves cardiac function through facilitation of autophagy and proteasomal protein processing. PMID:23608608

  11. Novel Investigational Agents & Clinical Trials

    E-print Network

    Kay, Mark A.

    mechanisms: Signal transduction/transcription activation pathways (e.g., ubiquitin-proteasome, AKT/PI3K/mTOR-proteasome, AKT/PI3K/mTOR, RAS/RAF/MEK, MAPK) Apoptotic pathways (e.g. Bcl/Bax, TNFR, Fas, miRNAs) Epigenetics (e.g., ubiquitin-proteasome, AKT/PI3K/mTOR, RAS/RAF/MEK, MAPK) Apoptotic pathways (e.g. Bcl/Bax, TNFR, Fas, mi

  12. Pathways and metabolites of microbial degradation of selected acidic pharmaceutical and their occurrence in municipal wastewater treated by a membrane bioreactor.

    PubMed

    Quintana, José Benito; Weiss, Stefan; Reemtsma, Thorsten

    2005-07-01

    Laboratory degradation tests with five acidic pharmaceuticals using activated sludge as inocculum under aerobic conditions were performed and microbial metabolites were analysed by liquid chromatography-mass spectrometry (LC-MS). Ketoprofen was partly mineralized as a sole source of carbon and energy and the metabolites determined by LC-MS suggest microbial ketoprofen degradation to proceed along the pathway known for biphenyls and related compounds. Bezafibrate, naproxen and ibuprofen were degraded only cometabolically whereas no transformation was obtained for diclofenac. Some biodegradation intermediates in these batch tests could be tentatively identified by means of LC-MS. The first step in microbial bezafibrate degradation appears to be the hydrolytic cleavage of the amide bond, generating well degradable 4-chlorobenzoic acid as one of the hydrolysis products. As previously found for mammals, ether cleavage and formation of desmethylnaproxen was the initial step in microbial degradation of naproxen. Two isomers of hydroxy-ibuprofen were detected as intermediates in the mineralization of ibuprofen. Laboratory studies suggest that naproxen and ibuprofen can be fully mineralized whereas more stable metabolites occur in microbial ketoprofen and bezafibrate transformation, that may deserve further attention. A LC-MS method for the trace analysis of these metabolites in water was developed and applied to municipal wastewater. Municipal wastewater treatment by a membrane bioreactor may gradually improve the removal of these pharmaceuticals. PMID:15979124

  13. Privileged delivery of polymer nanoparticles to the perinuclear region of live cells via a non-clathrin, non-degradative pathway

    Microsoft Academic Search

    Samuel K. Lai; Kaoru Hida; Stan T. Man; Clive Chen; Carolyn Machamer; Trina A. Schroer; Justin Hanes

    2007-01-01

    The efficacy of many therapeutic molecules could be greatly enhanced by polymer-based nanoparticle systems capable of delivering them to the direct vicinity of the cell nucleus. However, degradation of the particles and encapsulated drugs within the enzyme-rich and low-pH environments of the endo\\/lysosomal pathway of cells has dramatically limited the efficacy of such systems. In this paper, we discovered that

  14. PP2A inhibitors suppress migration and growth of PANC-1 pancreatic cancer cells through inhibition on the Wnt/?-catenin pathway by phosphorylation and degradation of ?-catenin.

    PubMed

    Wu, Meng-Yao; Xie, Xin; Xu, Ze-Kuan; Xie, Li; Chen, Zheng; Shou, Liu-Mei; Gong, Fei-Ran; Xie, Yu-Feng; Li, Wei; Tao, Min

    2014-08-01

    Cantharidin is an active constituent of mylabris, a traditional Chinese medicine, and presents strong anticancer activity in various cell lines. Cantharidin is a potent and selective inhibitor of serine/threonine protein phosphatase 2A (PP2A). Our previous studies revealed the prospect of application of cantharidin, as well as other PP2A inhibitors, in the treatment of pancreatic cancer. However, the mechanisms involved in the anticancer effect of PP2A inhibitors have not been fully explored. The Wnt/??catenin pathway is involved in cell migration and proliferation and participates in the progression of pancreatic cancer. If ??catenin is phosphorylated and degraded, the Wnt/??catenin pathway is blocked. PP2A dephosphorylates ??catenin and keeps the Wnt/??catenin pathway active. In the present study, we found that PP2A inhibitor treatment induced phosphorylation and degradation of ??catenin. The suppression on the migration and growth of PANC?1 pancreatic cancer cells could be attenuated by pretreatment with FH535, a ??catenin pathway inhibitor. Microarray showed that PP2A inhibitor treatment induced expression changes in 13 of 138 genes downstream of the ??catenin pathway. Real?time PCR further confirmed that FH535 attenuated the expression changes induced by PP2A inhibitors in 6 of these 13 candidate genes. These 6 genes, VEGFB, Dkk3, KRT8, NRP1, Cacnalg and WISP2, have been confirmed to participate in the migration and/or growth regulation in previous studies. Thus, the phosphorylation- and degradation-mediated suppression on ??catenin participates in the cytotoxicity of PP2A inhibitors. Our findings may provide insight into the treatment of pancreatic cancer using a targeting PP2A strategy. PMID:24926961

  15. Sulfamethoxazole in poultry wastewater: Identification, treatability and degradation pathway determination in a membrane-photocatalytic slurry reactor.

    PubMed

    Asha, Raju C; Kumar, Mathava

    2015-08-24

    The presence of sulfamethoxazole (SMX) in a real-time poultry wastewater was identified via HPLC analysis. Subsequently, SMX removal from the poultry wastewater was investigated using a continuous-mode membrane-photocatalytic slurry reactor (MPSR). The real-time poultry wastewater was found to have an SMX concentration of 0-2.3 mg L(-1). A granular activated carbon supported TiO2 (GAC-TiO2) was synthesized, characterized and used in MPSR experiments. The optimal MPSR condition, i.e., HRT ?125 min and catalyst dosage 529.3 mg L(-1), for complete SMX removal was found out using unconstrained optimization technique. Under the optimized condition, the effect of SMX concentration on MPSR performance was investigated by synthetic addition of SMX (i.e., 1, 25, 50, 75 and 100 mg L(-1)) into the wastewater. Interestingly, complete removals of total volatile solids (TVS), biochemical oxygen demand (BOD) and SMX were observed under all SMX concentrations investigated. However, a decline in SMX removal rate and proportionate increase in transmembrane-pressure (TMP) were observed when the SMX concentration was increased to higher levels. In the MPSR, the SMX mineralization was through one of the following degradation pathways: (i) fragmentation of the isoxazole ring and (ii) the elimination of methyl and amide moieties followed by the formation of phenyl sulfinate ion. These results show that the continuous-mode MPSR has great potential in the removal for SMX contaminated real-time poultry wastewater and similar organic micropollutants from wastewater. PMID:26121016

  16. A systems biology approach to identify molecular pathways altered by HDAC inhibition in osteosarcoma.

    PubMed

    Wittenburg, Luke A; Ptitsyn, Andrey A; Thamm, Douglas H

    2012-03-01

    Osteosarcoma (OS) is the most common primary tumor in humans and dogs affecting the skeleton, and spontaneously occurring OS in dogs serves as an extremely useful model. Unacceptable toxicities using current treatment protocols prevent further dose-intensification from being a viable option to improve patient survival and thus, novel treatment strategies must be developed. Histone deacetylase inhibitors (HDACi) have recently emerged as a promising class of therapeutics demonstrating an ability to enhance the anti-tumor activity of traditional chemotherapeutics. To date, gene expression analysis of OS cell lines treated with HDACi has not been reported, and evaluation of the resultant gene expression changes may provide insight into the mechanisms that lead to success of HDACi. Canine OS cells, treated with a clinically relevant concentration of the HDACi valproic acid (VPA), were used for expression analysis on the Affymetrix canine v2.0 genechip. Differentially expressed genes were grouped into pathways based upon functional annotation; pathway analysis was performed with MetaCore and Ingenuity Pathways Analysis software. Validation of microarray results was performed by a combination of qRT-PCR and functional/biochemical assays revealing oxidative phosphorylation, cytoskeleton remodeling, cell cycle, and ubiquitin-proteasome among those pathways most affected by HDACi. The mitomycin C-bioactivating enzyme NQ01 also demonstrated upregulation following VPA treatment, leading to synergistic reductions in cell viability. These results provide a better understanding of the mechanisms by which HDACi exert their effect in OS, and have the potential to identify biomarkers that may serve as novel targets and/or predictors of response to HDACi-containing combination therapies in OS. PMID:21976144

  17. Ubiquitin-dependent Protein Degradation at the Yeast Endoplasmic Reticulum and Nuclear Envelope

    PubMed Central

    Zattas, Dimitrios; Hochstrasser, Mark

    2014-01-01

    The endoplasmic reticulum (ER) is the primary organelle in eukaryotic cells where membrane and secreted proteins are inserted into or across cell membranes. Its membrane bilayer and luminal compartments provide a favorable environment for the folding and assembly of thousands of newly synthesized proteins. However, protein folding is intrinsically error-prone, and various stress conditions can further increase levels of protein misfolding and damage, particularly in the ER, which can lead to cellular dysfunction and disease. The ubiquitin-proteasome system (UPS) is responsible for the selective destruction of a vast array of protein substrates, either for protein quality control or to allow rapid changes in the levels of specific regulatory proteins. In this review, we will focus on the components and mechanisms of ER-associated protein degradation (ERAD), an important branch of the UPS. ER membranes extend from subcortical regions of the cell to the nuclear envelope, with its continuous outer and inner membranes; the nuclear envelope is a specialized subdomain of the ER. ERAD presents additional challenges to the UPS beyond those faced with soluble substrates of the cytoplasm and nucleus. These include recognition of sugar modifications that occur in the ER, retrotranslocation of proteins across the membrane bilayer, and transfer of substrates from the ER extraction machinery to the proteasome. Here we review characteristics of ERAD substrate degradation signals (degrons), mechanisms underlying substrate recognition and processing by the ERAD machinery, and ideas on the still unresolved problem of how substrate proteins are moved across and extracted from the ER membrane. PMID:25231236

  18. NGX6a is degraded through a proteasome-dependent pathway without ubiquitination mediated by ezrin, a cytoskeleton-membrane linker.

    PubMed

    Wang, Li; Li, Xiaoling; Xiang, Bo; Zhou, Ming; Li, Xiayu; Xiong, Wei; Niu, Man; Wei, Pingpin; Wang, Zeyou; Wang, Heran; Chen, Pan; Shen, Shourong; Peng, Shuping; Li, Guiyuan

    2014-12-26

    Our previous study demonstrated that the NGX6b gene acts as a suppressor in the invasion and migration of nasopharyngeal carcinoma (NPC). Recently, we identified the novel isoform NGX6a, which is longer than NGX6b. In this study, we first found that NGX6a was degraded in NPC cells and that this degradation was mediated by ezrin, a linker between membrane proteins and the cytoskeleton. Specific siRNAs against ezrin increase the protein level of NGX6a in these cells. During degradation, NGX6a is not ubiquitinated but is degraded through a proteasome-dependent pathway. The distribution pattern of ezrin was negatively associated with NGX6a in an immunochemistry analysis of a nasopharyngeal carcinoma tissue microarray and fetus multiple organ tissues and Western blot analysis in nasopharyngeal and NPC cell lines, suggesting that ezrin and NGX6a are associated and are involved in the progression and invasion of NPC. By mapping the interacting binding sites, the seven-transmembrane domain of NGX6a was found to be the critical region for the degradation of NGX6a, and the amino terminus of ezrin is required for the induction of NGX6a degradation. The knockdown of ezrin or transfection of the NGX6a mutant CO, which has an EGF-like domain and a transmembrane 1 domain, resulted in no degradation, significantly reducing the ability of invasion and migration of NPC cells. This study provides a novel molecular mechanism for the low expression of NGX6a in NPC cells and an important molecular event in the process of invasion and metastasis of nasopharyngeal carcinoma cells. PMID:25378401

  19. Dubble or Nothing? Is HAUSP Deubiquitylating Enzyme the Final Arbiter of p53 Levels?

    NSDL National Science Digital Library

    Stephen A. Wood (University of Adelaide; Child Health Research Institute and Centre for the Molecular Genetics of Development REV)

    2002-07-30

    Signal transduction processes can be regulated by biochemical modifications that affect protein activity or localization and by protein stability. Proteins implicated in cancer, such as ?-catenin and p53, are regulated by a combination of posttranslational modifications and protein degradation by the ubiquitin-proteasome pathway. Wood explores how ubiquitylation of these proteins may not be as unidirectional as previously thought. With the identification of substrate-specific deubiquitylating enzymes, ubiquitylation may not always lead to protein destruction, but may provide another finely tunable step for controlling protein activity.

  20. Ubiquitin domain proteins in disease

    PubMed Central

    Madsen, Louise; Schulze, Andrea; Seeger, Michael; Hartmann-Petersen, Rasmus

    2007-01-01

    The human genome encodes several ubiquitin-like (UBL) domain proteins (UDPs). Members of this protein family are involved in a variety of cellular functions and many are connected to the ubiquitin proteasome system, an essential pathway for protein degradation in eukaryotic cells. Despite their structural similarity, the UBL domains appear to have a range of different targets, resulting in a considerable diversity with respect to UDP function. Here, we give a short summary of the biochemical and physiological roles of the UDPs, which have been linked to human diseases including neurodegeneration and cancer. Publication history: Republished from Current BioData's Targeted Proteins database (TPdb; ). PMID:18047733

  1. Identification of an AAA ATPase VPS4B-Dependent Pathway That Modulates Epidermal Growth Factor Receptor Abundance and Signaling during Hypoxia

    PubMed Central

    Lin, H. Helen; Li, Xu; Chen, Jo-Lin; Sun, Xiuzhu; Cooper, Fariba Norouziyan; Chen, Yun-Ru; Zhang, Wenyu; Chung, Yiyin; Li, Angela; Cheng, Chun-Ting; Yang, Lixin; Deng, XuTao; Liu, Xiyong; Yen, Yun; Johnson, Deborah L.; Shih, Hsiu-Ming; Yang, Austin

    2012-01-01

    VPS4B, an AAA ATPase (ATPase associated with various cellular activities), participates in vesicular trafficking and autophagosome maturation in mammalian cells. In solid tumors, hypoxia is a common feature and an indicator of poor treatment outcome. Our studies demonstrate that exogenous or endogenous (assessed with anchorage-independent three-dimensional multicellular spheroid culture) hypoxia induces VPS4B downregulation by the ubiquitin-proteasome system. Inhibition of VPS4B function by short hairpin VPS4B (sh-VPS4B) or expression of dominant negative VPS4B(E235Q) promotes anchorage-independent breast cancer cell growth and resistance to gefitinib, U0126, and genotoxicity. Biochemically, hyperactivation of epidermal growth factor receptor (EGFR), a receptor tyrosine kinase essential for cell proliferation and survival, accompanied by increased EGFR accumulation and altered intracellular compartmentalization, is observed in cells with compromised VPS4B. Furthermore, enhanced FOS/JUN induction and AP-1 promoter activation are noted in EGF-treated cells with VPS4B knockdown. However, VPS4B depletion does not affect EGFRvIII stability or its associated signaling. An inverse correlation between VPS4B expression and EGFR abundance is observed in breast tumors, and high-grade or recurrent breast carcinomas exhibit lower VPS4B expression. Together, our findings highlight a potentially critical role of VPS4B downregulation or chronic-hypoxia-induced VPS4B degradation in promoting tumor progression, unveiling a nongenomic mechanism for EGFR overproduction in human breast cancer. PMID:22252323

  2. The Homogentisate Pathway: a Central Catabolic Pathway Involved in the Degradation of L-Phenylalanine, L-Tyrosine, and 3-Hydroxyphenylacetate in Pseudomonas putida

    Microsoft Academic Search

    Elsa Arias-Barrau; Elõ ´ as; R. Olivera; Cristina Fernandez; Beatriz Galan; Jose L. Garcõ ´; Eduardo Dõ; Baltasar Minambres

    Pseudomonas putida metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate. Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate.

  3. The Non-Canonical Wnt/PKC Pathway Regulates Mitochondrial Dynamics through Degradation of the Arm-Like Domain-Containing Protein Alex3

    PubMed Central

    Mirra, Serena; Quevedo, Martí; Garcia-Fernàndez, Jordi; Ulloa, Fausto; Burgaya, Ferrán; Soriano, Eduardo

    2013-01-01

    The regulation of mitochondrial dynamics is vital in complex cell types, such as neurons, that transport and localize mitochondria in high energy-demanding cell domains. The Armcx3 gene encodes a mitochondrial-targeted protein (Alex3) that contains several arm-like domains. In a previous study we showed that Alex3 protein regulates mitochondrial aggregation and trafficking. Here we studied the contribution of Wnt proteins to the mitochondrial aggregation and dynamics regulated by Alex3. Overexpression of Alex3 in HEK293 cells caused a marked aggregation of mitochondria, which was attenuated by treatment with several Wnts. We also found that this decrease was caused by Alex3 degradation induced by Wnts. While the Wnt canonical pathway did not alter the pattern of mitochondrial aggregation induced by Alex3, we observed that the Wnt/PKC non-canonical pathway regulated both mitochondrial aggregation and Alex3 protein levels, thereby rendering a mitochondrial phenotype and distribution similar to control patterns. Our data suggest that the Wnt pathway regulates mitochondrial distribution and dynamics through Alex3 protein degradation. PMID:23844091

  4. Use of 13C NMR and ftir for elucidation of degradation pathways during natural litter decomposition and composting I. early stage leaf degradation

    USGS Publications Warehouse

    Wershaw, R.L.; Leenheer, J.A.; Kennedy, K.R.; Noyes, T.I.

    1996-01-01

    Oxidative degradation of plant tissue leads to the formation of natural dissolved organic carbon (DOC) and humus. Infrared (IR) and 13C nuclear magnetic resonance (NMR) spectrometry have been used to elucidate the chemical reactions of the early stages of degradation that give rise to DOC derived from litter and compost. The results of this study indicate that oxidation of the lignin components of plant tissue follows the sequence of O-demethylation, and hydroxylation followed by ring-fission, chain-shortening, and oxidative removal of substituents. Oxidative ring-fission leads to the formation of carboxylic acid groups on the cleaved ends of the rings and, in the process, transforms phenolic groups into aliphatic alcoholic groups. The carbohydrate components are broken down into aliphatic hydroxy acids and aliphatic alcohols.

  5. The Arabidopsis F-Box Protein CORONATINE INSENSITIVE1 Is Stabilized by SCFCOI1 and Degraded via the 26S Proteasome Pathway[C][W

    PubMed Central

    Yan, Jianbin; Li, Haiou; Li, Shuhua; Yao, Ruifeng; Deng, Haiteng; Xie, Qi; Xie, Daoxin

    2013-01-01

    Jasmonate regulates critical aspects of plant development and defense. The F-box protein CORONATINE INSENSITIVE1 (COI1) functions as a jasmonate receptor and forms Skp1/Cullin1/F-box protein COI1 (SCFCOI1) complexes with Arabidopsis thaliana Cullin1 and Arabidopsis Skp1-like1 (ASK1) to recruit its substrate jasmonate ZIM-domain proteins for ubiquitination and degradation. Here, we reveal a mechanism regulating COI1 protein levels in Arabidopsis. Genetic and biochemical analysis and in vitro degradation assays demonstrated that the COI1 protein was initially stabilized by interacting with ASK1 and further secured by assembly into SCFCOI1 complexes, suggesting a function for SCFCOI1 in the stabilization of COI1 in Arabidopsis. Furthermore, we show that dissociated COI1 is degraded through the 26S proteasome pathway, and we identified the 297th Lys residue as an active ubiquitination site in COI1. Our data suggest that the COI1 protein is strictly regulated by a dynamic balance of SCFCOI1-mediated stabilization and 26S proteasome–mediated degradation and thus maintained at a protein level essential for proper biological functions in Arabidopsis development and defense responses. PMID:23386265

  6. How early studies on secreted and membrane protein quality control gave rise to the ER associated degradation (ERAD) pathway: The early history of ERAD

    PubMed Central

    Needham, Patrick G.; Brodsky, Jeffrey L.

    2013-01-01

    All newly synthesized proteins are subject to quality control check-points, which prevent aberrant polypeptides from harming the cell. For proteins that ultimately reside in the cytoplasm, components that also reside in the cytoplasm were known for many years to mediate quality control. Early biochemical and genetic data indicated that misfolded proteins were selected by molecular chaperones and then targeted to the proteasome (in eukaryotes) or to proteasome-like particles (in bacteria) for degradation. What was less clear was how secreted and integral membrane proteins, which in eukaryotes enter the endoplasmic reticulum (ER), were subject to quality control decisions. In this review, we highlight early studies that ultimately led to the discovery that secreted and integral membrane proteins also utilize several components that constitute the cytoplasmic quality machinery. This component of the cellular quality control pathway is known as ER associated degradation, or ERAD. PMID:23557783

  7. Catabolite repression of the toluene degradation pathway in Pseudomonas putida harboring pWW0 under various conditions of nutrient limitation in chemostat culture.

    PubMed Central

    Duetz, W A; Marqués, S; Wind, B; Ramos, J L; van Andel, J G

    1996-01-01

    In earlier studies, the pathway of toluene and m- and p-xylene degradation (TOL pathway) in Pseudomonas putida (pWW0) was found to be subject to catabolite repression when the strain was grown at the maximal rate on glucose or succinate in the presence of an inducer. This report describes catabolite repression of the TOL pathway by succinate in chemostat cultures run at a low dilution rate (D = 0.05 h-1) under different conditions of inorganic-nutrient limitation. The activity of benzylalcohol dehydrogenase (BADH) in cell extracts was used as a measure of the expression of the TOL upper pathway. When cells were grown in the presence of 10 to 15 mM succinate under conditions of phosphate or sulfate limitation, the BADH activity in response to the nonmetabolizable inducer o-xylene was less than 2% of that of cells grown under conditions of succinate limitation. Less repression was found under conditions of ammonium or oxygen limitation (2 to 10% and 20 to 35%, respectively, of the BADH levels under succinate limitation). The BADH expression levels determined under the different growth conditions appeared to correlate well with the mRNA transcript levels from the upper pathway promoter (Pu), which indicates that repression was due to a blockage at the transcriptional level. The meta-cleavage pathway was found to be less susceptible to catabolite repression. The results obtained suggest that the occurrence of catabolite repression is related to a high-energy status of the cells rather than to a high growth rate or directly to the presence of growth-saturating concentrations of a primary carbon and energy source. PMID:8593060

  8. The endosomal sorting complex required for transport pathway mediates chemokine receptor CXCR4-promoted lysosomal degradation of the mammalian target of rapamycin antagonist DEPTOR.

    PubMed

    Verma, Rita; Marchese, Adriano

    2015-03-13

    G protein-coupled receptor (GPCR) signaling mediates many cellular functions, including cell survival, proliferation, and cell motility. Many of these processes are mediated by GPCR-promoted activation of Akt signaling by mammalian target of rapamycin complex 2 (mTORC2) and the phosphatidylinositol 3-kinase (PI3K)/phosphoinositide-dependent kinase 1 (PDK1) pathway. However, the molecular mechanisms by which GPCRs govern Akt activation by these kinases remain poorly understood. Here, we show that the endosomal sorting complex required for transport (ESCRT) pathway mediates Akt signaling promoted by the chemokine receptor CXCR4. Pharmacological inhibition of heterotrimeric G protein G?i or PI3K signaling and siRNA targeting ESCRTs blocks CXCR4-promoted degradation of DEPTOR, an endogenous antagonist of mTORC2 activity. Depletion of ESCRTs by siRNA leads to increased levels of DEPTOR and attenuated CXCR4-promoted Akt activation and signaling, consistent with decreased mTORC2 activity. In addition, ESCRTs likely have a broad role in Akt signaling because ESCRT depletion also attenuates receptor tyrosine kinase-promoted Akt activation and signaling. Our data reveal a novel role for the ESCRT pathway in promoting intracellular signaling, which may begin to identify the signal transduction pathways that are important in the physiological roles of ESCRTs and Akt. PMID:25605718

  9. Blocking Phosphatidylcholine Utilization in Pseudomonas aeruginosa, via Mutagenesis of Fatty Acid, Glycerol and Choline Degradation Pathways, Confirms the Importance of This Nutrient Source In Vivo

    PubMed Central

    Norris, Michael H.; Troyer, Ryan M.; Son, Mike S.; Schweizer, Herbert P.; Dow, Steven W.; Hoang, Tung T.

    2014-01-01

    Pseudomonas aeruginosa can grow to very high-cell-density (HCD) during infection of the cystic fibrosis (CF) lung. Phosphatidylcholine (PC), the major component of lung surfactant, has been hypothesized to support HCD growth of P. aeruginosa in vivo. The phosphorylcholine headgroup, a glycerol molecule, and two long-chain fatty acids (FAs) are released by enzymatic cleavage of PC by bacterial phospholipase C and lipases. Three different bacterial pathways, the choline, glycerol, and fatty acid degradation pathways, are then involved in the degradation of these PC components. Here, we identified five potential FA degradation (Fad) related fadBA-operons (fadBA1-5, each encoding 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA thiolase). Through mutagenesis and growth analyses, we showed that three (fadBA145) of the five fadBA-operons are dominant in medium-chain and long-chain Fad. The triple fadBA145 mutant also showed reduced ability to degrade PC in vitro. We have previously shown that by partially blocking Fad, via mutagenesis of fadBA5 and fadDs, we could significantly reduce the ability of P. aeruginosa to replicate on FA and PC in vitro, as well as in the mouse lung. However, no studies have assessed the ability of mutants, defective in choline and/or glycerol degradation in conjunction with Fad, to grow on PC or in vivo. Hence, we constructed additional mutants (?fadBA145?glpD, ?fadBA145?betAB, and ?fadBA145?betAB?glpD) significantly defective in the ability to degrade FA, choline, and glycerol and, therefore, PC. The analysis of these mutants in the BALB/c mouse lung infection model showed significant inability to utilize PC in vitro, resulted in decreased replication fitness and competitiveness in vivo compared to the complement strain, although there was little to no variation in typical virulence factor production (e.g., hemolysin, lipase, and protease levels). This further supports the hypothesis that lung surfactant PC serves as an important nutrient for P. aeruginosa during CF lung infection. PMID:25068317

  10. The Extracellular Signal-regulated Kinase–Mitogen-activated Protein Kinase Pathway Phosphorylates and Targets Cdc25A for SCF?-TrCP-dependent Degradation for Cell Cycle Arrest

    PubMed Central

    Isoda, Michitaka; Kanemori, Yoshinori; Nakajo, Nobushige; Uchida, Sanae; Yamashita, Katsumi; Ueno, Hiroyuki

    2009-01-01

    The extracellular signal-regulated kinase (ERK) pathway is generally mitogenic, but, upon strong activation, it causes cell cycle arrest by a not-yet fully understood mechanism. In response to genotoxic stress, Chk1 hyperphosphorylates Cdc25A, a positive cell cycle regulator, and targets it for Skp1/Cullin1/F-box protein (SCF)?-TrCP ubiquitin ligase-dependent degradation, thereby leading to cell cycle arrest. Here, we show that strong ERK activation can also phosphorylate and target Cdc25A for SCF?-TrCP-dependent degradation. When strongly activated in Xenopus eggs, the ERK pathway induces prominent phosphorylation and SCF?-TrCP-dependent degradation of Cdc25A. p90rsk, the kinase downstream of ERK, directly phosphorylates Cdc25A on multiple sites, which, interestingly, overlap with Chk1 phosphorylation sites. Furthermore, ERK itself phosphorylates Cdc25A on multiple sites, a major site of which apparently is phosphorylated by cyclin-dependent kinase (Cdk) in Chk1-induced degradation. p90rsk phosphorylation and ERK phosphorylation contribute, roughly equally and additively, to the degradation of Cdc25A, and such Cdc25A degradation occurs during oocyte maturation in which the endogenous ERK pathway is fully activated. Finally, and importantly, ERK-induced Cdc25A degradation can elicit cell cycle arrest in early embryos. These results suggest that strong ERK activation can target Cdc25A for degradation in a manner similar to, but independent of, Chk1 for cell cycle arrest. PMID:19244340

  11. CHIP stabilizes amyloid precursor protein via proteasomal degradation and p53-mediated trans-repression of ?-secretase.

    PubMed

    Singh, Amir Kumar; Pati, Uttam

    2015-08-01

    In patient with Alzheimer's disease (AD), deposition of amyloid-beta A?, a proteolytic cleavage of amyloid precursor protein (APP) by ?-secretase/BACE1, forms senile plaque in the brain. BACE1 activation is caused due to oxidative stresses and dysfunction of ubiquitin-proteasome system (UPS), which is linked to p53 inactivation. As partial suppression of BACE1 attenuates A? generation and AD-related pathology, it might be an ideal target for AD treatment. We have shown that both in neurons and in HEK-APP cells, BACE1 is a new substrate of E3-ligase CHIP and an inverse relation exists between CHIP and BACE1 level. CHIP inhibits ectopic BACE1 level by promoting its ubiquitination and proteasomal degradation, thus reducing APP processing; it stabilizes APP in neurons, thus reducing A?. CHIP(U) (box) domain physically interacts with BACE1; however, both U-box and TPR domain are essential for ubiquitination and degradation of BACE1. Further, BACE1 is a downstream target of p53 and overexpression of p53 decreases BACE1 level. In HEK-APP cells, CHIP is shown to negatively regulate BACE1 promoter through stabilization of p53's DNA-binding conformation and its binding upon 5' UTR element (+127 to +150). We have thus discovered that CHIP regulates p53-mediated trans-repression of BACE1 at both transcriptional and post-translational level. We propose that a CHIP-BACE1-p53 feedback loop might control APP stabilization, which could further be utilized for new therapeutic intervention in AD. PMID:25773675

  12. Effects of 1-MCP on chlorophyll degradation pathway-associated genes expression and chloroplast ultrastructure during the peel yellowing of Chinese pear fruits in storage.

    PubMed

    Cheng, Yudou; Dong, Yu; Yan, Hongbo; Ge, Wenya; Shen, Chengguo; Guan, Junfeng; Liu, Liqin; Zhang, Yingying

    2012-11-15

    The peel yellowing is an important pigment physiological process of green fruit ripening, which mainly results from chlorophyll degradation in the fruit peel. In this work, two typical cultivars with different ripening speed, a slow ripening pear 'Emerald' (Pyrus bretschneideri Rehd. cv. Emerald) and a fast ripening 'Jingbai' (Pyrus ussuriensis Maxim. cv. Jingbai) were used to investigate the molecular mechanism of chlorophyll degradation in pear yellowing/ripening during postharvest storage. The fruits after harvest were treated with 1-methylcyclopropene (1-MCP), an ethylene action inhibitor at 1.0 ?Ll(-1) to determine its effect on chloroplast ultrastructure and the expression of chlorophyll degradation associated genes in peel tissues. Our results show that the pears treated with 1-MCP had a lower ethylene production rate and higher chlorophyll content compared to those of untreated fruit. The more intact chloroplasts with well-organised grana thylakoids and small plastoglobuli were maintained in the peel of 1-MCP treated fruit for up to 30 and 15 d in 'Emerald' and 'Jingbai', respectively. The expression of chlorophyll degradation associated genes: pheophorbide a oxygenase (PAO), non-yellow colouring (NYC), NYC1-like (NOL), stay-green 1(SGR1), was suppressed, while no significant change was found in chlorophyllase 1 (CHL1) and red chlorophyll catabolite reductase (RCCR) in both cultivar fruits treated with 1-MCP. These results suggest that 1-MCP can delay chlorophyll degradation by inhibiting ethylene production and suppressing the gene expression of PAO, NYC, NOL and SGR1, which are closely associated with chlorophyll catabolic pathway. PMID:22868108

  13. Photocatalytic degradation of herbicide bentazone in aqueous suspension of TiO2: mineralization, identification of intermediates and reaction pathways.

    PubMed

    Mir, Niyaz A; Haque, M M; Khan, A; Muneer, M; Vijayalakshmi, S

    2014-01-01

    Semiconductor-mediated hydrogen peroxide-assisted photocatalytic degradation of a selected herbicide, Bentazone (1) has been investigated in aqueous suspensions of TiO2 under a variety of conditions. The degradation was studied by monitoring the depletion in total organic carbon content as a function of irradiation time. The degradation kinetics was investigated under different conditions such as type of TiO2 (Anatase/Anatase-Rutile mixture), reaction pH, catalyst dosage and hydrogen peroxide (H202) concentration. The degradation rates were found to be strongly influenced by all the above parameters. Titanium dioxide Degussa P25 was found to be more efficient as compared with other two commercially available TiO2 powders like Hombikat UV100 and PC500 from Millennium Inorganic Chemicals. Gas Chromatography-Mass Spectrometry (GC-MS) analysis of the irradiated mixture of Bentazone (1) indicates the formation of several intermediate products which have been characterized on the basis of molecular ion/mass fragmentation pattern and also on comparison with the National Institute of Standards and Technology (NIST) library. Plausible mechanism for the formation of different products during photocatalytic treatment of Bentazone in the presence of TiO2 has been proposed. The use of H202 substantially increased the efficiency of TiO2 photocatalytic degradation. PMID:24600881

  14. The homogentisate pathway: a central catabolic pathway involved in the degradation of L-phenylalanine, L-tyrosine, and 3-hydroxyphenylacetate in Pseudomonas putida.

    PubMed

    Arias-Barrau, Elsa; Olivera, Elías R; Luengo, José M; Fernández, Cristina; Galán, Beatriz; García, José L; Díaz, Eduardo; Miñambres, Baltasar

    2004-08-01

    Pseudomonas putida metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate. Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate. Whereas the phh, tyr, and hpd genes are not linked in the P. putida genome, the hmgABC genes appear to form a single transcriptional unit. Gel retardation assays and lacZ translational fusion experiments have shown that hmgR encodes a specific repressor that controls the inducible expression of the divergently transcribed hmgABC catabolic genes, and homogentisate is the inducer molecule. Footprinting analysis revealed that HmgR protects a region in the Phmg promoter that spans a 17-bp palindromic motif and an external direct repetition from position -16 to position 29 with respect to the transcription start site. The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway. We engineered a broad-host-range mobilizable catabolic cassette harboring the hmgABC, hpd, and tyrB genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source. Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in P. putida U funnels also into the homogentisate central pathway, revealing that the hmg cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds. PMID:15262943

  15. The Homogentisate Pathway: a Central Catabolic Pathway Involved in the Degradation of l-Phenylalanine, l-Tyrosine, and 3-Hydroxyphenylacetate in Pseudomonas putida

    PubMed Central

    Arias-Barrau, Elsa; Olivera, Elías R.; Luengo, José M.; Fernández, Cristina; Galán, Beatriz; García, José L.; Díaz, Eduardo; Miñambres, Baltasar

    2004-01-01

    Pseudomonas putida metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate. Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate. Whereas the phh, tyr, and hpd genes are not linked in the P. putida genome, the hmgABC genes appear to form a single transcriptional unit. Gel retardation assays and lacZ translational fusion experiments have shown that hmgR encodes a specific repressor that controls the inducible expression of the divergently transcribed hmgABC catabolic genes, and homogentisate is the inducer molecule. Footprinting analysis revealed that HmgR protects a region in the Phmg promoter that spans a 17-bp palindromic motif and an external direct repetition from position ?16 to position 29 with respect to the transcription start site. The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway. We engineered a broad-host-range mobilizable catabolic cassette harboring the hmgABC, hpd, and tyrB genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source. Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in P. putida U funnels also into the homogentisate central pathway, revealing that the hmg cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds. PMID:15262943

  16. Water deficit induces chlorophyll degradation via the 'PAO/phyllobilin' pathway in leaves of homoio- (Craterostigma pumilum) and poikilochlorophyllous (Xerophyta viscosa) resurrection plants.

    PubMed

    Christ, Bastien; Egert, Aurélie; Süssenbacher, Iris; Kräutler, Bernhard; Bartels, Dorothea; Peters, Shaun; Hörtensteiner, Stefan

    2014-11-01

    Angiosperm resurrection plants exhibit poikilo- or homoiochlorophylly as a response to water deficit. Both strategies are generally considered as effective mechanisms to reduce oxidative stress associated with photosynthetic activity under water deficiency. The mechanism of water deficit-induced chlorophyll (Chl) degradation in resurrection plants is unknown but has previously been suggested to occur as a result of non-enzymatic photooxidation. We investigated Chl degradation during dehydration in both poikilochlorophyllous (Xerophyta viscosa) and homoiochlorophyllous (Craterostigma pumilum) species. We demonstrate an increase in the abundance of PHEOPHORBIDE?a?OXYGENASE (PAO), a key enzyme of Chl breakdown, together with an accumulation of phyllobilins, that is, products of PAO-dependent Chl breakdown, in both species. Phyllobilins and PAO levels diminished again in leaves from rehydrated plants. We conclude that water deficit-induced poikilochlorophylly occurs via the well-characterized PAO/phyllobilin pathway of Chl breakdown and that this mechanism also appears conserved in a resurrection species displaying homoiochlorophylly. The roles of the PAO/phyllobilin pathway during different plant developmental processes that involve Chl breakdown, such as leaf senescence and desiccation, fruit ripening and seed maturation, are discussed. PMID:24697723

  17. Evidence for isofunctional enzymes in the degradation of phenol, m- and p-toluate, and p-cresol via catechol meta-cleavage pathways in Alcaligenes eutrophus.

    PubMed Central

    Hughes, E J; Bayly, R C; Skurray, R A

    1984-01-01

    A study of the degradation of phenol, p-cresol, and m- and p-toluate by Alcaligenes eutrophus 345 has provided evidence that these compounds are metabolized via separate catechol meta-cleavage pathways. Analysis of the enzymes synthesized by wild-type and mutant strains and by strains cured of the plasmid pRA1000, which encodes m- and p-toluate degradation, indicated that two or more isofunctional enzymes mediated several steps in the pathway. The formation of three catechol 2,3-oxygenases and two 2-hydroxymuconic semialdehyde hydrolases was indicated from an examination of the ratio of the specific activities of these enzymes against various substrates. Evidence for two 2-hydroxymuconic semialdehyde dehydrogenases, two 4-oxalocrotonate isomerases and decarboxylases, and three 2-ketopent-4-enoate hydratases was derived from the induction of these enzymes under different growth conditions. Each activity was detected when the wild type was grown in the presence of m-toluate, but not when grown with phenol (except for a hydratase) or p-cresol, whereas in strains cured of pRA1000, growth with phenol or p-cresol, but not with m-toluate, induced these enzymes. Hydroxylation of phenol and p-cresol appears to be mediated by the same enzyme. PMID:6370966

  18. Effects of a Defective Endoplasmic Reticulum-Associated Degradation Pathway on the Stress Response, Virulence, and Antifungal Drug Susceptibility of the Mold Pathogen Aspergillus fumigatus

    PubMed Central

    Krishnan, Karthik; Feng, Xizhi; Powers-Fletcher, Margaret V.; Bick, Gregory; Richie, Daryl L.; Woollett, Laura A.

    2013-01-01

    Proteins that are destined for release outside the eukaryotic cell, insertion into the plasma membrane, or delivery to intracellular organelles are processed and folded in the endoplasmic reticulum (ER). An imbalance between the level of nascent proteins entering the ER and the organelle's ability to manage that load results in the accumulation of unfolded proteins. Terminally unfolded proteins are disposed of by ER-associated degradation (ERAD), a pathway that transports the aberrant proteins across the ER membrane into the cytosol for proteasomal degradation. The ERAD pathway was targeted in the mold pathogen Aspergillus fumigatus by deleting the hrdA gene, encoding the A. fumigatus ortholog of Hrd1, the E3 ubiquitin ligase previously shown to contribute to ERAD in other species. Loss of HrdA was associated with impaired degradation of a folding-defective ERAD substrate, CPY*, as well as activation of the unfolded-protein response (UPR). The ?hrdA mutant showed resistance to voriconazole and reduced thermotolerance but was otherwise unaffected by a variety of environmental stressors. A double-deletion mutant deficient in both HrdA and another component of the same ERAD complex, DerA, was defective in secretion and showed hypersensitivity to ER, thermal, and cell wall stress. However, the ?hrdA ?derA mutant remained virulent in mouse and insect infection models. These data demonstrate that HrdA and DerA support complementary ERAD functions that promote survival under conditions of ER stress but are dispensable for virulence in the host environment. PMID:23355008

  19. Amyotrophic Lateral Sclerosis-Linked Mutant VAPB Inclusions Do Not Interfere with Protein Degradation Pathways or Intracellular Transport in a Cultured Cell Model

    PubMed Central

    Genevini, Paola; Papiani, Giulia; Ruggiano, Annamaria; Cantoni, Lavinia; Navone, Francesca; Borgese, Nica

    2014-01-01

    VAPB is a ubiquitously expressed, ER-resident adaptor protein involved in interorganellar lipid exchange, membrane contact site formation, and membrane trafficking. Its mutant form, P56S-VAPB, which has been linked to a dominantly inherited form of Amyotrophic Lateral Sclerosis (ALS8), generates intracellular inclusions consisting in restructured ER domains whose role in ALS pathogenesis has not been elucidated. P56S-VAPB is less stable than the wild-type protein and, at variance with most pathological aggregates, its inclusions are cleared by the proteasome. Based on studies with cultured cells overexpressing the mutant protein, it has been suggested that VAPB inclusions may exert a pathogenic effect either by sequestering the wild-type protein and other interactors (loss-of-function by a dominant negative effect) or by a more general proteotoxic action (gain-of-function). To investigate P56S-VAPB degradation and the effect of the inclusions on proteostasis and on ER-to-plasma membrane protein transport in a more physiological setting, we used stable HeLa and NSC34 Tet-Off cell lines inducibly expressing moderate levels of P56S-VAPB. Under basal conditions, P56S-VAPB degradation was mediated exclusively by the proteasome in both cell lines, however, it could be targeted also by starvation-stimulated autophagy. To assess possible proteasome impairment, the HeLa cell line was transiently transfected with the ERAD (ER Associated Degradation) substrate CD3?, while autophagic flow was investigated in cells either starved or treated with an autophagy-stimulating drug. Secretory pathway functionality was evaluated by analyzing the transport of transfected Vesicular Stomatitis Virus Glycoprotein (VSVG). P56S-VAPB expression had no effect either on the degradation of CD3? or on the levels of autophagic markers, or on the rate of transport of VSVG to the cell surface. We conclude that P56S-VAPB inclusions expressed at moderate levels do not interfere with protein degradation pathways or protein transport, suggesting that the dominant inheritance of the mutant gene may be due mainly to haploinsufficiency. PMID:25409455

  20. Oxidation products and degradation pathways of 4-chlorophenol by catalytic ozonation with MnOx/?-Al2O3/TiO2 as catalyst in aqueous solution.

    PubMed

    Qi, Lili; Yao, Jie; You, Hong; Zhang, Ran; Feng, Chunhui; Van Agtmaal, Sjack

    2014-01-01

    To identify the intermediates of 4-chlorophenol (4-CP) and bring forward the degradation pathways in the process of catalytic ozonation of 4-CP, 4-CP was ozonated with MnOx/?-Al2O3/TiO2 (MAT) catalyst, and 4-CP was almost decomposed within 30 min, the mineralization reaching above 94.1% at 100 min. The evident reduction of the degradation with the addition of the radical scavenger tert-butanol (TBA) and the stronger spin-adduct signals of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) indicated that 4-CP was oxidized primarily by hydroxyl radical (·OH). Analysis of GC-MS, HPLC and IC confirmed that aromatic compounds and carboxylic acids were predominant oxidative organic intermediates of 4-CP in catalytic ozonation.The main degradation steps were hydroxylation of 4-CP and the formation of hydroquinone, 4-chlororesorcinol and 4-chlorocatechol. The low molecular weight (LMW) acids, such as malic, malonic, oxalic, acetic, and formic acid, were formed from the further oxidation of the intermediates. PMID:24279624

  1. The HIV-1 accessory protein Vpr induces the degradation of the anti-HIV-1 agent APOBEC3G through a VprBP-mediated proteasomal pathway.

    PubMed

    Zhou, Dawei; Wang, Yan; Tokunaga, Kenzo; Huang, Fang; Sun, Binlian; Yang, Rongge

    2015-01-01

    The host anti-HIV-1 factor APOBEC3G (A3G) plays a potential role in restricting HIV-1 replication, although this antagonist can be encountered and disarmed by the Vif protein. In this paper, we report that another HIV-1 accessory protein, viral protein R (Vpr), can interact with A3G and intervene in its antiviral behavior. The interaction of Vpr and A3G was predicted by computer-based screen and confirmed by a co-immunoprecipitation (Co-IP) approach. We found that Vpr could reduce the virion encapsidation of A3G to enhance viral replication. Subsequent experiments showed that Vpr downregulated A3G through Vpr-binding protein (VprBP)-mediated proteasomal degradation, and further confirmed that the reduction of A3G encapsidation associated with Vpr was due to Vpr's degradation-inducing activity. Our findings highlight the versatility of Vpr by unveiling the hostile relationship between Vpr and A3G. In addition, the observation that A3G is targeted to the proteasomal degradation pathway by Vpr in addition to Vif implicates the existence of crosstalk between different HIV-1-host ubiquitin ligase complex systems. PMID:25200749

  2. CRM1-mediated nuclear export is required for 26 S proteasome-dependent degradation of the TRIP-Br2 proto-oncoprotein.

    PubMed

    Cheong, Jit Kong; Gunaratnam, Lakshman; Hsu, Stephen I-Hong

    2008-04-25

    Overexpression of the proto-oncogene TRIP-Br2 (SERTAD2) has been shown to induce E2F activity and promote tumorigenesis, whereas ablation of TRIP-Br2 arrests cell proliferation. Timely degradation of many cell cycle regulators is fundamental to the maintenance of proper cell cycle progression. Here we report novel mechanism(s) that govern the tight regulation of TRIP-Br2 levels during cell cycle progression. TRIP-Br2 was observed to be a short-lived protein in which the expression level peaks at the G(1)/S boundary. TRIP-Br2 accumulated in cells treated with 26 S proteasome inhibitors. Co-immunoprecipitation studies revealed that TRIP-Br2 forms ubiquitin conjugates. In silico analysis identified a putative leucine-rich nuclear export signal (NES) motif that overlaps with the PHD-Bromo interaction domain in the acidic C-terminal transactivation domain (TAD) of TRIP-Br2. This NES motif is highly conserved in widely divergent species and in all TRIP-Br family members. TRIP-Br2 was shown to be stabilized in G(2)/M phase cells through nuclear entrapment, either by deletion of the acidic C-terminal TAD, which includes the NES motif, or by leptomycin B-mediated inhibition of the CRM1-dependent nuclear export machinery. Mutation of leucine residue 238 of this NES motif abolished the interaction between CRM1 and TRIP-Br2, as well as the nuclear export of TRIP-Br2 and its subsequent 26 S proteasome-dependent degradation. These data suggest that CRM1-mediated nuclear export may be required for the proper execution of ubiquitin-proteasome-dependent degradation of TRIP-Br2. PMID:18316374

  3. Radiation induced degradation pathways for poly (methyl methacrylate) and polystyrene polymers as models for polymer behavior in space environments

    Microsoft Academic Search

    Kenneth Henry Heffner

    2003-01-01

    Modeling methods are required for predicting the chemical stability of macromolecular materials used in critical spacecraft components of satellites orbiting in the high-energy radiation environment of near earth and deep space planetary magnetic belts. Methods for establishing degradation mechanisms and predicting and simulating the total absorbed dose and ionization for long term space missions are presented herein. This investigation evaluates

  4. Activation of a Membrane-Bound Transcription Factor by Regulated Ubiquitin\\/Proteasome-Dependent Processing

    Microsoft Academic Search

    Thorsten Hoppe; Kai Matuschewski; Michael Rape; Stephan Schlenker; Helle D Ulrich; Stefan Jentsch

    2000-01-01

    Processing of integral membrane proteins in order to liberate active proteins is of exquisite cellular importance. Examples are the processing events that govern sterol regulation, Notch signaling, the unfolded protein response, and APP fragmentation linked to Alzheimer's disease. In these cases, the proteins are thought to be processed by regulated intramembrane proteolysis, involving site-specific, membrane-localized proteases. Here we show that

  5. The role of the ubiquitin\\/proteasome system in cellular responses to radiation

    Microsoft Academic Search

    William H McBride; Keisuke S Iwamoto; Randi Syljuasen; Milena Pervan; Frank Pajonk

    2003-01-01

    In the last few years, the ubiquitin(Ub)\\/proteasome system has become increasingly recognized as a controller of numerous physiological processes, including signal transduction, DNA repair, chromosome maintenance, transcriptional activation, cell cycle progression, cell survival, and certain immune cell functions. This is in addition to its more established roles in the removal of misfolded, damaged, and effete proteins. This review examines the

  6. The Ubiquitin-Proteasome System Is a Key Component of the SUMO2\\/3 Cycle

    Microsoft Academic Search

    Joost Schimmel; Katja M. Larsen; Ivan Matic; Martijn van Hagen; Jurgen Cox; Matthias Mann; Jens S. Andersen; Alfred C. O. Vertegaal

    2008-01-01

    Many proteins are regulated by a variety of post-transla- tional modifications, and orchestration of these modifica- tions is frequently required for full control of activity. Cur- rently little is known about the combinatorial activity of different post-translational modifications. Here we show that extensive cross-talk exists between sumoylation and ubiquitination. We found that a subset of SUMO-2-conju- gated proteins is subsequently

  7. Isolation and characterization of neuronal substrates of the ubiquitin proteasome system

    E-print Network

    Keil, Jeffrey McCartney

    2011-01-01

    Rat primary dissociated hippocampal neurons were treated with either (A) DMSODMSO and MG-132 treated samples 43 Chapter II Figure 6. STIM1 is ubiquitinated in the ratRat cortical neurons (DIV 18) were treated with MG-132 (5 µM) or DMSO

  8. Caspase-dependent regulation of the ubiquitin–proteasome system through direct substrate targeting

    PubMed Central

    Yeh, Ting-Chun; Bratton, Shawn B.

    2013-01-01

    Drosophila inhibitor of apoptosis (IAP) 1 (DIAP1) is an E3 ubiquitin ligase that regulates apoptosis in flies, in large part through direct inhibition and/or ubiquitinylation of caspases. IAP antagonists, such as Reaper, Hid, and Grim, are thought to induce cell death by displacing active caspases from baculovirus IAP repeat domains in DIAP1, but can themselves become targets of DIAP1-mediated ubiquitinylation. Herein, we demonstrate that Grim self-associates in cells and is ubiquitinylated by DIAP1 at Lys136 in an UbcD1-dependent manner, resulting in its rapid turnover. K48-linked ubiquitin chains are added almost exclusively to BIR2-bound Grim as a result of its structural proximity to DIAP1’s RING domain. However, active caspases can simultaneously cleave Grim at Asp132, removing the lysine necessary for ubiquitinylation as well as any existing ubiquitin conjugates. Cleavage therefore enhances the stability of Grim and initiates a feed-forward caspase amplification loop, resulting in greater cell death. In summary, Grim is a caspase substrate whose cleavage promotes apoptosis by limiting, in a target-specific fashion, its ubiquitinylation and turnover by the proteasome. PMID:23940367

  9. Differential Regulation of N-Myc and c-Myc Synthesis, Degradation, and Transcriptional Activity by the Ras/Mitogen-activated Protein Kinase Pathway*

    PubMed Central

    Kapeli, Katannya; Hurlin, Peter J.

    2011-01-01

    Myc transcription factors are important regulators of proliferation and can promote oncogenesis when deregulated. Deregulated Myc expression in cancers can result from MYC gene amplification and translocation but also from alterations in mitogenic signaling pathways that affect Myc levels through both transcriptional and post-transcription mechanisms. For example, mutations in Ras family GTPase proteins that cause their constitutive activation can increase cellular levels of c-Myc by interfering with its rapid proteasomal degradation. Although enhanced protein stability is generally thought to be applicable to other Myc family members, here we show that c-Myc and its paralog N-Myc respond to oncogenic H-Ras (H-RasG12V) in very different ways. H-RasG12V promotes accumulation of both c-Myc and N-Myc, but although c-Myc accumulation is achieved by enhanced protein stability, N-Myc accumulation is associated with an accelerated rate of translation that overcomes a surprising H-RasG12V-mediated destabilization of N-Myc. We show that H-RasG12V-mediated degradation of N-Myc functions independently of key phosphorylation sites in the highly conserved Myc homology box I region that controls c-Myc protein stability by oncogenic Ras. Finally, we found that N-Myc and c-Myc transcriptional activity is associated with their proteasomal degradation but that N-Myc may be uniquely dependent on Ras-stimulated proteolysis for target gene expression. Taken together, these studies provide mechanistic insight into how oncogenic Ras augments N-Myc levels in cells and suggest that enhanced N-Myc translation and degradation-coupled transactivation may contribute to oncogenesis. PMID:21908617

  10. AGE/RAGE/Akt pathway contributes to prostate cancer cell proliferation by promoting Rb phosphorylation and degradation.

    PubMed

    Bao, Ji-Ming; He, Min-Yi; Liu, Ya-Wei; Lu, Yong-Jie; Hong, Ying-Qia; Luo, Hai-Hua; Ren, Zhong-Lu; Zhao, Shan-Chao; Jiang, Yong

    2015-01-01

    Metabolomic research has revealed that metabolites play an important role in prostate cancer development and progression. Previous studies have suggested that prostate cancer cell proliferation is induced by advanced glycation end products (AGEs) exposure, but the mechanism of this induction remains unknown. This study investigated the molecular mechanisms underlying the proliferative response of prostate cancer cell to the interaction of AGEs and the receptor for advanced glycation end products (RAGE). To investigate this mechanism, we used Western blotting to evaluate the responses of the retinoblastoma (Rb), p-Rb and PI3K/Akt pathway to AGEs stimulation. We also examined the effect of knocking down Rb and blocking the PI3K/Akt pathway on AGEs induced PC-3 cell proliferation. Our results indicated that AGE-RAGE interaction enhanced Rb phosphorylation and subsequently decreased total Rb levels. Bioinformatics analysis further indicated a negative correlation between RAGE and RB1 expression in prostate cancer tissue. Furthermore, we observed that AGEs stimulation activated the PI3K/Akt signaling pathway and that blocking PI3K/Akt signaling abrogated AGEs-induced cell proliferation. We report, for the first time, that AGE-RAGE interaction enhances prostate cancer cell proliferation by phosphorylation of Rb via the PI3K/Akt signaling pathway. PMID:26175942

  11. Roles of the divergent branches of the meta-cleavage pathway in the degradation of benzoate and substituted benzoates.

    PubMed Central

    Harayama, S; Mermod, N; Rekik, M; Lehrbach, P R; Timmis, K N

    1987-01-01

    The TOL plasmid-specified meta-cleavage pathway for the oxidative catabolism of benzoate and toluates branches at the ring cleavage products of catechols and reconverges later at 2-oxopent-4-enoate or its corresponding substituted derivatives. The hydrolytic branch of the pathway involves the direct formation of 2-oxopent-4-enoate or its derivatives, whereas the oxalocrotonate branch involves three enzymatic steps effected by a dehydrogenase, an isomerase, and a decarboxylase, which produce the same compounds. Evidence is presented which shows that benzoate and p-toluate can, under certain circumstances, be catabolized by the hydrolytic branch. However, in a fully functional pathway, only m-toluate is dissimilated via this branch, and benzoate and p-toluate are catabolized almost exclusively by the oxalocrotonate branch. The biochemical basis of this selectivity was found to reside in the high affinity of the dehydrogenase for ring fission products derived from benzoate and p-toluate and its inability to attack the ring fission product derived from m-toluate. Although isomerization of 4-oxalocrotonate occurs spontaneously in vitro, enzymatic isomerization was found to be essential for effective functioning of this branch of the pathway in vivo. PMID:3542963

  12. Oxidation products and pathway of ceramic honeycomb-catalyzed ozonation for the degradation of nitrobenzene in aqueous solution

    Microsoft Academic Search

    Lei Zhao; Jun Ma; Zhi-zhong Sun

    2008-01-01

    Semi-continuous experiments on catalytic ozonation of nitrobenzene (NB) in aqueous solution using ceramic honeycomb as catalyst have been investigated. Experimental results showed that the presence of ceramic honeycombs significantly increased the ozonation degradation rate of NB compared to the case of non-catalytic ozonation. With addition of the radical scavenger tert-butanol, the evident reduction of NB removal indicated that NB was

  13. Degradation of o-Nitrobenzoate via Anthranilic Acid ( o-Aminobenzoate) by Arthrobacter protophormiae: A Plasmid-Encoded New Pathway

    Microsoft Academic Search

    Ashvini Chauhan; Rakesh K. Jain

    2000-01-01

    An Arthrobacter protophormiae strain RKJ100, isolated by selective enrichment, was capable of utilizing o-nitrobenzoate (ONB+) as the sole carbon, nitrogen, and energy source. The degradation of ONB proceeds through an oxygen insensitive reductive route as shown by the release of ammonia in the culture medium aerobically rather than nitrite ions. Thin-layer chromatography, gas chromatography, and gas chromatography-mass spectrometry of the

  14. Posttranslational Modification of Bcl2 Facilitates Its Proteasome-Dependent Degradation: Molecular Characterization of the Involved Signaling Pathway

    Microsoft Academic Search

    KRISTIN BREITSCHOPF; JUDITH HAENDELER; PHILIPP MALCHOW; ANDREAS M. ZEIHER; STEFANIE DIMMELER

    2000-01-01

    The ratio of proapoptotic versus antiapoptotic Bcl-2 members is a critical determinant that plays a signif- icant role in altering susceptibility to apoptosis. Therefore, a reduction of antiapoptotic protein levels in response to proximal signal transduction events may switch on the apoptotic pathway. In endothelial cells, tumor necrosis factor alpha (TNF-a) induces dephosphorylation and subsequent ubiquitin-dependent degra- dation of the

  15. Gene mdpC plays a regulatory role in the methyl-tert-butyl ether degradation pathway of Methylibium petroleiphilum strain PM1.

    PubMed

    Joshi, Geetika; Schmidt, Radomir; Scow, Kate M; Denison, Michael S; Hristova, Krassimira R

    2015-04-01

    Among the few bacteria known to utilize methyl tert-butyl ether (MTBE) as a sole carbon source, Methylibium petroleiphilum PM1 is a well-characterized organism with a sequenced genome; however, knowledge of the genetic regulation of its MTBE degradation pathway is limited. We investigated the role of a putative transcriptional activator gene, mdpC, in the induction of MTBE-degradation genes mdpA (encoding MTBE monooxygenase) and mdpJ (encoding tert-butyl alcohol hydroxylase) of strain PM1 in a gene-knockout mutant mdpC(-). We also utilized quantitative reverse transcriptase PCR assays targeting genes mdpA, mdpJ and mdpC to determine the effects of the mutation on transcription of these genes. Our results indicate that gene mdpC is involved in the induction of both mdpA and mdpJ in response to MTBE and tert-butyl alcohol (TBA) exposure in PM1. An additional independent mechanism may be involved in the induction of mdpJ in the presence of TBA. PMID:25724531

  16. Monascin and AITC attenuate methylglyoxal-induced PPAR? phosphorylation and degradation through inhibition of the oxidative stress/PKC pathway depending on Nrf2 activation.

    PubMed

    Hsu, Wei-Hsuan; Lee, Bao-Hong; Li, Chih-Heng; Hsu, Ya-Wen; Pan, Tzu-Ming

    2013-06-26

    Abnormal cellular accumulation of the dicarbonyl metabolite methylglyoxal (MG) results in cell damage, inflammation, and oxidative stress. It is also associated with increased protein linkage to form advanced glycation end products (AGEs) or induce DNA strand breaks. The association between peroxisome proliferator-activated receptor-? (PPAR?) and nuclear factor-erythroid 2-related factor 2 (Nrf2) is unclear. This study investigated Nrf2 activator protection against PPAR? phosphorylation and degradation to maintain pancreatic function. MG was used at a noncytotoxic concentration (200 ?M) to induce protein kinase C (PKC) and PPAR? phosphorylation in pancreatic RINm5F cells. For in vivo studies, MG (60 mg/kg bw) was intraperitoneally (IP) injected into Balb/C mice for 28 d to induce pancreas damage, at which point we investigated the effect of monascin protection (PPAR? and Nrf2 activator), rosiglitazone (PPAR? activator), allyl isothiocyanate (AITC; Nrf2 activator), or N-acetylcysteine (NAC) on pancreatic function. The in vitro and in vivo results indicated that MG leads to marked PPAR? phosphorylation (serine 82); this effect led to reduction in pancreatic and duodenal homeobox-1 (PDX-1), glucokinase (GCK), and insulin expression. However, monascin and rosiglitazone may protect PPAR? degradation by elevating PDX-1, GCK, and as a result, insulin expression. Monascin and AITC can attenuate PKC activation to suppress PPAR? phosphorylation caused by oxidative stress through the Nrf2 pathway. Similarly, the N-acetylcysteine (NAC) antioxidant also improved oxidative stress and pancreatic function. This study examined whether MG caused impairment of PDX-1, GCK, and insulin through PPAR? phosphorylation and degradation. MG and AGE accumulation improved on Nrf2 activation, thereby protecting against pancreas damage. Taken together, PPAR? activation maintained pancreatic PDX-1, GCK, and insulin expression levels to regulate blood glucose levels. PMID:23731245

  17. The Protein Quality Control Machinery Regulates Its Misassembled Proteasome Subunits

    PubMed Central

    David-Kadoch, Galit; Hazan, Rotem; Yu, Tzenlin; Glickman, Michael H.; Ben-Aroya, Shay

    2015-01-01

    Cellular toxicity introduced by protein misfolding threatens cell fitness and viability. Failure to eliminate these polypeptides is associated with various aggregation diseases. In eukaryotes, the ubiquitin proteasome system (UPS) plays a vital role in protein quality control (PQC), by selectively targeting misfolded proteins for degradation. While the assembly of the proteasome can be naturally impaired by many factors, the regulatory pathways that mediate the sorting and elimination of misassembled proteasomal subunits are poorly understood. Here, we reveal how the dysfunctional proteasome is controlled by the PQC machinery. We found that among the multilayered quality control mechanisms, UPS mediated degradation of its own misassembled subunits is the favored pathway. We also demonstrated that the Hsp42 chaperone mediates an alternative pathway, the accumulation of these subunits in cytoprotective compartments. Thus, we show that proteasome homeostasis is controlled through probing the level of proteasome assembly, and the interplay between UPS mediated degradation or their sorting into distinct cellular compartments. PMID:25919710

  18. IL-1-induced ERK1/2 activation up-regulates p21{sup Waf1/Cip1} protein by inhibition of degradation via ubiquitin-independent pathway in human melanoma cells A375

    SciTech Connect

    Arakawa, Tomohiro [Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan)] [Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Hayashi, Hidetoshi [Department of Drug Metabolism and Disposition, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan)] [Department of Drug Metabolism and Disposition, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Itoh, Saotomo; Takii, Takemasa [Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan)] [Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Onozaki, Kikuo, E-mail: konozaki@phar.nagoya-cu.ac.jp [Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan)] [Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan)

    2010-02-12

    IL-1 inhibits the proliferation of human melanoma cells A375 by arresting the cell cycle at G0/G1 phase, which accompanies the increase of p21{sup Waf1/Cip1} (p21) protein. Here, we demonstrate that IL-1 induces the stabilization of p21 protein via ERK1/2 pathway. The degradation of p21 was inhibited by IL-1, however the ubiquitination level of p21 was not affected. In addition, the degradation of non-ubiquitinated form of lysine less mutant p21-K6R was also inhibited by IL-1, suggesting that IL-1 stabilized p21 protein via ubiquitin-independent pathway. Furthermore, the inhibition of p21 protein degradation was prevented by a selective inhibitor of ERK1/2 pathway, PD98059. These results suggest that IL-1-induced ERK1/2 activation leads to the up-regulation of p21 by inhibiting degradation via ubiquitin-independent pathway in human melanoma cells A375.

  19. Degradation of dioxins by cyclic ether degrading fungus, Cordyceps sinensis

    Microsoft Academic Search

    Kunichika Nakamiya; Shyunji Hashimoto; Hiroyasu Ito; John S. Edmonds; Akio Yasuhara; Masatoshi Morita

    2005-01-01

    Use of the cyclic ether degrading fungus, Cordyceps sinensis strain A to degrade dibenzo-p-dioxin (DD), 2,3,7-trichlorodibenzo-p-dioxin (2,3,7-triCDD) and octachlorodibenzo-p-dioxin (octaCDD) has revealed a new degradation pathway for dioxins. Catechols and other possible degradation products were synthesized to facilitate the identification, detection and quantification of these products, and phenylboronate was used for the derivatization and analysis of dihydroxylated degradation products. Degradation

  20. Miswiring the brain: ?9-tetrahydrocannabinol disrupts cortical development by inducing an SCG10/stathmin-2 degradation pathway

    PubMed Central

    Tortoriello, Giuseppe; Morris, Claudia V; Alpar, Alan; Fuzik, Janos; Shirran, Sally L; Calvigioni, Daniela; Keimpema, Erik; Botting, Catherine H; Reinecke, Kirstin; Herdegen, Thomas; Courtney, Michael; Hurd, Yasmin L; Harkany, Tibor

    2014-01-01

    Children exposed in utero to cannabis present permanent neurobehavioral and cognitive impairments. Psychoactive constituents from Cannabis spp., particularly ?9-tetrahydrocannabinol (THC), bind to cannabinoid receptors in the fetal brain. However, it is unknown whether THC can trigger a cannabinoid receptor-driven molecular cascade to disrupt neuronal specification. Here, we show that repeated THC exposure disrupts endocannabinoid signaling, particularly the temporal dynamics of CB1 cannabinoid receptor, to rewire the fetal cortical circuitry. By interrogating the THC-sensitive neuronal proteome we identify Superior Cervical Ganglion 10 (SCG10)/stathmin-2, a microtubule-binding protein in axons, as a substrate of altered neuronal connectivity. We find SCG10 mRNA and protein reduced in the hippocampus of midgestational human cannabis-exposed fetuses, defining SCG10 as the first cannabis-driven molecular effector in the developing cerebrum. CB1 cannabinoid receptor activation recruits c-Jun N-terminal kinases to phosphorylate SCG10, promoting its rapid degradation in situ in motile axons and microtubule stabilization. Thus, THC enables ectopic formation of filopodia and alters axon morphology. These data highlight the maintenance of cytoskeletal dynamics as a molecular target for cannabis, whose imbalance can limit the computational power of neuronal circuitries in affected offspring. PMID:24469251

  1. A model for the catabolism of rhizopine in Rhizobium leguminosarum involves a ferredoxin oxygenase complex and the inositol degradative pathway.

    PubMed

    Bahar, M; de Majnik, J; Wexler, M; Fry, J; Poole, P S; Murphy, P J

    1998-11-01

    Rhizopines are nodule-specific compounds that confer an intraspecies competitive nodulation advantage to strains that can catabolize them. The rhizopine (3-O-methyl-scyllo-inosamine, 3-O-MSI) catabolic moc gene cluster mocCABRDE(F) in Rhizobium leguminosarum bv. viciae strain 1a is located on the Sym plasmid. MocCABR are homologous to the mocCABR gene products from Sinorhizobium meliloti. MocD and MocE contain motifs corresponding to a TOL-like oxygenase and a [2Fe-2S] Rieske-like ferredoxin, respectively. The mocF gene encodes a ferredoxin reductase that would complete the oxygenase system, but is not essential for rhizopine catabolism. We propose a rhizopine catabolic model whereby MocB transports rhizopine into the cell and MocDE and MocF (or a similar protein elsewhere in the genome), under the regulation of MocR, act in concert to form a ferredoxin oxygenase system that demethylates 3-O-MSI to form scyllo-inosamine (SI). MocA, an NAD(H)-dependent dehydrogenase, and MocC continue the catabolic process. Compounds formed then enter the inositol catabolic pathway. PMID:9805393

  2. The mll6786 gene encodes a repressor protein controlling the degradation pathway for vitamin B6 in Mesorhizobium loti.

    PubMed

    Nagase, Takayuki; Mugo, Andrew N; Chu, Huy Nhat; Yoshikane, Yu; Ohnishi, Kouhei; Yagi, Toshiharu

    2012-04-01

    Pyridoxine is converted to succinic semialdehyde, acetate, ammonia and CO(2) through the actions of eight enzymes. The genes encoding the enzymes occur as a cluster on the chromosomal DNA of Mesorhizobium loti, a symbiotic nitrogen-fixing bacterium. Here, it was found that disruption of the mll6786 gene, which is located between the genes encoding the first and eighth enzymes of the pathway, caused constitutive expression of the eight enzymes. The protein encoded by the mll6786 gene is a member of the GntR family and is designated as PyrR. PyrR comprises 223 amino acid residues and is a dimeric protein with a subunit molecular mass of 25 kDa. The purified PyrR with a C-terminal His(6) -tag could bind to an intergenic 67-bp DNA region, which contains a palindrome sequence and a deduced promoter sequence, between the mll6786 and mlr6787 genes, encoding PyrR and AAMS amidohydrolase, respectively. PMID:22276911

  3. Cellular Processing of Myocilin

    PubMed Central

    Qiu, Ye; Shen, Xiang; Shyam, Rajalekshmy; Yue, Beatrice Y. J. T.; Ying, Hongyu

    2014-01-01

    Background Myocilin (MYOC) is a gene linked directly to juvenile- and adult-onset open angle glaucoma. Mutations including Pro370Leu (P370L) and Gln368stop (Q368X) have been identified in patients. In the present study, we investigated the processing of myocilin in human trabecular meshwork (TM) cells as well as in inducible, stable RGC5 cell lines. Methodology/Principal Findings The turnover and photoactivation experiments revealed that the endogenous myocilin in human trabecular meshwork (TM) cells was a short-lived protein. It was found that the endogenous myocilin level in TM cells was increased by treatment of lysosomal and proteasomal inhibitors, but not by autophagic inhibitor. Multiple bands immunoreactive to anti-ubiquitin were seen in the myocilin pull down, indicating that myocilin was ubiquitinated. In inducible cell lines, the turnover rate of overexpressed wild-type and mutant P370L and Q368X myocilin-GFP fusion proteins was much prolonged. The proteasome function was compromised and autophagy was induced. A decreased PSMB5 level and an increased level of autophagic marker, LC3, were demonstrated. Conclusions/Significance The current study provided evidence that in normal homeostatic situation, the turnover of endogenous myocilin involves ubiquitin-proteasome and lysosomal pathways. When myocilin was upregulated or mutated, the ubiquitin-proteasome function is compromised and autophagy is induced. Knowledge of the degradation pathways acting on myocilin can help in design of novel therapeutic strategies for myocilin-related glaucoma. PMID:24732711

  4. ?–Hydroxy ?–Methylbutyrate Improves Dexamethasone-Induced Muscle Atrophy by Modulating the Muscle Degradation Pathway in SD Rat

    PubMed Central

    Choi, Yeon Ja; Park, Min Hi; Jang, Eun Ji; Park, Chan Hum; Yoon, Changshin; Kim, Nam Deuk; Kim, Mi Kyung; Chung, Hae Young

    2014-01-01

    Skeletal muscle atrophy results from various conditions including high levels of glucocorticoids, and ?–hydroxy ?–methylbutyrate (HMB; a metabolite of leucine) is a potent therapeutical supplement used to treat various muscle disorders. Recent studies have demonstrated that HMB inhibits dexamethasone-induced atrophy in cultured myotubes, but its effect on dexamethasone-induced muscle atrophy has not been determined in vivo. In the present study, we investigated the effect of HMB on dexamethasone-induced muscle atrophy in rats. Treatment with dexamethasone weakened grip strengths and increased muscle damage as determined by increased serum creatine kinase levels and by histological analysis. Dexamethasone treatment also reduced both soleus and gastrocnemius muscle masses. However, HMB supplementation significantly prevented reductions in grip strengths, reduced muscle damage, and prevented muscle mass and protein concentration decrease in soleus muscle. Biochemical analysis demonstrated that dexamethasone markedly increased levels of MuRF1 protein, which causes the ubiquitination and degradation of MyHC. Indeed, dexamethasone treatment decreased MyHC protein expression and increased the ubiquitinated-MyHC to MyHC ratio. However, HMB supplementation caused the down-regulations of MuRF1 protein and of ubiquitinated-MyHC. Furthermore, additional experiments provided evidence that HMB supplementation inhibited the nuclear translocation of FOXO1 induced by dexamethasone, and showed increased MyoD expression in the nuclear fractions of soleus muscles. These findings suggest that HMB supplementation attenuates dexamethasone-induced muscle wasting by regulating FOXO1 transcription factor and subsequent MuRF1 expression. Accordingly, our results suggest that HMB supplementation could be used to prevent steroid myopathy. PMID:25032690

  5. ?-Hydroxy ?-methylbutyrate improves dexamethasone-induced muscle atrophy by modulating the muscle degradation pathway in SD rat.

    PubMed

    Noh, Kyung Kyun; Chung, Ki Wung; Choi, Yeon Ja; Park, Min Hi; Jang, Eun Ji; Park, Chan Hum; Yoon, Changshin; Kim, Nam Deuk; Kim, Mi Kyung; Chung, Hae Young

    2014-01-01

    Skeletal muscle atrophy results from various conditions including high levels of glucocorticoids, and ?-hydroxy ?-methylbutyrate (HMB; a metabolite of leucine) is a potent therapeutical supplement used to treat various muscle disorders. Recent studies have demonstrated that HMB inhibits dexamethasone-induced atrophy in cultured myotubes, but its effect on dexamethasone-induced muscle atrophy has not been determined in vivo. In the present study, we investigated the effect of HMB on dexamethasone-induced muscle atrophy in rats. Treatment with dexamethasone weakened grip strengths and increased muscle damage as determined by increased serum creatine kinase levels and by histological analysis. Dexamethasone treatment also reduced both soleus and gastrocnemius muscle masses. However, HMB supplementation significantly prevented reductions in grip strengths, reduced muscle damage, and prevented muscle mass and protein concentration decrease in soleus muscle. Biochemical analysis demonstrated that dexamethasone markedly increased levels of MuRF1 protein, which causes the ubiquitination and degradation of MyHC. Indeed, dexamethasone treatment decreased MyHC protein expression and increased the ubiquitinated-MyHC to MyHC ratio. However, HMB supplementation caused the down-regulations of MuRF1 protein and of ubiquitinated-MyHC. Furthermore, additional experiments provided evidence that HMB supplementation inhibited the nuclear translocation of FOXO1 induced by dexamethasone, and showed increased MyoD expression in the nuclear fractions of soleus muscles. These findings suggest that HMB supplementation attenuates dexamethasone-induced muscle wasting by regulating FOXO1 transcription factor and subsequent MuRF1 expression. Accordingly, our results suggest that HMB supplementation could be used to prevent steroid myopathy. PMID:25032690

  6. Protein/protein interactions in the mammalian heme degradation pathway: heme oxygenase-2, cytochrome P450 reductase, and biliverdin reductase.

    PubMed

    Spencer, Andrea L M; Bagai, Ireena; Becker, Donald F; Zuiderweg, Erik R P; Ragsdale, Stephen W

    2014-10-24

    Heme oxygenase (HO) catalyzes the rate-limiting step in the O2-dependent degradation of heme to biliverdin, CO, and iron with electrons delivered from NADPH via cytochrome P450 reductase (CPR). Biliverdin reductase (BVR) then catalyzes conversion of biliverdin to bilirubin. We describe mutagenesis combined with kinetic, spectroscopic (fluorescence and NMR), surface plasmon resonance, cross-linking, gel filtration, and analytical ultracentrifugation studies aimed at evaluating interactions of HO-2 with CPR and BVR. Based on these results, we propose a model in which HO-2 and CPR form a dynamic ensemble of complex(es) that precede formation of the productive electron transfer complex. The (1)H-(15)N TROSY NMR spectrum of HO-2 reveals specific residues, including Leu-201, near the heme face of HO-2 that are affected by the addition of CPR, implicating these residues at the HO/CPR interface. Alanine substitutions at HO-2 residues Leu-201 and Lys-169 cause a respective 3- and 22-fold increase in K(m) values for CPR, consistent with a role for these residues in CPR binding. Sedimentation velocity experiments confirm the transient nature of the HO-2 · CPR complex (K(d) = 15.1 ?M). Our results also indicate that HO-2 and BVR form a very weak complex that is only captured by cross-linking. For example, under conditions where CPR affects the (1)H-(15)N TROSY NMR spectrum of HO-2, BVR has no effect. Fluorescence quenching experiments also suggest that BVR binds HO-2 weakly, if at all, and that the previously reported high affinity of BVR for HO is artifactual, resulting from the effects of free heme (dissociated from HO) on BVR fluorescence. PMID:25196843

  7. Loquat (Eriobotrya japonica) extract prevents dexamethasone-induced muscle atrophy by inhibiting the muscle degradation pathway in Sprague Dawley rats.

    PubMed

    Noh, Kyung Kyun; Chung, Ki Wung; Sung, Bokyung; Kim, Min Jo; Park, Chan Hum; Yoon, Changshin; Choi, Jae Sue; Kim, Mi Kyung; Kim, Cheol Min; Kim, Nam Deuk; Chung, Hae Young

    2015-09-01

    In the Orient, loquat (Eriobotrya japonica) extract (LE) is widely used in teas, food and folk medicines. The leaves of the loquat tree have been used for generations to treat chronic bronchitis, coughs, phlegm production, high fever and gastroenteric disorders. One of the major active components of loquat leaves is ursolic acid, which was recently investigated in the context of preventing muscle atrophy. The present study investigated the therapeutic potential of LE on dexamethasone?induced muscle atrophy in rats. Daily intraperitoneal injections of dexamethasone caused muscle atrophy and evidence of muscle atrophy prevention by LE was demonstrated using various assays. In particular, dexamethasone?induced grip strength loss was alleviated by LE and the increase in serum creatine kinase activity, a surrogate marker of muscle damage, caused by dexamethasone injection was reduced by LE. Western blot analysis and immunoprecipitation demonstrated that dexamethasone markedly increased the protein expression levels of muscle ring finger 1 (MuRF1), which causes the ubiquitination and degradation of myosin heavy chain (MyHC), and decreased the protein expression levels of MyHC as well as increased the ubiquitinated MyHC to MyHC ratio. However, LE reduced the dexamethasone?induced protein expression levels of MuRF1 and ubiquitinated MyHC. Additional experiments revealed that LE supplementation inhibited the nuclear translocation of FoxO1 induced by dexamethasone. These findings suggested that LE prevented dexamethasone?induced muscle atrophy by regulating the FoxO1 transcription factor and subsequently the expression of MuRF1. PMID:26004741

  8. 17?-Estradiol regulates insulin-degrading enzyme expression via an ER?/PI3-K pathway in hippocampus: relevance to Alzheimer’s prevention

    PubMed Central

    Zhao, Liqin; Yao, Jia; Mao, Zisu; Chen, Shuhua; Wang, Yan; Brinton, Roberta Diaz

    2010-01-01

    Insulin-degrading enzyme (IDE), an enzyme that primarily degrades insulin, has recently been demonstrated to play a significant role in the catabolism of amyloid ? (A?) protein in the brain. Reduced IDE expression and/or activity have been associated with the etiology and development of Alzheimer’s disease (AD). Using three model systems, the present investigation provides the first documentation indicating that estrogen robustly regulates the expression of IDE in normal, menopausal and early-stage AD brains. In vitro analyses in primary cultures of rat hippocampal neurons revealed that 17?-estradiol (17?-E2) increased IDE in both mRNA and protein levels in a time-dependent manner. Further pharmacological analyses indicated that 17?-E2-induced IDE expression was dependent upon estrogen receptor (ER) ? and required activation of phosphatidylinositol 3-kinase (PI3-K). In vivo analyses in adult female rats revealed a brain region-specific responsive profile. Ovariectomy (OVX) induced a significant decline in IDE expression in the hippocampus, which was prevented by 17?-E2. However, both OVX and 17?-E2 did not exert a significant effect in the cerebellum. In vivo analyses in triple transgenic AD (3xTg-AD) female mice revealed an inverse correlation between the age-related increase in A? load and the decrease in IDE expression in the hippocampal formation. Treatment with 17?-E2 attenuated A? accumulation/plaque formation and elevated hippocampal IDE expression in 12-month-old 3xTg-AD OVX mice. Collectively, these findings indicate that 17?-E2 regulates IDE expression in a brain region-specific manner and such a regulatory role in the hippocampus, mediated by an ER?/PI3-K pathway, could serve as a direct mechanism underlying estrogen-mediated preventative effect against AD when timely initiated at the onset of menopause. PMID:20053478

  9. Aucubin prevents interleukin-1 beta induced inflammation and cartilage matrix degradation via inhibition of NF-?B signaling pathway in rat articular chondrocytes.

    PubMed

    Wang, Sheng-Nan; Xie, Guo-Ping; Qin, Cheng-He; Chen, Yi-Rong; Zhang, Kai-Rui; Li, Xue; Wu, Qian; Dong, Wei-Qiang; Yang, Jun; Yu, Bin

    2015-02-01

    Proinflammatory cytokine interleukin-1? (IL-1?) plays a crucial role in the pathogenesis of Osteoarthritis (OA) by stimulating several mediators contributed to cartilage degradation. Aucubin, a natural compound derived from plants which has been shown to possess diverse biological activities including anti-inflammatory property, may benefit the IL-1? stimulated chondrocytes. The present study was aimed to investigate the effects of Aucubin on IL-1? stimulated rat chondrocytes. Rat chondrocytes were cultured and pretreated with Aucubin (1, 10, 20, 50?M), and then stimulated with or without IL-1? (10ng/ml). Gene and protein expression of MMP-3, MMP-9, MMP-13, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) was determined by real-time PCR and Western blotting respectively. Nitric oxide (NO) production was quantified by Griess reagent. Phosphorylation and nuclear translocation of p65 were detected by western blotting and immunofluorescence, respectively. We found that Aucubin significantly reversed the elevated gene and protein expression of MMP-3, MMP-9, MMP-13, iNOS, COX-2 and the production of NO induced by IL-1? challenge in rat chondrocytes. Furthermore, Aucubin was able to suppress the IL-1?-mediated phosphorylation and nuclear translocation of p65, indicating Aucubin may possibly act via the NF-?B signaling pathway. The present study proposes that Aucubin may be a potential therapeutic choice in the treatment of OA due to its anti-inflammatory and chondroprotective features. PMID:25576403

  10. The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control

    PubMed Central

    Millerioux, Yoann; Ebikeme, Charles; Biran, Marc; Morand, Pauline; Bouyssou, Guillaume; Vincent, Isabel M; Mazet, Muriel; Riviere, Loïc; Franconi, Jean-Michel; Burchmore, Richard J S; Moreau, Patrick; Barrett, Michael P; Bringaud, Frédéric

    2013-01-01

    The Trypanosoma brucei procyclic form resides within the digestive tract of its insect vector, where it exploits amino acids as carbon sources. Threonine is the amino acid most rapidly consumed by this parasite, however its role is poorly understood. Here, we show that the procyclic trypanosomes grown in rich medium only use glucose and threonine for lipid biosynthesis, with threonine's contribution being ??2.5 times higher than that of glucose. A combination of reverse genetics and NMR analysis of excreted end-products from threonine and glucose metabolism, shows that acetate, which feeds lipid biosynthesis, is also produced primarily from threonine. Interestingly, the first enzymatic step of the threonine degradation pathway, threonine dehydrogenase (TDH, EC 1.1.1.103), is under metabolic control and plays a key role in the rate of catabolism. Indeed, a trypanosome mutant deleted for the phosphoenolpyruvate decarboxylase gene (PEPCK, EC 4.1.1.49) shows a 1.7-fold and twofold decrease of TDH protein level and activity, respectively, associated with a 1.8-fold reduction in threonine-derived acetate production. We conclude that TDH expression is under control and can be downregulated in response to metabolic perturbations, such as in the PEPCK mutant in which the glycolytic metabolic flux was redirected towards acetate production. PMID:23899193

  11. Biodegradation and metabolic pathway of sulfamethoxazole by Pseudomonas psychrophila HA-4, a newly isolated cold-adapted sulfamethoxazole-degrading bacterium.

    PubMed

    Jiang, Benchao; Li, Ang; Cui, Di; Cai, Rui; Ma, Fang; Wang, Yingning

    2014-05-01

    Sulfamethoxazole is a common antibiotic that is frequently detected in wastewater and surface water. This study investigated the biodegradation and metabolic pathway of sulfamethoxazole by Pseudomonas psychrophila HA-4, a cold-adapted bacterium. Strain HA-4, which uses sulfamethoxazole as its sole source of carbon and energy, was isolated at a low temperature (10 °C) and identified as P. psychrophila by physico-biochemical characterization and 16S rRNA gene sequence analysis. Strain HA-4 removed sulfamethoxazole at temperatures ranging from 5.0 °C to 30 °C, with the maximal removal rate at 10 °C. The maximal removal rate of sulfamethoxazole by strain HA-4 was 34.30 % after 192 h at 10 °C. The highest percentage of unsaturated fatty acid was determined to be 23.03 % at 10 °C, which adheres to the characteristic for cold-adapted psychrophiles and psychrotrophs. At low concentrations of sulfamethoxazole, the growth kinetics correlated well with the Haldane model. The single-substrate parameter values of sulfamethoxazole on cell growth were determined to be ? max?=?0.01 h(-1), K s?=?20.91 mg/l and K i?=?170.60 mg/l. Additionally, the major intermediates from sulfamethoxazole biodegradation by strain HA-4, including aniline, 3-amino-5-methylisoxazole, 4-aminothiophenol and sulfanilamide, were identified by GC-MS and high-resolution mass spectrometry (HR-MS) analysis. The results demonstrate that strain HA-4 has the potential to degrade sulfamethoxazole at low temperatures. PMID:24522726

  12. Toward an Integrated Structural Model of the 26S Proteasome*

    PubMed Central

    Förster, Friedrich; Lasker, Keren; Nickell, Stephan; Sali, Andrej; Baumeister, Wolfgang

    2010-01-01

    The 26S proteasome is the end point of the ubiquitin-proteasome pathway and degrades ubiquitylated substrates. It is composed of the 20S core particle (CP), where degradation occurs, and the 19S regulatory particle (RP), which ensures substrate specificity of degradation. Whereas the CP is resolved to atomic resolution, the architecture of the RP is largely unknown. We provide a comprehensive analysis of the current structural knowledge on the RP, including structures of the RP subunits, physical protein-protein interactions, and cryoelectron microscopy data. These data allowed us to compute an atomic model for the CP-AAA-ATPase subcomplex. In addition to this atomic model, further subunits can be mapped approximately, which lets us hypothesize on the substrate path during its degradation. PMID:20467039

  13. A mechanistic insight into a proteasome-independent constitutive inhibitor kappaBalpha (IkappaBalpha) degradation and nuclear factor kappaB (NF-kappaB) activation pathway in WEHI-231 B-cells.

    PubMed Central

    Shumway, Stuart D; Miyamoto, Shigeki

    2004-01-01

    Inducible activation of the transcription factor NF-kappaB (nuclear factor kappaB) is classically mediated by proteasomal degradation of its associated inhibitors, IkappaBalpha (inhibitory kappaBalpha) and IkappaBbeta. However, certain B-lymphocytes maintain constitutively nuclear NF-kappaB activity (a p50-c-Rel heterodimer) which is resistant to inhibition by proteasome inhibitors. This activity in the WEHI-231 B-cell line is associated with continual and preferential degradation of IkappaBalpha, which is also unaffected by proteasome inhibitors. Pharmacological studies indicated that there was a correlation between inhibition of IkappaBalpha degradation and constitutive p50-c-Rel activity. Domain analysis of IkappaBalpha by deletion mutagenesis demonstrated that an N-terminal 36-amino-acid sequence of IkappaBalpha represented an instability determinant for constitutive degradation. Moreover, domain grafting studies indicated that this sequence was sufficient to cause IkappaBbeta, but not chloramphenicol acetyltransferase, to be rapidly degraded in WEHI-231 B-cells. However, this sequence was insufficient to target IkappaBbeta to the non-proteasome degradation pathway, suggesting that there was an additional cis-element(s) in IkappaBalpha that was required for complete targeting. Nevertheless, the NF-kappaB pool associated with IkappaBbeta now became constitutively active by virtue of IkappaBbeta instability in these cells. These findings further support the notion that IkappaB instability governs the maintenance of constitutive p50-c-Rel activity in certain B-cells via a unique degradation pathway. PMID:14763901

  14. KINETICS OF CHEMICAL DEGRADATION OF MALATHION IN WATER

    EPA Science Inventory

    Acid degradation of malathion is too slow to be important under environmental reaction conditions. However, alkaline degradation is fast enough to be a competitive degradation pathway in the environment. The products of alkaline degradation are temperature dependent. Oxidation by...

  15. Cell cycle regulation as a mechanism for functional separation of the apparently redundant uracil DNA glycosylases TDG and UNG2

    PubMed Central

    Hardeland, Ulrike; Kunz, Christophe; Focke, Frauke; Szadkowski, Marta; Schär, Primo

    2007-01-01

    Human Thymine-DNA Glycosylase (TDG) is a member of the uracil DNA glycosylase (UDG) superfamily. It excises uracil, thymine and a number of chemical base lesions when mispaired with guanine in double-stranded DNA. These activities are not unique to TDG; at least three additional proteins with similar enzymatic properties are present in mammalian cells. The successful co-evolution of these enzymes implies the existence of non-redundant biological functions that must be coordinated. Here, we report cell cycle regulation as a mechanism for the functional separation of apparently redundant DNA glycosylases. We show that cells entering S-phase eliminate TDG through the ubiquitin–proteasome system and then maintain a TDG-free condition until G2. Incomplete degradation of ectopically expressed TDG impedes S-phase progression and cell proliferation. The mode of cell cycle regulation of TDG is strictly inverse to that of UNG2, which peaks in and throughout S-phase and then declines to undetectable levels until it appears again just before the next S-phase. Thus, TDG- and UNG2-dependent base excision repair alternates throughout the cell cycle, and the ubiquitin–proteasome pathway constitutes the underlying regulatory system. PMID:17526518

  16. Towards a Biocatalyst for (S)-Styrene Oxide Production: Characterization of the Styrene Degradation Pathway of Pseudomonas sp. Strain VLB120

    PubMed Central

    Panke, Sven; Witholt, Bernard; Schmid, Andreas; Wubbolts, Marcel G.

    1998-01-01

    In order to design a biocatalyst for the production of optically pure styrene oxide, an important building block in organic synthesis, the metabolic pathway and molecular biology of styrene degradation in Pseudomonas sp. strain VLB120 was investigated. A 5.7-kb XhoI fragment, which contained on the same strand of DNA six genes involved in styrene degradation, was isolated from a gene library of this organism in Escherichia coli by screening for indigo formation. T7 RNA polymerase expression experiments indicated that this fragment coded for at least five complete polypeptides, StyRABCD, corresponding to five of the six genes. The first two genes encoded the potential carboxy-terminal part of a sensor, named StySc, and the complete response regulator StyR. Fusion of the putative styAp promoter to a lacZ reporter indicated that StySc and StyR together regulate expression of the structural genes at the transcriptional level. Expression of styScR also alleviated a block that prevented translation of styA mRNA when a heterologous promoter was used. The structural genes styA and styB produced a styrene monooxygenase that converted styrene to styrene oxide, which was then converted to phenylacetaldehyde by StyC. Sequence homology analysis of StyD indicated a probable function as a phenylacetaldehyde dehydrogenase. To assess the usefulness of the enzymes for the production of enantiomerically pure styrene oxide, we investigated the enantiospecificities of the reactions involved. Kinetic resolution of racemic styrene oxide by styrene oxide isomerase was studied with E. coli recombinants carrying styC, which converted styrene oxide at a very high rate but with only a slight preference for the S enantiomer. However, recombinants producing styrene monooxygenase catalyzed the formation of (S)-styrene oxide from inexpensive styrene with an excellent enantiomeric excess of more than 99% at rates up to 180 U g (dry weight) of cells?1. PMID:9603811

  17. Genetic and Chemical Activation of TFEB Mediates Clearance of Aggregated ?-Synuclein

    PubMed Central

    Kilpatrick, Kiri; Zeng, Yimeng; Hancock, Tommy; Segatori, Laura

    2015-01-01

    Aggregation of ?-synuclein (?-syn) is associated with the development of a number of neurodegenerative diseases, including Parkinson’s disease (PD). The formation of ?-syn aggregates results from aberrant accumulation of misfolded ?-syn and insufficient or impaired activity of the two main intracellular protein degradation systems, namely the ubiquitin-proteasome system and the autophagy-lysosomal pathway. In this study, we investigated the role of transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in preventing the accumulation of ?-syn aggregates in human neuroglioma cells. We found that TFEB overexpression reduces the accumulation of aggregated ?-syn by inducing autophagic clearance of ?-syn. Furthermore, we showed that pharmacological activation of TFEB using 2-hydroxypropyl-?-cyclodextrin promotes autophagic clearance of aggregated ?-syn. In summary, our findings demonstrate that TFEB modulates autophagic clearance of ?-syn and suggest that pharmacological activation of TFEB is a promising strategy to enhance the degradation of ?-syn aggregates. PMID:25790376

  18. Cathepsin D in a murine model of frontotemporal dementia with Parkinsonism-linked to chromosome 17.

    PubMed

    Fernández-Montoya, Julia; Pérez, Mar

    2015-01-01

    Tauopathies, such as Alzheimer's disease (AD) and Frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), are characterized by tau accumulation. This accumulation could result from alterations in tau degradation by either the ubiquitin-proteasome system or the autophagy-lysosomal pathway. To analyze a possible alteration of the autophagy-lysosomal pathway in transgenic mice expressing human tau with three FTDP-17 missense mutations (TauVLW mice), we studied the lysosomal enzyme Cathepsin D. The hippocampi of TauVLW mice, where the human mutant tau accumulates, showed both increased Cathepsin D and partial colocalization of Cathepsin D with human mutant tau. At the ultrastructural level, some multivesicular bodies showed human mutant tau-immunopositive vesicles. This finding could provide insights into the molecular mechanisms of tau degradation in human tauopathies. PMID:25428250

  19. Regulation by mitophagy.

    PubMed

    Hattori, Nobutaka; Saiki, Shinji; Imai, Yuzuru

    2014-08-01

    Eukaryotes employ elaborate mitochondrial quality control to maintain the function of the power-generating organelle. Mitochondrial quality control is particularly important for the maintenance of neural and muscular tissues. Mitophagy is specialized version of the autophagy pathway. Mitophagy delivers damaged mitochondria to lysosomes for degradation. Recently, a series of elegant studies have demonstrated that two Parkinson's disease-associated genes PINK1 and parkin are involved in the maintenance of healthy mitochondria as mitophagy. Parkin in co-operation with PINK1 specifically recognizes damaged mitochondria with reduced mitochondrial membrane potential (??m), rapidly isolates them from the mitochondrial network and eliminates them through the ubiquitin-proteasome and autophagy pathways. Here we introduce and review recent studies that contribute to understanding the molecular mechanisms of mitophagy such as PINK1 and Parkin-mediated mitochondrial regulation. We also discuss how defects in the PINK1-Parkin pathway may cause neurodegeneration in Parkinson's disease. PMID:24842103

  20. Degradation Kinetics of VX

    SciTech Connect

    Gary S. Groenewold

    2010-12-01

    O-ethyl S-(2-diisopropylaminoethyl)phosphonothiolate (VX) is the most toxic of the conventional chemical warfare agents. It is a persistent compound, an attribute derived from its relative involatility and slow rates of hydrolysis. These properties suggest that VX can linger in an exposed environment for extended periods of time long after the air has cleared. Concern over prolonged risk from VX exposure is exacerbated by the fact that it poses a dermal contact hazard. Hence a detailed understanding of volatilization rates, and degradation pathways and rates occurring in various environments is needed. Historically, volatilization has not been considered to be an important mechanism for VX depletion, but recent studies have shown that a significant fraction of VX may volatilize, depending on the matrix. A significant body of research has been conducted over the years to unravel VX degradation reaction pathways and to quantify the rates at which they proceed. Rigorous measurement of degradation rates is frequently difficult, and thus in many cases the degradation of VX has been described in terms of half lives, while in fewer instances rate constants have been measured. This variable approach to describing degradation kinetics reflects uncertainty regarding the exact nature of the degradation mechanisms. In this review, rates of VX degradation are compared on the basis of pseudo-first order rate constants, in order to provide a basis for assessing likelihood of VX persistence in a given environment. An issue of specific concern is that one VX degradation pathway produces S-2-(diisopropylaminoethyl) methylphosphonothioic acid (known as EA2192), which is a degradation product that retains much of the original toxicity of VX. Consequently degradation pathways and rates for EA2192 are also discussed.

  1. Lindane degradation by Candida VITJzN04, a newly isolated yeast strain from contaminated soil: kinetic study, enzyme analysis and biodegradation pathway.

    PubMed

    Salam, Jaseetha Abdul; Das, Nilanjana

    2014-04-01

    A new yeast strain was isolated from sugarcane cultivation field which was able to utilize lindane as sole carbon source for growth in mineral medium. The yeast was identified and named as Candida sp. VITJzN04 based on a polyphasic approach using morphological, biochemical and 18S rDNA, D1/D2 and ITS sequence analysis. The isolated yeast strain efficiently degraded 600 mg L?¹ of lindane within 6 days in mineral medium under the optimal conditions (pH 7; temperature 30 °C and inoculum dosage 0.06 g L?¹) with the least half-life of 1.17 days and degradation constant of 0.588 per day. Lindane degradation was tested with various kinetic models and results revealed that the reaction could be described best by first-order and pseudo first-order models. In addition, involvement of the enzymes viz. dechlorinase, dehalogenase, dichlorohydroquinone reductive dechlorinase, lignin peroxidase and manganese peroxidase was noted during lindane degradation. Addition of H2O2 in the mineral medium showed 32 % enhancement of lindane degradation within 3 days. Based on the metabolites identified by GC-MS and FTIR analysis, sequential process of lindane degradation by Candida VITJzN04 was proposed. To the best of our knowledge, this is the first report of isolation and characterization of lindane-degrading Candida sp. and elucidation of enzyme systems during the degradation process. PMID:24217897

  2. BIOCHEMISTRY: Cargo Load Reduction

    NSDL National Science Digital Library

    Ineke Braakman (Utrecht University; Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Faculty of Science)

    2008-07-25

    Access to the article is free, however registration and sign-in are required. A specific enzyme that reduces protein disulfide bonds is part of a complex that eliminates aggregated, misfolded proteins. In eukaryotic cells, most newly synthesized secretory proteins are first translocated into the endoplasmic reticulum (ER) and transit through organelles that constitute a secretory pathway. However, proteins that misfold in the ER are retrotranslocated out of this organelle to the cytoplasm, where they are degraded by the ubiquitin-proteasome system (a process called ER-associated degradation). For efficient retrotranslocation, the disulfide bonds of misfolded proteins must be reduced, and on page 569 in this issue, Ushioda et al. (1) report that this reaction is catalyzed by ERdj5, the first dedicated reductase identified in the ER.

  3. VPS29-VPS35 intermediate of retromer is stable and may be involved in the retromer complex assembly process.

    PubMed

    Fuse, Atsuhito; Furuya, Norihiko; Kakuta, Soichiro; Inose, Aki; Sato, Masumi; Koike, Masato; Saiki, Shinji; Hattori, Nobutaka

    2015-06-01

    Retromer is a complex of proteins that functions in the endosome-to-Golgi retrieval cargo transport pathway. VPS35 works as the central subunit of retromer to recognize the cargos and binds with VPS29 and VPS26 via distinct domains. We show that deficiency of VPS35 or VPS29 accompanies degradation of other subunits, whereas VPS26 deficiency had no effect on VPS29 and VPS35 levels. Although VPS35 forms VPS26-VPS35 and VPS29-VPS35 sub-complexes with similar efficiency in vitro, VPS26-VPS35 was more easily degradable by the ubiquitin-proteasome-system than VPS29-VPS35. These results indicate that VPS29 and VPS35 form a biologically stable sub-complex in vivo. PMID:25937119

  4. TOF-SIMS investigation of degradation pathways occurring in a variety of organic photovoltaic devices--the ISOS-3 inter-laboratory collaboration.

    PubMed

    Andreasen, Birgitta; Tanenbaum, David M; Hermenau, Martin; Voroshazi, Eszter; Lloyd, Matthew T; Galagan, Yulia; Zimmernann, Birger; Kudret, Suleyman; Maes, Wouter; Lutsen, Laurence; Vanderzande, Dirk; Würfel, Uli; Andriessen, Ronn; Rösch, Roland; Hoppe, Harald; Teran-Escobar, Gerardo; Lira-Cantu, Monica; Rivaton, Agnès; Uzuno?lu, Gül?ah Y; Germack, David S; Hösel, Markus; Dam, Henrik F; Jørgensen, Mikkel; Gevorgyan, Suren A; Madsen, Morten V; Bundgaard, Eva; Krebs, Frederik C; Norrman, Kion

    2012-09-01

    The present work is the fourth (and final) contribution to an inter-laboratory collaboration that was planned at the 3rd International Summit on Organic Photovoltaic Stability (ISOS-3). The collaboration involved six laboratories capable of producing seven distinct sets of OPV devices that were degraded under well-defined conditions in accordance with the ISOS-3 protocols. The degradation experiments lasted up to 1830 hours and involved more than 300 cells on more than 100 devices. The devices were analyzed and characterized at different points of their lifetimes by a large number of non-destructive and destructive techniques in order to identify specific degradation mechanisms responsible for the deterioration of the photovoltaic response. Work presented herein involves time-of-flight secondary ion mass spectrometry (TOF-SIMS) in order to study chemical degradation in-plane as well as in-depth in the organic solar cells. Various degradation mechanisms were investigated and correlated with cell performance. For example, photo-oxidation of the active material was quantitatively studied as a function of cell performance. The large variety of cell architectures used (some with and some without encapsulation) enabled valuable comparisons and important conclusions to be drawn on degradation behaviour. This comprehensive investigation of OPV stability has significantly advanced the understanding of degradation behaviour in OPV devices, which is an important step towards large scale application of organic solar cells. PMID:22829118

  5. The degradation (by distinct pathways) of human D-amino acid oxidase and its interacting partner pLG72--two key proteins in D-serine catabolism in the brain.

    PubMed

    Cappelletti, Pamela; Campomenosi, Paola; Pollegioni, Loredano; Sacchi, Silvia

    2014-02-01

    Human D-amino acid oxidase (EC 1.4.3.3; hDAAO) is a peroxisomal flavoenzyme significantly enriched in the mammalian brain. hDAAO has been proposed to play (with serine racemase; EC 5.1.1.18) an essential role in the catabolism of D-serine, an 'atypical' key signalling molecule that acts as allosteric activator of the N-methyl-D-aspartate-type glutamate receptor (NMDAr). hDAAO and its interacting partner pLG72 have been related to schizophrenia, a highly disabling psychiatric disorder in which a dysfunction of NMDA-mediated neurotransmission is widely assumed to occur. We previously demonstrated that the D-serine cellular concentration depends on hDAAO and pLG72 expression levels and that newly-synthesized hDAAO interacts with its modulator in the cytosol, being progressively destabilized and inactivated. To obtain insight into the mechanisms regulating cellular D-serine levels, we investigated the degradation pathways of hDAAO and pLG72 in U87 glioblastoma cells stably expressing enhanced yellow fluorescent protein-hDAAO (peroxisomal), hDAAO-enhanced yellow fluorescent protein (cytosolic) or pLG72-enhanced cyan fluorescent protein (mitochondrial) proteins. hDAAO is a long-lived protein: the peroxisomal fraction of this flavoprotein is degraded via the lysosomal/endosomal pathway (and blocking this pathway increases the cellular hDAAO activity and decreases D-serine levels), whereas the cytosolic portion is ubiquitinated and targeted to the proteasome. By contrast, pLG72 shows a rapid turnover (t(1/2) ? 25-40 min) and is degraded via the proteasome system, albeit not ubiquitinated. Overexpression of pLG72 increases the turnover of hDAAO, in turn playing a protective role against excessive D-serine depletion. PMID:24237903

  6. Pin1, a new player in the fate of HIF-1? degradation: an hypothetical mechanism inside vascular damage as Alzheimer’s disease risk factor

    PubMed Central

    Lonati, Elena; Brambilla, Anna; Milani, Chiara; Masserini, Massimo; Palestini, Paola; Bulbarelli, Alessandra

    2014-01-01

    Aetiology of neurodegenerative mechanisms underlying Alzheimer’s disease (AD) are still under elucidation. The contribution of cerebrovascular deficiencies (such as cerebral ischemia/stroke) has been strongly endorsed in recent years. Reduction of blood supply leading to hypoxic condition is known to activate cellular responses mainly controlled by hypoxia-inducible transcription factor-1 (HIF-1). Thus alterations of oxygen responsive HIF-1? subunit in the central nervous system may contribute to the cognitive decline, especially influencing mechanisms associated to amyloid precursor protein (APP) amyloidogenic metabolism. Although HIF-1? protein level is known to be regulated by von Hippel-Lindau (VHL) ubiquitin-proteasome system, it has been recently suggested that glycogen synthase kinase-3? (Gsk-3?) promotes a VHL-independent HIF-1? degradation. Here we provide evidences that in rat primary hippocampal cell cultures, HIF-1? degradation might be mediated by a synergic action of Gsk-3? and peptidyl-prolyl cis/trans isomerase (Pin1). In post-ischemic conditions, such as those mimicked with oxygen glucose deprivation (OGD), HIF-1? protein level increases remaining unexpectedly high for long time after normal condition restoration jointly with the increase of lactate dehydrogenase (LDH) and ?-secretase 1 (BACE1) protein expression (70 and 140% respectively). Interestingly the Pin1 activity decreases about 40–60% and Pin1S16 inhibitory phosphorylation significantly increases, indicating that Pin1 binding to its substrate and enzymatic activity are reduced by treatment. Co-immunoprecipitation experiments demonstrate that HIF-1?/Pin1 in normoxia are associated, and that in presence of specific Pin1 and Gsk-3? inhibitors their interaction is reduced in parallel to an increase of HIF-1? protein level. Thus we suggest that in post-OGD neurons the high level of HIF-1? might be due to Pin1 binding ability and activity reduction which affects HIF-1? degradation: an event that may highlight the relevance of ischemia/HIF-1? as a risk factor in AD pathogenesis. PMID:24478626

  7. Roles of Horizontal Gene Transfer and Gene Integration in Evolution of 1,3-Dichloropropene- and 1,2-Dibromoethane-Degradative Pathways

    Microsoft Academic Search

    Michael J. Larkin; Leonid A. Kulakov; Gerrit J. Poelarends; Dick B. Janssen

    2000-01-01

    The haloalkane-degrading bacteria Rhodococcus rhodochrous NCIMB13064, Pseudomonas pavonaceae 170, and Mycobacterium sp. strain GP1 share a highly conserved haloalkane dehalogenase gene (dhaA). Here, we describe the extent of the conserved dhaA segments in these three phylogenetically distinct bacteria and an analysis of their flanking sequences. The dhaA gene of the 1-chlorobutane-degrading strain NCIMB13064 was found to reside within a 1-chlorobutane

  8. The Ubiquitin-Specific Protease 14 (USP14) Is a Critical Regulator of Long-Term Memory Formation

    ERIC Educational Resources Information Center

    Jarome, Timothy J.; Kwapis, Janine L.; Hallengren, Jada J.; Wilson, Scott M.; Helmstetter, Fred J.

    2014-01-01

    Numerous studies have suggested a role for ubiquitin-proteasome-mediated protein degradation in learning-dependent synaptic plasticity; however, very little is known about how protein degradation is regulated at the level of the proteasome during memory formation. The ubiquitin-specific protease 14 (USP14) is a proteasomal deubiquitinating enzyme…

  9. ERAD: the long road to destruction

    Microsoft Academic Search

    Birgit Meusser; Christian Hirsch; Ernst Jarosch; Thomas Sommer

    2005-01-01

    Endoplasmic reticulum (ER)-associated protein degradation (ERAD) eliminates misfolded or unassembled proteins from the ER. ERAD targets are selected by a quality control system within the ER lumen and are ultimately destroyed by the cytoplasmic ubiquitin–proteasome system (UPS). The spatial separation between substrate selection and degradation in ERAD requires substrate transport from the ER to the cytoplasm by a process termed

  10. Can tonsillectomy modify the innate and adaptive immunity pathways involved in IgA nephropathy?

    PubMed

    Vergano, Luca; Loiacono, Elisa; Albera, Roberto; Coppo, Rosanna; Camilla, Roberta; Peruzzi, Licia; Amore, Alessandro; Donadio, Maria Elena; Chiale, Federica; Boido, Alberto; Mariano, Filippo; Mazzucco, Gianna; Ravera, Sara; Cancarini, Giovanni; Magistroni, Riccardo; Beltrame, Giulietta; Rollino, Cristiana; Stratta, Piero; Quaglia, Marco; Bergia, Roberto; Cravero, Raffaella; Cusinato, Stefano; Benozzi, Luisa; Savoldi, Silvana; Licata, Carola

    2015-02-01

    The benefits of tonsillectomy in IgA nephropathy (IgAN) are still debated. Tonsillectomy may remove pathogen sources and reduce the mucosal associated lymphoid tissue (MALT), limiting degalactosylated IgA1 (deGal-IgA1) production, which is considered to be the initiating pathogenetic event leading to IgA glomerular deposition. In the European network VALIGA, 62/1147 IgAN patients underwent tonsillectomy (TxIgAN). In a cross-sectional study 15 of these patients were tested and compared to 45 non-tonsillectomized IgAN (no-TxIgAN) and healthy controls (HC) regarding levels of deGal-IgA1, and markers of innate immunity and oxidative stress, including toll-like receptors (TLR)2, 3, 4 and 9 mRNAs, proteasome (PS) and immunoproteasome (iPS) mRNAs in peripheral blood mononuclear cells (PBMC), and advanced oxidation protein products (AOPP). Levels of deGal-IgA1 were lower in TxIgAN than in no-TxIgAN (p = 0.015), but higher than in HC (p = 0.003). TLR mRNAs were more expressed in TxIgAN than in HC (TLR4, p = 0.021; TLR9, p = 0.027), and higher in TxIgAN than in no-TxIgAN (p ? 0.001 for TLR2, 4, 9). A switch from PS to iPS was detected in PBMC of TxIgAN in comparison to HC and it was higher than in no-TxIgAN [large multifunctional peptidase (LMP)2/?1, p = 0.039; LPM7/?5, p < 0.0001]. The levels of AOPP were significantly higher in TxIgAN than HC (p < 0.001) and no-TxIgAN (p = 0.033). In conclusion, the activation of innate immunity via TLRs and ubiquitin-proteasome pathways and the pro-oxidative milieu were not affected by tonsillectomy, even though the levels of aberrantly galactosylated IgA1 were lower in patients with IgAN who had tonsillectomy. The residual hyperactivation of innate immunity in tonsillectomized patients may result from extra-tonsillar MALT. PMID:24756968

  11. Novel Gene Clusters and Metabolic Pathway Involved in 3,5,6-Trichloro-2-Pyridinol Degradation by Ralstonia sp. Strain T6

    PubMed Central

    Li, Jingquan; Huang, Yan; Hou, Ying; Li, Xiangmin; Cao, Hui

    2013-01-01

    3,5,6-Trichloro-2-pyridinol (TCP) is a widespread pollutant. Some bacteria and fungi have been reported to degrade TCP, but the gene clusters responsible for TCP biodegradation have not been characterized. In this study, a fragment of the reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase gene tcpA was amplified from the genomic DNA of Ralstonia sp. strain T6 with degenerate primers. The tcpA disruption mutant strain T6-?tcpA could not degrade TCP but could degrade the green intermediate metabolite 3,6-dihydroxypyridine-2,5-dione (DHPD), which was generated during TCP biodegradation by strain T6. The flanking sequences of tcpA were obtained by self-formed adaptor PCR. tcpRXA genes constitute a gene cluster. TcpR and TcpX are closely related to the LysR family transcriptional regulator and flavin reductase, respectively. T6-?tcpA-com, the complementation strain for the mutant strain T6-?tcpA, recovered the ability to degrade TCP, and the strain Escherichia coli DH10B-tcpRXA, which expressed the tcpRXA gene cluster, had the ability to transform TCP to DHPD, indicating that tcpA is a key gene in the initial step of TCP degradation and that TcpA dechlorinates TCP to DHPD. A library of DHPD degradation-deficient mutants of strain T6 was obtained by random transposon mutagenesis. The fragments flanking the Mariner transposon were amplified and sequenced, and the dhpRIJK gene cluster was cloned. DhpJ could transform DHPD to yield an intermediate product, 5-amino-2,4,5-trioxopentanoic acid (ATOPA), which was further degraded by DhpI. DhpR and DhpK are closely related to the AraC family transcriptional regulator and the MFS family transporter, respectively. PMID:24056464

  12. Ubiquitin-proteasome system is involved in induction of LFA-1\\/ICAM-1-dependent adhesion of HL60 cells

    Microsoft Academic Search

    Koko Katagiri; Hideyoshi Yokosawa; Tatsuo Kinashi; Seiichi Kawashima; Shinkichi Irie; Keiji Tanaka; Takuya Katagiri

    1999-01-01

    Membrane-permeable proteasome in- hibitors, lactacystin (LC) and N-acetyl-Leu-Leu- norleucinal (ALLN), but not calpain inhibitor Z-Leu- leucinal (ZLL), prevented LFA-1\\/ICAM-1-dependent cellular adhesion of TPA-stimulated HL-60 cells. These proteasome inhibitors affected neither the induction of monocytic differentiation nor the ac- companying protein-tyrosine phosphorylation. They suppressed the increase in the avidity of LFA-1 to ICAM-1 without changing the expression of these molecules. Immunoblotting

  13. Metabolic analysis of the soil microbe Dechloromonas aromatica str. RCB: indications of a surprisingly complex life-style and cryptic anaerobic pathways for aromatic degradation

    Microsoft Academic Search

    Kennan Kellaris Salinero; Keith Keller; William S Feil; Helene Feil; Stephan Trong; Genevieve Di Bartolo; Alla Lapidus

    2009-01-01

    BACKGROUND: Initial interest in Dechloromonas aromatica strain RCB arose from its ability to anaerobically degrade benzene. It is also able to reduce perchlorate and oxidize chlorobenzoate, toluene, and xylene, creating interest in using this organism for bioremediation. Little physiological data has been published for this microbe. It is considered to be a free-living organism. RESULTS: The a priori prediction that

  14. Unveiling the long-held secrets of the 26S proteasome.

    PubMed

    Förster, Friedrich; Unverdorben, Pia; Sled?, Pawe?; Baumeister, Wolfgang

    2013-09-01

    The 26S proteasome is a 2.5 MDa molecular machine for the degradation of substrates of the ubiquitin-proteasome pathway with a key role in cellular proteostasis. Until recently, only the structure of its core particle, the 20S proteasome, could be studied in detail, whereas the 19S regulatory particle or the holocomplex remained elusive. Novel integrative approaches have now revealed the molecular architecture of the entire complex and provided the first insights into the conformational changes during its functional cycle. Here we review the problems in structural studies of the 26S proteasome, the methods that made possible its structure determination, the architectural principles of the holocomplex, and its conformational space. These advances provide valuable insights into the mechanism of substrate recruitment and processing preceding their destruction in the 20S core particle. PMID:24010714

  15. Promiscuous Interactions of gp78 E3 ligase CUE domain with polyubiquitin chains

    PubMed Central

    Liu, Shan; Chen, Yinghua; Huang, Tao; Tarasov, Sergey; King, Aaren; Li, Jess; Weissman, Allan M.; Byrd, R. Andrew; Das, Ranabir

    2012-01-01

    Recognition of ubiquitin and polyubiquitin chains by ubiquitin-binding domains (UBDs) is vital for ubiquitin-mediated signaling pathways. The endoplasmic reticulum resident RING finger ubiquitin ligase (E3) gp78 regulates critical proteins via the ubiquitin-proteasome system to maintain cellular homeostasis and includes a UBD known as the CUE domain, which is essential for function. A probable role of this domain is to recognize ubiquitin modified substrates, enabling gp78 to assemble polyubiquitin chains on these substrates and mark them for degradation. Here, we report the molecular details of the interaction of gp78CUE domain with ubiquitin and diubiquitin. The gp78CUE domain exhibits a well-defined set of interactions with ubiquitin and a dynamic, promiscuous interaction with diubiquitin chains. This leads to a model where the CUE domain functions to both facilitate substrate binding and enables switching between adjacent ubiquitin molecules of a growing chain to facilitate processivity in ubiquitination. PMID:23123110

  16. Hydrazino-aza and N-azapeptoids with therapeutic potential as anticancer agents.

    PubMed

    Bouget, Karine; Aubin, Sandrine; Delcros, Jean Guy; Arlot-Bonnemains, Yannick; Baudy-Floc'h, Michèle

    2003-11-17

    The ubiquitin-proteasome-mediated degradation pathway plays an important role in regulating protein turnover in eucaryotic cells and, consequently, regulates both cell proliferation and cell death. The proteasome influences many cellular regulatory signals and is thus a potential target for pharmacological agents. The study of proteasome function has led to the identification of several natural and synthetic compounds that can act as tumor cell growth inhibitors. In this study, we have developed a series of hydrazino-aza and N-azapeptoids, analogues of Ac-Leucyl-Leucyl-Norleucinal (ALLN) a non-specific peptidyl aldehyde inhibitor of the proteasome. These peptide analogues share a common backbone and bear different C- and N-terminal functions. Their antiproliferative activity on murine leukemia L1210 cells is reported here. PMID:14604649

  17. An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome

    SciTech Connect

    Foerster, Friedrich [Department of Structural Biology, Max-Planck-Institute of Biochemistry, D-82152 Martinsried (Germany) [Department of Structural Biology, Max-Planck-Institute of Biochemistry, D-82152 Martinsried (Germany); Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, and California Institute for Quantitative Biosciences (QB3), University of California at San Francisco, San Francisco (United States); Lasker, Keren [Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, and California Institute for Quantitative Biosciences (QB3), University of California at San Francisco, San Francisco (United States) [Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, and California Institute for Quantitative Biosciences (QB3), University of California at San Francisco, San Francisco (United States); Blavatnik School of Computer Science, Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv (Israel); Beck, Florian; Nickell, Stephan [Department of Structural Biology, Max-Planck-Institute of Biochemistry, D-82152 Martinsried (Germany)] [Department of Structural Biology, Max-Planck-Institute of Biochemistry, D-82152 Martinsried (Germany); Sali, Andrej [Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, and California Institute for Quantitative Biosciences (QB3), University of California at San Francisco, San Francisco (United States)] [Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, and California Institute for Quantitative Biosciences (QB3), University of California at San Francisco, San Francisco (United States); Baumeister, Wolfgang, E-mail: baumeist@biochem.mpg.de [Department of Structural Biology, Max-Planck-Institute of Biochemistry, D-82152 Martinsried (Germany)] [Department of Structural Biology, Max-Planck-Institute of Biochemistry, D-82152 Martinsried (Germany)

    2009-10-16

    The 26S proteasome is the most downstream element of the ubiquitin-proteasome pathway of protein degradation. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The RP consists of 6 AAA-ATPases and at least 13 non-ATPase subunits. Based on a cryo-EM map of the 26S proteasome, structures of homologs, and physical protein-protein interactions we derive an atomic model of the AAA-ATPase-CP sub-complex. The ATPase order in our model (Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5) is in excellent agreement with the recently identified base-precursor complexes formed during the assembly of the RP. Furthermore, the atomic CP-AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP-RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners.

  18. Insights into the relationship between the proteasome and autophagy in human and yeast cells.

    PubMed

    Athané, Axel; Buisson, Anthony; Challier, Marion; Beaumatin, Florian; Manon, Stéphen; Bhatia-Kiššová, Ingrid; Camougrand, Nadine

    2015-07-01

    In eukaryotes, the ubiquitin-proteasome system (UPS) and autophagy are two major intracellular protein degradation pathways. Several lines of evidence support the emerging concept of a coordinated and complementary relationship between these two processes, and a particularly interesting finding is that the inhibition of the proteasome induces autophagy. Yet, there is limited knowledge of the regulation of the UPS by autophagy. In this study, we show that the disruption of ATG5 and ATG32 genes in yeast cells under both nutrient-deficient conditions as well as stress that causes mitochondrial dysfunction leads to an activation of proteasome. The same scenario occurs after pharmacological inhibition of basal autophagy in cultured human cells. Our findings underline the view that the two processes are interconnected and tend to compensate, to some extent, for each other's functions. PMID:25882491

  19. Studies on the degradation pathway of iron-sulfur centers during unfolding of a hyperstable ferredoxin: cluster dissociation, iron release and protein stability

    Microsoft Academic Search

    Sónia S. Leal; Miguel Teixeira; Cláudio M. Gomes

    2004-01-01

    The ferredoxin from the thermoacidophile Acidianus ambivalens is a representative of the archaeal family of di-cluster [3Fe-4S][4Fe-4S] ferredoxins. Previous studies have shown that these ferredoxins are intrinsically very stable and led to the suggestion that upon protein unfolding the iron-sulfur clusters degraded via linear three-iron sulfur center species, with 610 and 520 nm absorption bands, resembling those observed in purple aconitase.

  20. GPG-NH2 acts via the metabolite ?HGA to target HIV1 Env to the ER-associated protein degradation pathway

    Microsoft Academic Search

    Alenka Jejcic; Stefan Höglund; Anders Vahlne

    2010-01-01

    ABSTRACT: BACKGROUND: The synthetic peptide glycyl-prolyl-glycine amide (GPG-NH2) was previously shown to abolish the ability of HIV-1 particles to fuse with the target cells, by reducing the content of the viral envelope glycoprotein (Env) in progeny HIV-1 particles. The loss of Env was found to result from GPG-NH2 targeting the Env precursor protein gp160 to the ER-associated protein degradation (ERAD)

  1. Coherent anti-Stokes Raman scattering imaging of myelin degradation reveals a calcium-dependent pathway in lyso-PtdCho-induced demyelination.

    PubMed

    Fu, Yan; Wang, Haifeng; Huff, Terry B; Shi, Riyi; Cheng, Ji-Xin

    2007-10-01

    Coherent anti-Stokes Raman scattering (CARS) microscopy, which allows vibrational imaging of myelin sheath in its natural state, was applied to characterize lysophosphatidylcholine (lyso-PtdCho)-induced myelin degradation in tissues and in vivo. After the injection of lyso-PtdCho into ex vivo spinal tissues or in vivo mouse sciatic nerves, myelin swelling characterized by the decrease of CARS intensity and loss of excitation polarization dependence was extensively observed. The swelling corresponds to myelin vesiculation and splitting observed by electron microscopy. The demyelination dynamics were quantified by the increase of g ratio measured from the CARS images. Treating spinal tissues with Ca2+ ionophore A23187 resulted in the same kind of myelin degradation as lyso-PtdCho. Moreover, the demyelination lesion size was significantly reduced upon preincubation of the spinal tissue with Ca2+ free Krebs' solution or a cytosolic phospholipase A2 (cPLA(2)) inhibitor or a calpain inhibitor. In accordance with the imaging results, removal of Ca2+ or addition of cPLA(2) inhibitor or calpain inhibitor in the Krebs' solution remarkably increased the mean compound action potential amplitude in lyso-PtdCho treated spinal tissues. Our results suggest that lyso-PtdCho induces myelin degradation via Ca(2+) influx into myelin and subsequent activation of cPLA(2) and calpain, which break down the myelin lipids and proteins. The current work also shows that CARS microscopy is a potentially powerful tool for the study of demyelination. PMID:17551984

  2. Apoprotein B100 has a prolonged interaction with the translocon during which its lipidation and translocation change from dependence on the microsomal triglyceride transfer protein to independence

    PubMed Central

    Mitchell, Deborah M.; Zhou, Mingyue; Pariyarath, Rajalakshmi; Wang, Hongxing; Aitchison, John D.; Ginsberg, Henry N.; Fisher, Edward A.

    1998-01-01

    When lipid synthesis is limited in HepG2 cells, apoprotein B100 (apoB100) is not secreted but rapidly degraded by the ubiquitin-proteasome pathway. To investigate apoB100 biosynthesis and secretion further, the physical and functional states of apoB100 destined for either degradation or lipoprotein assembly were studied under conditions in which lipid synthesis, proteasomal activity, and microsomal triglyceride transfer protein (MTP) lipid-transfer activity were varied. Cells were pretreated with a proteasomal inhibitor (which remained with the cells throughout the experiment) and radiolabeled for 15 min. During the chase period, labeled apoB100 remained associated with the microsomes. Furthermore, by crosslinking sec61? to apoB100, we showed that apoB100 remained close to the translocon at the same time apoB100–ubiquitin conjugates could be detected. When lipid synthesis and lipoprotein assembly/secretion were stimulated by adding oleic acid (OA) to the chase medium, apoB100 was deubiquitinated, and its interaction with sec61? was disrupted, signifying completion of translocation concomitant with the formation of lipoprotein particles. MTP participates in apoB100 translocation and lipoprotein assembly. In the presence of OA, when MTP lipid-transfer activity was inhibited at the end of pulse labeling, apoB100 secretion was abolished. In contrast, when the labeled apoB100 was allowed to accumulate in the cell for 60 min before adding OA and the inhibitor, apoB100 lipidation and secretion were no longer impaired. Overall, the data imply that during most of its association with the endoplasmic reticulum, apoB100 is close to or within the translocon and is accessible to both the ubiquitin-proteasome and lipoprotein-assembly pathways. Furthermore, MTP lipid-transfer activity seems to be necessary only for early translocation and lipidation events. PMID:9843958

  3. A TNF and c-Cbl-dependent FLIPS-degradation pathway and its function in Mycobacterium tuberculosis–induced macrophage apoptosis

    Microsoft Academic Search

    Manikuntala Kundu; Sushil Kumar Pathak; Kuldeep Kumawat; Sanchita Basu; Gargi Chatterjee; Shresh Pathak; Takuya Noguchi; Kohsuke Takeda; Hidenori Ichijo; Christine B F Thien; Wallace Y Langdon; Joyoti Basu

    2009-01-01

    Apoptosis is central to the interaction between pathogenic mycobacteria and host macrophages. Caspase-8-dependent apoptosis of infected macrophages, which requires activation of the mitogen-activated protein (MAP) kinase p38, lowers the spread of mycobacteria. Here we establish a link between the release of tumor necrosis factor (TNF) and mycobacteria-mediated macrophage apoptosis. TNF activated a pathway involving the kinases ASK1, p38 and c-Abl.

  4. Pharmacological strategies in lung cancer-induced cachexia: effects on muscle proteolysis, autophagy, structure, and weakness.

    PubMed

    Chacon-Cabrera, Alba; Fermoselle, Clara; Urtreger, Alejandro J; Mateu-Jimenez, Mercè; Diament, Miriam J; de Kier Joffé, Elisa D Bal; Sandri, Marco; Barreiro, Esther

    2014-11-01

    Cachexia is a relevant comorbid condition of chronic diseases including cancer. Inflammation, oxidative stress, autophagy, ubiquitin-proteasome system, nuclear factor (NF)-?B, and mitogen-activated protein kinases (MAPK) are involved in the pathophysiology of cancer cachexia. Currently available treatment is limited and data demonstrating effectiveness in in vivo models are lacking. Our objectives were to explore in respiratory and limb muscles of lung cancer (LC) cachectic mice whether proteasome, NF-?B, and MAPK inhibitors improve muscle mass and function loss through several molecular mechanisms. Body and muscle weights, limb muscle force, protein degradation and the ubiquitin-proteasome system, signaling pathways, oxidative stress and inflammation, autophagy, contractile and functional proteins, myostatin and myogenin, and muscle structure were evaluated in the diaphragm and gastrocnemius of LC (LP07 adenocarcinoma) bearing cachectic mice (BALB/c), with and without concomitant treatment with NF-?B (sulfasalazine), MAPK (U0126), and proteasome (bortezomib) inhibitors. Compared to control animals, in both respiratory and limb muscles of LC cachectic mice: muscle proteolysis, ubiquitinated proteins, autophagy, myostatin, protein oxidation, FoxO-1, NF-?B and MAPK signaling pathways, and muscle abnormalities were increased, while myosin, creatine kinase, myogenin, and slow- and fast-twitch muscle fiber size were decreased. Pharmacological inhibition of NF-?B and MAPK, but not the proteasome system, induced in cancer cachectic animals, a substantial restoration of muscle mass and force through a decrease in muscle protein oxidation and catabolism, myostatin, and autophagy, together with a greater content of myogenin, and contractile and functional proteins. Attenuation of MAPK and NF-?B signaling pathway effects on muscles is beneficial in cancer-induced cachexia. PMID:24615622

  5. Newborn mouse lens proteome and its alteration by lysine 6 mutant ubiquitin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  6. Channel Catfish, Ictalurus punctatus, ubiquitin carboxy-terminal hydrolase L5 cDNA

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome cycle is a complex, non-lysosomal biochemical process for intracellular protein degradation. This process involves many enzymes. One of them is ubiquitin carboxy-terminal hydrolase (UCT). In this report, we cloned, sequenced and characterized the channel catfish UCT L5 cDNA....

  7. Identification of proteins that interact with mammalian peptide:N-glycanase and implicate this hydrolase in the proteasome-dependent pathway for protein degradation

    PubMed Central

    Park, Hangil; Suzuki, Tadashi; Lennarz, William J.

    2001-01-01

    Peptide:N-glycanase (PNGase) cleaves oligosaccharide chains from glycopeptides and glycoproteins. Recently the deduced amino acid sequence of a cytoplasmic PNGase has been identified in various eukaryotes ranging from yeast to mammals, suggesting that deglycosylation may play a central role in some catabolic process. Several lines of evidence indicate that the cytoplasmic enzyme is involved in the quality control system for newly synthesized glycoproteins. Two-hybrid library screening by using mouse PNGase as the target yielded several PNGase-interacting proteins that previously had been implicated in proteasome-dependent protein degradation: mHR23B, ubiquitin, a regulatory subunit of the 19S proteasome, as well as a protein containing an ubiquitin regulatory motif (UBX) and an ubiquitin-associated motif (UBA). These findings by using the two-hybrid system were further confirmed either by in vitro binding assays or size fractionation assays. These results suggest that PNGase may be required for efficient proteasome-mediated degradation of misfolded glycoproteins in mammalian cells. PMID:11562482

  8. Genomic Analysis of the Basal Lineage Fungus Rhizopus oryzae Reveals a Whole-Genome Duplication

    E-print Network

    associated with respiratory electron transport chains, the V-ATPase, and the ubiquitin­proteasome systems pathway, especially the major azole target, lanosterol 14a- demethylase (ERG11), could contribute families of cell-wall synthesis enzymes, essential for fungal cell integrity but absent in mammalian hosts

  9. Elevated Proteasome Capacity Extends Replicative Lifespan in Saccharomyces cerevisiae

    Microsoft Academic Search

    Undine Kruegel; Brett Robison; Thomas Dange; Günther Kahlert; Joe R. Delaney; Soumya Kotireddy; Mitsuhiro Tsuchiya; Scott Tsuchiyama; Christopher J. Murakami; Jennifer Schleit; George Sutphin; Daniel Carr; Krisztina Tar; Gunnar Dittmar; Matt Kaeberlein; Brian K. Kennedy; Marion Schmidt

    2011-01-01

    Aging is characterized by the accumulation of damaged cellular macromolecules caused by declining repair and elimination pathways. An integral component employed by cells to counter toxic protein aggregates is the conserved ubiquitin\\/proteasome system (UPS). Previous studies have described an age-dependent decline of proteasomal function and increased longevity correlates with sustained proteasome capacity in centenarians and in naked mole rats, a

  10. Conversion of chlorobiphenyls into phenylhexasdienoates and benzoates by the enzymes of the upper pathway for polychlorobiphenyl degradation encoded by the bph locus of pseudomonas sp. strain LB400

    SciTech Connect

    Seeger, M. [Gesellschaft fuer Biotechnologische Forschung-National Research Centre for Biotechnology, Braunschweig (Germany)]|[Universidad de Chile, Santiago (Chile); Timmis, K.N.; Hofer, B. [Gesellschaft fuer Biotechnologische Forschung-National Research Centre for Biotechnology, Braunschweig (Germany)

    1995-07-01

    Metabolism of 21 chlorobiphenyls by the enzymes of the upper biphenyl catabolic pathway encoded by the bph locus of Pseudomonas sp. strain LB400 was investigated by using recombinant strains harboring gene cassettes containing bphABC or bphABCD. The enzymes of the upper pathway were generally able to metabolize mono- and dischlorinated biphenyls but only partially transform most trichlorinated congeners investigated: 14 of 15 mono- and dichlorinated and 2 of 6 trichlorinated congeners were converted into benzoates. All mono- and at leas 8 of 12 dichlorinated congeners were attacked by the bphA-encoded biphenyl dioxygenase virtually exclusively at ortho and meta carbons. This enzyme exhibited a high degree of selectivity for the aromatic ring to be attacked, with the order or ring preference being non- > ortho- > meta- > para-substituted for mono- and dichlorinated congeners. The influence of the chlorine substitution pattern of the metabolized ring on benzoate formation resembled its influence on the reactivity of initial dioxygenation, suggesting that the rate of benzoate formation may frequently be determined by the rate of initial attack. The absorption spectra of phenylhexadienoates formed correlated with the presence or absence of a chlorine substituent at an ortho position. 24 refs., 3 figs., 2 tabs.

  11. Arsenite induces endothelial cytotoxicity by down-regulation of vascular endothelial nitric oxide synthase

    SciTech Connect

    Tsou, T.-C. [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, 100 Shih-Chuan 1st Road, Kaohsiung 807 (China)]. E-mail: tctsou@nhri.org.tw; Tsai, F.-Y. [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, 100 Shih-Chuan 1st Road, Kaohsiung 807 (China); Hsieh, Y.-W. [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, 100 Shih-Chuan 1st Road, Kaohsiung 807 (China); Li, L.-A. [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, 100 Shih-Chuan 1st Road, Kaohsiung 807 (China); Yeh, S.C [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, 100 Shih-Chuan 1st Road, Kaohsiung 807 (China); Chang, L.W. [Division of Environmental Health and Occupational Medicine, National Health Research Institutes, 100 Shih-Chuan 1st Road, Kaohsiung 807 (China)

    2005-11-01

    Epidemiological studies have demonstrated a high association of inorganic arsenic exposure with vascular diseases. Recent research has also linked this vascular damage to impairment of endothelial nitric oxide synthase (eNOS) function by arsenic exposure. However, the role of eNOS in regulating the arsenite-induced vascular dysfunction still remains to be clarified. In our present study, we investigated the effect of arsenite on Akt1 and eNOS and its involvement in cytotoxicity of vascular endothelial cells. Our study demonstrated that arsenite decreased the protein levels of both Akt1 and eNOS accompanied with increased levels of ubiquitination of total cell lysates. We found that inhibition of the ubiquitin-proteasome pathway by MG-132 could partially protect Akt1 and eNOS from degradation by arsenite together with a proportional protection from the arsenite-induced cytoxicity. Moreover, up-regulation of eNOS protein expression significantly attenuated the arsenite-induced cytotoxicity and eNOS activity could be significantly inhibited after incubation with arsenite for 24 h in a cell-free system. Our study indicated that endothelial eNOS activity could be attenuated by arsenite via the ubiquitin-proteasome-mediated degradation of Akt1/eNOS as well as via direct inhibition of eNOS activity. Our study also demonstrated that eNOS actually played a protective role in arsenite-induced cytoxicity. These observations supported the hypothesis that the impairment of eNOS function by arsenite is one of the mechanisms leading to vascular changes and diseases.

  12. Prokaryotic Homologs of the Eukaryotic 3-Hydroxyanthranilate 3,4-Dioxygenase and 2-Amino-3-Carboxymuconate-6-Semialdehyde Decarboxylase in the 2-Nitrobenzoate Degradation Pathway of Pseudomonas fluorescens Strain KU-7†

    PubMed Central

    Muraki, Takamichi; Taki, Masami; Hasegawa, Yoshie; Iwaki, Hiroaki; Lau, Peter C. K.

    2003-01-01

    The 2-nitrobenzoic acid degradation pathway of Pseudomonas fluorescens strain KU-7 proceeds via a novel 3-hydroxyanthranilate intermediate. In this study, we cloned and sequenced a 19-kb DNA locus of strain KU-7 that encompasses the 3-hydroxyanthranilate meta-cleavage pathway genes. The gene cluster, designated nbaEXHJIGFCDR, is organized tightly and in the same direction. The nbaC and nbaD gene products were found to be novel homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase, respectively. The NbaC enzyme carries out the oxidation of 3-hydroxyanthranilate to 2-amino-3-carboxymuconate-6-semialdehyde, while the NbaD enzyme catalyzes the decarboxylation of the latter compound to 2-aminomuconate-6-semialdehyde. The NbaC and NbaD proteins were overexpressed in Escherichia coli and characterized. The substrate specificity of the 23.8-kDa NbaC protein was found to be restricted to 3-hydroxyanthranilate. In E. coli, this enzyme oxidizes 3-hydroxyanthranilate with a specific activity of 8 U/mg of protein. Site-directed mutagenesis experiments revealed the essential role of two conserved histidine residues (His52 and His96) in the NbaC sequence. The NbaC activity is also dependent on the presence of Fe2+ but is inhibited by other metal ions, such as Zn2+, Cu2+, and Cd2+. The NbaD protein was overproduced as a 38.7-kDa protein, and its specific activity towards 2-amino-3-carboxymuconate-6-semialdehyde was 195 U/mg of protein. Further processing of 2-aminomuconate-6-semialdehyde to pyruvic acid and acetyl coenzyme A was predicted to proceed via the activities of NbaE, NbaF, NbaG, NbaH, NbaI, and NbaJ. The predicted amino acid sequences of these proteins are highly homologous to those of the corresponding proteins involved in the metabolism of 2-aminophenol (e.g., AmnCDEFGH in Pseudomonas sp. strain AP-3). The NbaR-encoding gene is predicted to have a regulatory function of the LysR family type. The function of the product of the small open reading frame, NbaX, like the homologous sequences in the nitrobenzene or 2-aminophenol metabolic pathway, remains elusive. PMID:12620844

  13. Use of liquid chromatography/electrospray ionization tandem mass spectrometry to study the degradation pathways of terbuthylazine (TER) by Typha latifolia in constructed wetlands: identification of a new TER metabolite.

    PubMed

    Gikas, Evagelos; Papadopoulos, Nikolaos G; Bazoti, Fotini N; Zalidis, Georgios; Tsarbopoulos, Anthony

    2012-01-30

    S-Triazines are used worldwide as herbicides for agricultural and non-agricultural purposes. Although terbuthylazine (TER) is the second most frequently used S-triazine, there is limited information on its metabolism. For this reason, an analytical method based on liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI MS/MS) has been developed aiming at the identification of TER and its five major metabolites (desisopropyl-hydroxy-atrazine, desethyl-hydroxy-terbuthylazine, desisopropyl-atrazine, hydroxy-terbuthylazine and desethyl-terbuthylazine) in constructed wetland water samples. The separation of TER and its major metabolites was performed by reversed-phase high-performance liquid chromatography (HPLC) on a C(8) column using a gradient elution of aqueous acetic acid 1% (solvent A) and acetonitrile (solvent B), followed by MS/MS analysis on a triple quadrupole mass spectrometer. The data-depended analysis (DDA) scan approach has been employed and the main degradation pathways of both hydroxyl and chloro (dealkylated and alkylated) metabolites are elucidated through the tandem mass spectral (MS/MS) interpretation of triazine fragments under CID conditions. In addition, another major metabolite of TER, namely N2-tert-butyl-N4-ethyl-6-methoxy-1,3,5-triazine-2,4-diamine, has been identified. This methodology can be further employed in biodegradation studies of TER, thus assisting the assessment of its environmental impact. PMID:22173806

  14. Proteasome inhibitors and knockdown of SMG1 cause accumulation of Upf1 and Upf2 in human cells.

    PubMed

    Zhao, Xia; Nogawa, Atsushi; Matsunaga, Tsukasa; Takegami, Tsutomu; Nakagawa, Hideaki; Ishigaki, Yasuhito

    2014-01-01

    The ubiquitin-proteasome system (UPS) is one of the most promising anticancer drug targets of the century. However, the involved molecular mechanisms are still unclear. The nonsense-mediated mRNA decay (NMD) pathway is a highly conserved pathway which degrades nonsense mutation?containing mRNA selectively and efficiently. In this pathway, the SMG1-Upf1-eRF (SURF) complex binds to Upf2 on the exon junction complex and finally causes degradation of nonsense-containing mRNA. To reveal the relationship between the UPS and NMD pathways, we analyzed the effects of proteasome inhibitors on Upf1 and Upf2. The data showed that treatment with proteasome inhibitors caused the accumulation of the Upf1 and Upf2 proteins in A549 cells. In addition, we found that knockdown of SMG1 also caused the upregulation of Upf1 and Upf2 proteins, which was confirmed by different target sequences of siRNA. SMG1 and UPS appear to participate in different pathways of the degradation of Upf1 and Upf2, since simultaneous treatment with both of them caused additive effects. This study demonstrated the quantitative regulation of Upf1 and Upf2 proteins by UPS and SMG1. PMID:24173962

  15. SOIL DEGRADATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soil degradation can be defined as loss in the quality or productivity of soil, and is often the result of human activities, such as agriculture, deforestation, mining, waste disposal, or chemical spills. Degradation is attributed to changes in soil nutrient status, biota, loss of organic matter, d...

  16. Proteolytic pathways involved in modulation of CD20 levels.

    PubMed

    Winiarska, Magdalena; Bil, Jacek; Nowis, Dominika; Golab, Jakub

    2010-08-01

    Recent observations indicate that rituximab-resistant lymphoma cells exhibit upregulation of components of the ubiquitin-proteasome system (UPS). Therefore, proteasome inhibitors including the clinically approved bortezomib might influence the levels of CD20, a rituximab target antigen. We observed that incubation of tumor cells with rituximab leads to increased levels of ubiquitinated CD20. However, inhibition of the UPS is not associated with upregulation, but rather with a counterintuitive downregulation of surface CD20 levels that increases resistance of tumor cells to rituximab-mediated cytotoxicity. Although preliminary observations indicate that CD20 might be a substrate for two proteolytic systems, the mechanisms as well as significance of these findings require further studies. PMID:20574159

  17. Sulforaphane enhances proteasomal and autophagic activities in mice and is a potential therapeutic reagent for Huntington’s disease

    PubMed Central

    Liu, Yanying; Hettinger, Casey L.; Zhang, Dong; Rezvani, Khosrow; Wang, Xuejun; Wang, Hongmin

    2014-01-01

    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 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 SFN promotes mutant huntingtin (mHtt) degradation, we treated HD 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 since 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 HD and other intractable disorders. PMID:24383989

  18. Proteasomal degradation in plant–pathogen interactions

    Microsoft Academic Search

    Vitaly Citovsky; Adi Zaltsman; Stanislav V. Kozlovsky; Yedidya Gafni; Alexander Krichevsky

    2009-01-01

    The ubiquitin\\/26S proteasome pathway is a basic biological mechanism involved in the regulation of a multitude of cellular processes. Increasing evidence indicates that plants utilize the ubiquitin\\/26S proteasome pathway in their immune response to pathogen invasion, emphasizing the role of this pathway during plant–pathogen interactions. The specific functions of proteasomal degradation in plant–pathogen interactions are diverse, and do not always

  19. Protein Quality Control in the Nucleus

    PubMed Central

    Nielsen, Sofie V.; Poulsen, Esben G.; Rebula, Caio A.; Hartmann-Petersen, Rasmus

    2014-01-01

    In their natural environment, cells are regularly exposed to various stress conditions that may lead to protein misfolding, but also in the absence of stress, misfolded proteins occur as the result of mutations or failures during protein synthesis. Since such partially denatured proteins are prone to aggregate, cells have evolved several elaborate quality control systems to deal with these potentially toxic proteins. First, various molecular chaperones will seize the misfolded protein and either attempt to refold the protein or target it for degradation via the ubiquitin-proteasome system. The degradation of misfolded proteins is clearly compartmentalized, so unique degradation pathways exist for misfolded proteins depending on whether their subcellular localization is ER/secretory, mitochondrial, cytosolic or nuclear. Recent studies, mainly in yeast, have shown that the nucleus appears to be particularly active in protein quality control. Thus, specific ubiquitin-protein ligases located in the nucleus, target not only misfolded nuclear proteins, but also various misfolded cytosolic proteins which are transported to the nucleus prior to their degradation. In comparison, much less is known about these mechanisms in mammalian cells. Here we highlight recent advances in our understanding of nuclear protein quality control, in particular regarding substrate recognition and proteasomal degradation. PMID:25010148

  20. Cullin E3 Ligases and Their Rewiring by Viral Factors

    PubMed Central

    Mahon, Cathal; Krogan, Nevan J.; Craik, Charles S.; Pick, Elah

    2014-01-01

    The ability of viruses to subvert host pathways is central in disease pathogenesis. Over the past decade, a critical role for the Ubiquitin Proteasome System (UPS) in counteracting host immune factors during viral infection has emerged. This counteraction is commonly achieved by the expression of viral proteins capable of sequestering host ubiquitin E3 ligases and their regulators. In particular, many viruses hijack members of the Cullin-RING E3 Ligase (CRL) family. Viruses interact in many ways with CRLs in order to impact their ligase activity; one key recurring interaction involves re-directing CRL complexes to degrade host targets that are otherwise not degraded within host cells. Removal of host immune factors by this mechanism creates a more amenable cellular environment for viral propagation. To date, a small number of target host factors have been identified, many of which are degraded via a CRL-proteasome pathway. Substantial effort within the field is ongoing to uncover the identities of further host proteins targeted in this fashion and the underlying mechanisms driving their turnover by the UPS. Elucidation of these targets and mechanisms will provide appealing anti-viral therapeutic opportunities. This review is focused on the many methods used by viruses to perturb host CRLs, focusing on substrate sequestration and viral regulation of E3 activity. PMID:25314029

  1. The Hippo Signaling Pathway Interactome

    PubMed Central

    Kwon, Young; Vinayagam, Arunachalam; Sun, Xiaoyun; Dephoure, Noah; Gygi, Steven P.; Hong, Pengyu; Perrimon, Norbert

    2014-01-01

    The Hippo pathway controls metazoan organ growth by regulating cell proliferation and apoptosis. Many components have been identified, but our knowledge of the composition and structure of this pathway is still incomplete. Using existing pathway components as baits, we generated by mass spectrometry a high-confidence Drosophila Hippo protein-protein interaction network (Hippo-PPIN) consisting of 153 proteins and 204 interactions. Depletion of 67% of the proteins by RNA interference regulated the transcriptional coactivator Yorkie (Yki) either positively or negatively. We selected for further characterization a new member of the alpha-arrestin family, Leash, and show that it promotes degradation of Yki through the lysosomal pathway. Given the importance of the Hippo pathway in tumor development, the Hippo-PPIN will contribute to our understanding of this network in both normal growth and cancer. PMID:24114784

  2. O-GlcNAc Modification Is an Endogenous Inhibitor of the Proteasome

    Microsoft Academic Search

    Fengxue Zhang; Kaihong Su; Xiaoyong Yang; Damon B. Bowe; Andrew J. Paterson; Jeffrey E. Kudlow

    2003-01-01

    The ubiquitin proteasome system classically selects its substrates for degradation by tagging them with ubiquitin. Here, we describe another means of controlling proteasome function in a global manner. The 26S proteasome can be inhibited by modification with the enzyme, O-GlcNAc transferase (OGT). This reversible modification of the proteasome inhibits the proteolysis of the transcription factor Sp1 and a hydrophobic peptide

  3. WWP2: a multifunctional ubiquitin ligase gene.

    PubMed

    Chen, Wei; Jiang, Xiaofei; Luo, Zhuang

    2014-10-01

    The ubiquitin-proteasome system plays an important role in various celluar processes. WWP2, a recently identified ubiquitin E3 ligase, has been proved a multifunctional gene by degradation a series of targets via ubiquitin-dependent proteasome system, including PETN, Smads, Oct4, EGR2, TIRF and so. Hereafter, we reviewed the recent research process about the function of WWP2. PMID:25216927

  4. Mechanisms, biology and inhibitors of deubiquitinating enzymes

    Microsoft Academic Search

    Kerry Routenberg Love; André Catic; Christian Schlieker; Hidde L Ploegh

    2007-01-01

    The addition of ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers to proteins serves to modulate function and is a key step in protein degradation, epigenetic modification and intracellular localization. Deubiquitinating enzymes and Ubl-specific proteases, the proteins responsible for the removal of Ub and Ubls, act as an additional level of control over the ubiquitin-proteasome system. Their conservation and widespread occurrence in

  5. New Roles of Glycosaminoglycans in ?-Synuclein Aggregation in a Cellular Model of Parkinson Disease

    PubMed Central

    Lehri-Boufala, Sonia; Ouidja, Mohand-Ouidir; Barbier-Chassefière, Véronique; Hénault, Emilie; Raisman-Vozari, Rita; Garrigue-Antar, Laure; Papy-Garcia, Dulce; Morin, Christophe

    2015-01-01

    The causes of Parkinson disease (PD) remain mysterious, although some evidence supports mitochondrial dysfunctions and ?-synuclein accumulation in Lewy bodies as major events. The abnormal accumulation of ?-synuclein has been associated with a deficiency in the ubiquitin-proteasome system and the autophagy-lysosomal pathway. Cathepsin D (cathD), the major lysosomal protease responsible of ?-synuclein degradation was described to be up-regulated in PD model. As glycosaminoglycans (GAGs) regulate cathD activity, and have been recently suggested to participate in PD physiopathology, we investigated their role in ?-synuclein accumulation by their intracellular regulation of cathD activity. In a classical neuroblastoma cell model of PD induced by MPP+, the genetic expression of GAGs-biosynthetic enzymes was modified, leading to an increase of GAGs amounts whereas intracellular level of ?-synuclein increased. The absence of sulfated GAGs increased intracellular cathD activity and limited ?-synuclein accumulation. GAGs effects on cathD further suggested that specific sequences or sulfation patterns could be responsible for this regulation. The present study identifies, for the first time, GAGs as new regulators of the lysosome degradation pathway, regulating cathD activity and affecting two main biological processes, ?-synuclein aggregation and apoptosis. Finally, this opens new insights into intracellular GAGs functions and new fields of investigation for glycobiological approaches in PD and neurobiology. PMID:25617759

  6. Structural Insights into Proteasome Activation by the 19S Regulatory Particle

    PubMed Central

    Ehlinger, Aaron; Walters, Kylie J.

    2013-01-01

    Since its discovery in the late 1970s, the ubiquitin-proteasome system (UPS) has become recognized as the major pathway for regulated cellular proteolysis. Processes ranging from cell cycle control, pathogen resistance, and protein quality control rely on selective protein degradation at the proteasome for homeostatic function. Perhaps as a consequence of the importance of this pathway, and the genesis of severe diseases upon its dysregulation, protein degradation by the UPS is highly controlled from the level of substrate recognition to proteolysis. Technological advances over the last decade have created an explosion of structural and mechanistic information that has underscored the complexity of the proteasome and its upstream regulatory factors. Significant insights have come from study of the 19S proteasome regulatory particle (RP) responsible for recognition and processing of ubiquitinated substrates destined for proteolysis. Established as a highly dynamic proteasome activator, a large number of both permanent and transient RP components with specialized functional roles are critical for proteasome function. In this review, we highlight recent mechanistic developments in the study of proteasome activation by the RP and how they provide context to our current understanding of the UPS. PMID:23672618

  7. The Interplay between Alpha-Synuclein Clearance and Spreading

    PubMed Central

    Lopes da Fonseca, Tomás; Villar-Piqué, Anna; Outeiro, Tiago Fleming

    2015-01-01

    Parkinson’s Disease (PD) is a complex neurodegenerative disorder classically characterized by movement impairment. Pathologically, the most striking features of PD are the loss of dopaminergic neurons and the presence of intraneuronal protein inclusions primarily composed of alpha-synuclein (?-syn) that are known as Lewy bodies and Lewy neurites in surviving neurons. Though the mechanisms underlying the progression of PD pathology are unclear, accumulating evidence suggests a prion-like spreading of ?-syn pathology. The intracellular homeostasis of ?-syn requires the proper degradation of the protein by three mechanisms: chaperone-mediated autophagy, macroautophagy and ubiquitin-proteasome. Impairment of these pathways might drive the system towards an alternative clearance mechanism that could involve its release from the cell. This increased release to the extracellular space could be the basis for ?-syn propagation to different brain areas and, ultimately, for the spreading of pathology and disease progression. Here, we review the interplay between ?-syn degradation pathways and its intercellular spreading. The understanding of this interplay is indispensable for obtaining a better knowledge of the molecular basis of PD and, consequently, for the design of novel avenues for therapeutic intervention. PMID:25874605

  8. A Fragment-Based Ligand Screen Against Part of a Large Protein Machine: The ND1 Domains of the AAA+ ATPase p97/VCP.

    PubMed

    Chimenti, Michael S; Bulfer, Stacie L; Neitz, R Jeffrey; Renslo, Adam R; Jacobson, Matthew P; James, Thomas L; Arkin, Michelle R; Kelly, Mark J S

    2015-07-01

    The ubiquitous AAA+ ATPase p97 functions as a dynamic molecular machine driving several cellular processes. It is essential in regulating protein homeostasis, and it represents a potential drug target for cancer, particularly when there is a greater reliance on the endoplasmic reticulum-associated protein degradation pathway and ubiquitin-proteasome pathway to degrade an overabundance of secreted proteins. Here, we report a case study for using fragment-based ligand design approaches against this large and dynamic hexamer, which has multiple potential binding sites for small molecules. A screen of a fragment library was conducted by surface plasmon resonance (SPR) and followed up by nuclear magnetic resonance (NMR), two complementary biophysical techniques. Virtual screening was also carried out to examine possible binding sites for the experimental hits and evaluate the potential utility of fragment docking for this target. Out of this effort, 13 fragments were discovered that showed reversible binding with affinities between 140 µM and 1 mM, binding stoichiometries of 1:1 or 2:1, and good ligand efficiencies. Structural data for fragment-protein interactions were obtained with residue-specific [U-(2)H] (13)CH3-methyl-labeling NMR strategies, and these data were compared to poses from docking. The combination of virtual screening, SPR, and NMR enabled us to find and validate a number of interesting fragment hits and allowed us to gain an understanding of the structural nature of fragment binding. PMID:25690569

  9. Ubiquibodies, Synthetic E3 Ubiquitin Ligases Endowed with Unnatural Substrate Specificity for Targeted Protein Silencing*

    PubMed Central

    Portnoff, Alyse D.; Stephens, Erin A.; Varner, Jeffrey D.; DeLisa, Matthew P.

    2014-01-01

    The ubiquitin-proteasome pathway (UPP) is the main route of protein degradation in eukaryotic cells and is a common mechanism through which numerous cellular pathways are regulated. To date, several reverse genetics techniques have been reported that harness the power of the UPP for selectively reducing the levels of otherwise stable proteins. However, each of these approaches has been narrowly developed for a single substrate and cannot be easily extended to other protein substrates of interest. To address this shortcoming, we created a generalizable protein knock-out method by engineering protein chimeras called “ubiquibodies” that combine the activity of E3 ubiquitin ligases with designer binding proteins to steer virtually any protein to the UPP for degradation. Specifically, we reprogrammed the substrate specificity of a modular human E3 ubiquitin ligase called CHIP (carboxyl terminus of Hsc70-interacting protein) by replacing its natural substrate-binding domain with a single-chain Fv (scFv) intrabody or a fibronectin type III domain monobody that target their respective antigens with high specificity and affinity. Engineered ubiquibodies reliably transferred ubiquitin to surface exposed lysines on target proteins and even catalyzed the formation of biologically relevant polyubiquitin chains. Following ectopic expression of ubiquibodies in mammalian cells, specific and systematic depletion of desired target proteins was achieved, whereas the levels of a natural substrate of CHIP were unaffected. Taken together, engineered ubiquibodies offer a simple, reproducible, and customizable means for directly removing specific cellular proteins through accelerated proteolysis. PMID:24474696

  10. Proteomics Propels Protein Degradation Studies in San Diego*

    PubMed Central

    Bennett, Eric J.; Mayor, Thibault

    2012-01-01

    Exquisite in vitro biochemical examinations of protein ubiquitylation and degradation have historically been the dominant methods for unraveling the mechanisms of protein destruction. The study of protein abundance alterations and protein modifications, a cornerstone of protein degradation pathways, naturally lends itself to global and systematic proteomic methods to decipher the emerging complexity of protein degradation pathways. Advances in proteomic technologies have fueled an explosion of systematic and quantitative studies aimed at understanding how the proteome is shaped and regulated by ubiquitin-dependent processes. These types of studies, as well as targeted analyses of cellular pathways, have revealed that alterations in protein degradation function can have a severe impact on human health and disease. The fusion of these two themes was the focus of the January 2012 conference on proteomics of protein degradation and ubiquitin pathways (PPDUP) held in San Diego. To gain insights into both the current state-of-the-art proteomic methods to investigate protein turnover, and how protein degradation function is altered within a range of human disorders a variety of speakers revealed the many connections between altered protein degradation function and human disease. Many of the sessions were framed by a consistent focus aimed at the discovery and development of novel therapeutics targeting protein degradation pathway components to treat various human maladies ranging from cancer to heart disease. PMID:22798279

  11. Lignin-degrading enzymes.

    PubMed

    Pollegioni, Loredano; Tonin, Fabio; Rosini, Elena

    2015-04-01

    A main goal of green biotechnology is to reduce our dependence on fossil reserves and to increase the use of renewable materials. For this, lignocellulose, which is composed of cellulose, hemicellulose and lignin, represents the most promising feedstock. The latter is a complex aromatic heteropolymer formed by radical polymerization of guaiacyl, syringyl, and p-hydroxyphenyl units linked by ?-aryl ether linkages, biphenyl bonds and heterocyclic linkages. Accordingly, lignin appears to be a potentially valuable renewable aromatic chemical, thus representing a main pillar in future biorefinery. The resistance of lignin to breakdown is the main bottleneck in this process, although a variety of white-rot fungi, as well as bacteria, have been reported to degrade lignin by employing different enzymes and catabolic pathways. Here, recent investigations have expanded the range of natural biocatalysts involved in lignin degradation/modification and significant progress related to enzyme engineering and recombinant expression has been made. The present review is focused primarily on recent trends in ligninolytic green biotechnology to suggest the potential (industrial) application of ligninolytic enzymes. Future perspectives could include synergy between natural enzymes from different sources (as well as those obtained by protein engineering) and other pretreatment methods that may be required for optimal results in enzyme-based, environmentally friendly, technologies. PMID:25649492

  12. A proteomics study of auxin effects in Arabidopsis thaliana.

    PubMed

    Xing, Meiqing; Xue, Hongwei

    2012-09-01

    Many phytohormones regulate plant growth and development through modulating protein degradation. In this study, a proteome study based on multidimensional non-gel shotgun approach was performed to analyze the auxin-induced protein degradation via ubiquitin-proteasome pathway of Arabidopsis thaliana, with the emphasis to study the overall protein changes after auxin treatment (1 nM or 1 µM indole-3-acetic acid for 6, 12, or 24 h). More than a thousand proteins were detected by using label-free shotgun method, and 386 increased proteins and 370 decreased ones were identified after indole-3-acetic acid treatment. By using the auxin receptor-deficient mutant, tir1-1, as control, comparative analysis revealed that 69 and 79 proteins were significantly decreased and increased, respectively. Detailed analysis showed that among the altered proteins, some were previously reported to be associated with auxin regulation and others are potentially involved in mediating the auxin effects on specific cellular and physiological processes by regulating photosynthesis, chloroplast development, cytoskeleton, and intracellular signaling. Our results demonstrated that label-free shotgun proteomics is a powerful tool for large-scale protein identification and the analysis of the proteomic profiling of auxin-regulated biological processes will provide informative clues of underlying mechanisms of auxin effects. These results will help to expand the understanding of how auxin regulates plant growth and development via protein degradation. PMID:22814249

  13. Sirtuins and Proteolytic Systems: Implications for Pathogenesis of Synucleinopathies

    PubMed Central

    Sampaio-Marques, Belém; Ludovico, Paula

    2015-01-01

    Insoluble and fibrillar forms of ?-synuclein are the major components of Lewy bodies, a hallmark of several sporadic and inherited neurodegenerative diseases known as synucleinopathies. ?-Synuclein is a natural unfolded and aggregation-prone protein that can be degraded by the ubiquitin-proteasomal system and the lysosomal degradation pathways. ?-Synuclein is a target of the main cellular proteolytic systems, but it is also able to alter their function further, contributing to the progression of neurodegeneration. Aging, a major risk for synucleinopathies, is associated with a decrease activity of the proteolytic systems, further aggravating this toxic looping cycle. Here, the current literature on the basic aspects of the routes for ?-synuclein clearance, as well as the consequences of the proteolytic systems collapse, will be discussed. Finally, particular focus will be given to the sirtuins’s role on proteostasis regulation, since their modulation emerged as a promising therapeutic strategy to rescue cells from ?-synuclein toxicity. The controversial reports on the potential role of sirtuins in the degradation of ?-synuclein will be discussed. Connection between sirtuins and proteolytic systems is definitely worth of further studies to increase the knowledge that will allow its proper exploration as new avenue to fight synucleinopathies. PMID:25946078

  14. JAZ8 lacks a canonical degron and has an EAR motif that mediates transcriptional repression of jasmonate responses in Arabidopsis.

    PubMed

    Shyu, Christine; Figueroa, Pablo; Depew, Cody L; Cooke, Thomas F; Sheard, Laura B; Moreno, Javier E; Katsir, Leron; Zheng, Ning; Browse, John; Howe, Gregg A

    2012-02-01

    The lipid-derived hormone jasmonoyl-L-Ile (JA-Ile) initiates large-scale changes in gene expression by stabilizing the interaction of JASMONATE ZIM domain (JAZ) repressors with the F-box protein CORONATINE INSENSITIVE1 (COI1), which results in JAZ degradation by the ubiquitin-proteasome pathway. Recent structural studies show that the JAZ1 degradation signal (degron) includes a short conserved LPIAR motif that seals JA-Ile in its binding pocket at the COI1-JAZ interface. Here, we show that Arabidopsis thaliana JAZ8 lacks this motif and thus is unable to associate strongly with COI1 in the presence of JA-Ile. As a consequence, JAZ8 is stabilized against jasmonate (JA)-mediated degradation and, when ectopically expressed in Arabidopsis, represses JA-regulated growth and defense responses. These findings indicate that sequence variation in a hypervariable region of the degron affects JAZ stability and JA-regulated physiological responses. We also show that JAZ8-mediated repression depends on an LxLxL-type EAR (for ERF-associated amphiphilic repression) motif at the JAZ8 N terminus that binds the corepressor TOPLESS and represses transcriptional activation. JAZ8-mediated repression does not require the ZIM domain, which, in other JAZ proteins, recruits TOPLESS through the EAR motif-containing adaptor protein NINJA. These findings show that EAR repression domains in a subgroup of JAZ proteins repress gene expression through direct recruitment of corepressors to cognate transcription factors. PMID:22327740

  15. Sirtuins and proteolytic systems: implications for pathogenesis of synucleinopathies.

    PubMed

    Sampaio-Marques, Belém; Ludovico, Paula

    2015-01-01

    Insoluble and fibrillar forms of ?-synuclein are the major components of Lewy bodies, a hallmark of several sporadic and inherited neurodegenerative diseases known as synucleinopathies. ?-Synuclein is a natural unfolded and aggregation-prone protein that can be degraded by the ubiquitin-proteasomal system and the lysosomal degradation pathways. ?-Synuclein is a target of the main cellular proteolytic systems, but it is also able to alter their function further, contributing to the progression of neurodegeneration. Aging, a major risk for synucleinopathies, is associated with a decrease activity of the proteolytic systems, further aggravating this toxic looping cycle. Here, the current literature on the basic aspects of the routes for ?-synuclein clearance, as well as the consequences of the proteolytic systems collapse, will be discussed. Finally, particular focus will be given to the sirtuins's role on proteostasis regulation, since their modulation emerged as a promising therapeutic strategy to rescue cells from ?-synuclein toxicity. The controversial reports on the potential role of sirtuins in the degradation of ?-synuclein will be discussed. Connection between sirtuins and proteolytic systems is definitely worth of further studies to increase the knowledge that will allow its proper exploration as new avenue to fight synucleinopathies. PMID:25946078

  16. Mobilization of Processed, Membrane-Tethered SPT23 Transcription Factor by CDC48 UFD1\\/NPL4, a Ubiquitin-Selective Chaperone

    Microsoft Academic Search

    Michael Rape; Thorsten Hoppe; Ingo Gorr; Marian Kalocay; Holger Richly; Stefan Jentsch

    2001-01-01

    The OLE pathway of yeast regulates the level of the ER-bound enzyme ?9-fatty acid desaturase OLE1, thereby controlling membrane fluidity. A central component of this regulon is the transcription factor SPT23, a homolog of mammalian NF-?B. SPT23 is synthesized as an inactive, ER membrane-anchored precursor that is activated by regulated ubiquitin\\/proteasome-dependent processing (RUP). We now show that SPT23 dimerizes prior

  17. Insulin Resistance and Muscle Metabolism in Chronic Kidney Disease

    PubMed Central

    Bailey, James L.

    2013-01-01

    Insulin resistance is a common finding in chronic kidney disease (CKD) and is manifested by mild fasting hyperglycemia and abnormal glucose tolerance testing. Circulating levels of glucocorticoids are high. In muscle, changes in the insulin signaling pathway occur. An increase in the regulatory p85 subunit of Class I phosphatidylinositol 3-Kinase enzyme leads to decreased activation of the downstream effector protein kinase B (Akt). Mechanisms promoting muscle proteolysis and atrophy are unleashed. The link of Akt to the ubiquitin proteasome pathway, a major degradation pathway in muscle, is discussed. Another factor associated with insulin resistance in CKD is angiotensin II (Ang II) which appears to induce its intracellular effects through inflammatory cytokines or reactive oxygen species. Skeletal muscle ATP is depleted and the ability of AMP-activated protein kinase (AMPK) to replenish energy stores is blocked. How this can be reversed is discussed. Interleukin-6 (IL-6) levels are elevated in CKD and impair insulin signaling at the level of IRS-1. With exercise, IL-6 levels are reduced; glucose uptake and utilization are increased. For patients with CKD, exercise may improve insulin signaling and build up muscle. Treatment strategies for preventing muscle atrophy are discussed. PMID:23431467

  18. Autophagy in Dementias

    PubMed Central

    Kragh, Christine Lund; Ubhi, Kiren; Wyss-Corey, Tony; Masliah, Eliezer

    2011-01-01

    Dementias are a varied group of disorders typically associated with memory loss, impaired judgment and/or language and by symptoms affecting other cognitive and social abilities to a degree that interferes with daily functioning. Alzheimer’s disease (AD) is the most common cause of a progressive dementia, followed by dementia with Lewy bodies (DLB), frontotemporal dementia (FTD), vascular dementia (VaD) and HIV associated neurocognitive disorders (HAND). The pathogenesis of this group of disorders has been linked to the abnormal accumulation of proteins in the brains of affected individuals, which in turn has been related to deficits in protein clearance. Autophagy is a key cellular protein clearance pathway with proteolytic cleavage and degradation via the ubiquitin-proteasome pathway representing another important clearance mechanism. Alterations in the levels of autophagy and the proteins associated with the autophagocytic pathway have been reported in various types of dementias. This review will examine recent literature across these disorders and highlight a common theme of altered autophagy across the spectrum of the dementias. PMID:22150925

  19. Global Analysis of DELLA Direct Targets in Early Gibberellin Signaling in Arabidopsis[W

    PubMed Central

    Zentella, Rodolfo; Zhang, Zhong-Lin; Park, Mehea; Thomas, Stephen G.; Endo, Akira; Murase, Kohji; Fleet, Christine M.; Jikumaru, Yusuke; Nambara, Eiji; Kamiya, Yuji; Sun, Tai-ping

    2007-01-01

    Bioactive gibberellins (GAs) are phytohormones that regulate growth and development throughout the life cycle of plants. DELLA proteins are conserved growth repressors that modulate all aspects of GA responses. These GA-signaling repressors are nuclear localized and likely function as transcriptional regulators. Recent studies demonstrated that GA, upon binding to its receptor, derepresses its signaling pathway by binding directly to DELLA proteins and targeting them for rapid degradation via the ubiquitin-proteasome pathway. Therefore, elucidating the signaling events immediately downstream of DELLA is key to our understanding of how GA controls plant development. Two sets of microarray studies followed by quantitative RT-PCR analysis allowed us to identify 14 early GA-responsive genes that are also early DELLA-responsive in Arabidopsis thaliana seedlings. Chromatin immunoprecipitation provided evidence for in vivo association of DELLA with promoters of eight of these putative DELLA target genes. Expression of all 14 genes was downregulated by GA and upregulated by DELLA. Our study reveals that DELLA proteins play two important roles in GA signaling: (1) they help establish GA homeostasis by direct feedback regulation on the expression of GA biosynthetic and GA receptor genes, and (2) they promote the expression of downstream negative components that are putative transcription factors/regulators or ubiquitin E2/E3 enzymes. In addition, one of the putative DELLA targets, XERICO, promotes accumulation of abscisic acid (ABA) that antagonizes GA effects. Therefore, DELLA may restrict GA-promoted processes by modulating both GA and ABA pathways. PMID:17933900

  20. ROLE OF FUNGAL LIGNINOLYTIC ENZYMES IN POLLUTANT DEGRADATION

    EPA Science Inventory

    Lignin-degrading fungi have potential applications in programs for organopollutant biotreatment. he metabolic pathways that they employ for ligninolysis appear to have unusual xenobiotic capabilities, and there is some preliminary evidence that their extracellular lignin peroxida...