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Sample records for promoting proteasomal degradation

  1. Parkin promotes proteasomal degradation of misregulated BAX.

    PubMed

    Cakir, Zeynep; Funk, Kathrin; Lauterwasser, Joachim; Todt, Franziska; Zerbes, Ralf M; Oelgeklaus, Aline; Tanaka, Atsushi; van der Laan, Martin; Edlich, Frank

    2017-09-01

    The pro-apoptotic BCL-2 protein BAX commits human cells to apoptosis by permeabilizing the outer mitochondrial membrane. BAX activation has been suggested to require the separation of helix α5 from α6 - the 'latch' from the 'core' domain - among other conformational changes. Here, we show that conformational changes in this region impair BAX translocation to the mitochondria and retrotranslocation back into the cytosol, and therefore BAX inhibition, but not activation. Redirecting misregulated BAX to the mitochondria revealed an alternative mechanism of BAX inhibition. The E3 ligase parkin, which is known to trigger mitochondria-specific autophagy, ubiquitylates BAX K128 and targets the pro-apoptotic BCL-2 protein for proteasomal degradation. Retrotranslocation-deficient BAX is completely degraded in a parkin-dependent manner. Although only a minor pool of endogenous BAX escapes retrotranslocation into the cytosol, parkin-dependent targeting of misregulated BAX on the mitochondria provides substantial protection against BAX apoptotic activity. © 2017. Published by The Company of Biologists Ltd.

  2. Parkin promotes proteasomal degradation of synaptotagmin IV by accelerating polyubiquitination.

    PubMed

    Kabayama, Hiroyuki; Tokushige, Naoko; Takeuchi, Makoto; Kabayama, Miyuki; Fukuda, Mitsunori; Mikoshiba, Katsuhiko

    2017-04-01

    Parkin is an E3 ubiquitin ligase whose mutations cause autosomal recessive juvenile Parkinson's disease (PD). Unlike the human phenotype, parkin knockout (KO) mice show no apparent dopamine neuron degeneration, although they demonstrate reduced expression and activity of striatal mitochondrial proteins believed to be necessary for neuronal survival. Instead, parkin-KO mice show reduced striatal evoked dopamine release, abnormal synaptic plasticity, and non-motor symptoms, all of which appear to mimic the preclinical features of Parkinson's disease. Extensive studies have screened candidate synaptic proteins responsible for reduced evoked dopamine release, and synaptotagmin XI (Syt XI), an isoform of Syt family regulating membrane trafficking, has been identified as a substrate of parkin in humans. However, its expression level is unaltered in the striatum of parkin-KO mice. Thus, the target(s) of parkin and the molecular mechanisms underlying the impaired dopamine release in parkin-KO mice remain unknown. In this study, we focused on Syt IV because of its highly homology to Syt XI, and because they share an evolutionarily conserved lack of Ca(2+)-binding capacity; thus, Syt IV plays an inhibitory role in Ca(2+)-dependent neurotransmitter release in PC12 cells and neurons in various brain regions. We found that a proteasome inhibitor increased Syt IV protein, but not Syt XI protein, in neuron-like, differentiated PC12 cells, and that parkin interacted with and polyubiquitinated Syt IV, thereby accelerating its protein turnover. Parkin overexpression selectively degraded Syt IV protein, but not Syt I protein (indispensable for Ca(2+)-dependent exocytosis), thus enhancing depolarization-dependent exocytosis. Furthermore, in parkin-KO mice, the level of striatal Syt IV protein was increased. Our data indicate a crucial role for parkin in the proteasomal degradation of Syt IV, and provide a potential mechanism of parkin-regulated, evoked neurotransmitter release

  3. Polyamines directly promote antizyme-mediated degradation of ornithine decarboxylase by the proteasome

    PubMed Central

    Beenukumar, R. R.; Gödderz, Daniela; Palanimurugan, R.; Dohmen, R. J.

    2015-01-01

    Ornithine decarboxylase (ODC), a ubiquitin-independent substrate of the proteasome, is a homodimeric protein with a rate-limiting function in polyamine biosynthesis. Polyamines regulate ODC levels by a feedback mechanism mediated by ODC antizyme (OAZ). Higher cellular polyamine levels trigger the synthesis of OAZ and also inhibit its ubiquitin-dependent proteasomal degradation. OAZ binds ODC monomers and targets them to the proteasome. Here, we report that polyamines, aside from their role in the control of OAZ synthesis and stability, directly enhance OAZ-mediated ODC degradation by the proteasome. Using a stable mutant of OAZ, we show that polyamines promote ODC degradation in Saccharomyces cerevisiae cells even when OAZ levels are not changed. Furthermore, polyamines stimulated the in vitro degradation of ODC by the proteasome in a reconstituted system using purified components. In these assays, spermine shows a greater effect than spermidine. By contrast, polyamines do not have any stimulatory effect on the degradation of ubiquitin-dependent substrates. PMID:28357293

  4. CHIP facilitates ubiquitination of inducible nitric oxide synthase and promotes its proteasomal degradation.

    PubMed

    Chen, Li; Kong, Xiuqin; Fu, Jin; Xu, Yimiao; Fang, Shuping; Hua, Peng; Luo, Lan; Yin, Zhimin

    2009-01-01

    Inducible nitric oxide synthase (iNOS) is responsible for nitric oxide (NO) synthesis from l-arginine in response to inflammatory mediators. It is reported that iNOS is degraded mainly by the ubiquitin-proteasome pathway in RAW264.7 cells and human embryonic kidney (HEK) 293 cells. In this study, we showed that iNOS was ubiquitinated and degraded dependent on CHIP (COOH terminus of heat shock protein 70-interacting protein), a chaperone-dependent ubiquitin ligase. The results from overexpression and RNAi experiments demonstrated that CHIP decreased the protein level of iNOS, shortened the half-life of iNOS and attenuated the production of NO. Furthermore, CHIP promoted ubiquitination and proteasomal degradation of iNOS by associating with iNOS. These results suggest that CHIP plays an important role in regulation iNOS activity.

  5. Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility

    PubMed Central

    Young, Barry P.; Loewen, Christopher J.; Mayor, Thibault

    2016-01-01

    Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations. PMID:27448207

  6. Prefoldin Promotes Proteasomal Degradation of Cytosolic Proteins with Missense Mutations by Maintaining Substrate Solubility.

    PubMed

    Comyn, Sophie A; Young, Barry P; Loewen, Christopher J; Mayor, Thibault

    2016-07-01

    Misfolded proteins challenge the ability of cells to maintain protein homeostasis and can accumulate into toxic protein aggregates. As a consequence, cells have adopted a number of protein quality control pathways to prevent protein aggregation, promote protein folding, and target terminally misfolded proteins for degradation. In this study, we employed a thermosensitive allele of the yeast Guk1 guanylate kinase as a model misfolded protein to investigate degradative protein quality control pathways. We performed a flow cytometry based screen to identify factors that promote proteasomal degradation of proteins misfolded as the result of missense mutations. In addition to the E3 ubiquitin ligase Ubr1, we identified the prefoldin chaperone subunit Gim3 as an important quality control factor. Whereas the absence of GIM3 did not impair proteasomal function or the ubiquitination of the model substrate, it led to the accumulation of the poorly soluble model substrate in cellular inclusions that was accompanied by delayed degradation. We found that Gim3 interacted with the Guk1 mutant allele and propose that prefoldin promotes the degradation of the unstable model substrate by maintaining the solubility of the misfolded protein. We also demonstrated that in addition to the Guk1 mutant, prefoldin can stabilize other misfolded cytosolic proteins containing missense mutations.

  7. RNF20 promotes the polyubiquitination and proteasome-dependent degradation of AP-2α protein.

    PubMed

    Ren, Peng; Sheng, Zhifeng; Wang, Yijun; Yi, Xin; Zhou, Qiuzhi; Zhou, Jianlin; Xiang, Shuanglin; Hu, Xiang; Zhang, Jian

    2014-02-01

    Transcription factor activator protein 2α (AP-2α) is a negative regulator of adipogenesis by repressing the transcription of CCAAT/enhancer binding protein (C/EBPα) gene. During adipogenesis, AP-2α is degraded, leading to transcriptional up-regulation of C/EBPα. However, the mechanism for AP-2α degradation is not clear. Here, using immunoprecipitation assay and mass spectrometry, we identified ring finger protein 20 (RNF20) as an AP-2α-interacting protein in 3T3-L1 preadipocytes. RNF20 has been proved to be an E3 ubiquitin ligase for both histone H2B and tumor suppressor ErbB3-binding protein 1 (Ebp1). In this study, we demonstrated that RNF20 co-localized and interacted with AP-2α, and promoted its polyubiquitination and proteasome-dependent degradation. Over-expression of RNF20 inhibited the activity of AP-2α and rescued the C/EBPα expression which was inhibited by AP-2α. These results suggested that RNF20 may play roles in adipocyte differentiation by stimulating ubiquitin-proteasome-dependent degradation of AP-2α.

  8. SIRT6 delays cellular senescence by promoting p27Kip1 ubiquitin-proteasome degradation

    PubMed Central

    Zhao, Ganye; Wang, Hui; Xu, Chenzhong; Wang, Pan; Chen, Jun; Wang, Pengfeng; Sun, Zhaomeng; Su, Yuanyuan; Wang, Zhao; Han, Limin; Tong, Tanjun

    2016-01-01

    Sirtuin6 (SIRT6) has been implicated as a key factor in aging and aging-related diseases. However, the role of SIRT6 in cellular senescence has not been fully understood. Here, we show that SIRT6 repressed the expression of p27Kip1 (p27) in cellular senescence. The expression of SIRT6 was reduced during cellular senescence, whereas enforced SIRT6 expression promoted cell proliferation and antagonized cellular senescence. In addition, we demonstrated that SIRT6 promoted p27 degradation by proteasome and SIRT6 decreased the acetylation level and protein half-life of p27. p27 acetylation increased its protein stability. Furthermore, SIRT6 directly interacted with p27. Importantly, p27 was strongly acetylated and had a prolonged protein half-life with the reduction of SIRT6 when cells were senescent, compared with those young cells. Finally, SIRT6 markedly rescued senescence induced by p27. Our findings indicate that SIRT6 decreases p27 acetylation, leading to its degradation via ubiquitin-proteasome pathway and then delays cellular senescence. PMID:27794562

  9. Abscisic acid promotes proteasome-mediated degradation of the transcription coactivator NPR1 in Arabidopsis thaliana.

    PubMed

    Ding, Yezhang; Dommel, Matthew; Mou, Zhonglin

    2016-04-01

    Proteasome-mediated turnover of the transcription coactivator NPR1 is pivotal for efficient activation of the broad-spectrum plant immune responses known as localized acquired resistance (LAR) and systemic acquired resistance (SAR) in adjacent and systemic tissues, respectively, and requires the CUL3-based E3 ligase and its adaptor proteins, NPR3 and NPR4, which are receptors for the signaling molecule salicylic acid (SA). It has been shown that SA prevents NPR1 turnover under non-inducing and LAR/SAR-inducing conditions, but how cellular NPR1 homeostasis is maintained remains unclear. Here, we show that the phytohormone abscisic acid (ABA) and SA antagonistically influence cellular NPR1 protein levels. ABA promotes NPR1 degradation via the CUL3(NPR) (3/) (NPR) (4) complex-mediated proteasome pathway, whereas SA may protect NPR1 from ABA-promoted degradation through phosphorylation. Furthermore, we demonstrate that the timing and strength of SA and ABA signaling are critical in modulating NPR1 accumulation and target gene expression. Perturbing ABA or SA signaling in adjacent tissues alters the temporal dynamic pattern of NPR1 accumulation and target gene transcription. Finally, we show that sequential SA and ABA treatment leads to dynamic changes in NPR1 protein levels and target gene expression. Our results revealed a tight correlation between sequential SA and ABA signaling and dynamic changes in NPR1 protein levels and NPR1-dependent transcription in plant immune responses. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  10. RHOBTB3 promotes proteasomal degradation of HIFα through facilitating hydroxylation and suppresses the Warburg effect.

    PubMed

    Zhang, Chen-Song; Liu, Qi; Li, Mengqi; Lin, Shu-Yong; Peng, Yongying; Wu, Di; Li, Terytty Yang; Fu, Qiang; Jia, Weiping; Wang, Xinjun; Ma, Teng; Zong, Yue; Cui, Jiwen; Pu, Chengfei; Lian, Guili; Guo, Huiling; Ye, Zhiyun; Lin, Sheng-Cai

    2015-09-01

    Hypoxia-inducible factors (HIFs) are master regulators of adaptive responses to low oxygen, and their α-subunits are rapidly degraded through the ubiquitination-dependent proteasomal pathway after hydroxylation. Aberrant accumulation or activation of HIFs is closely linked to many types of cancer. However, how hydroxylation of HIFα and its delivery to the ubiquitination machinery are regulated remains unclear. Here we show that Rho-related BTB domain-containing protein 3 (RHOBTB3) directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation. RHOBTB3 also directly interacts with the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin ligase complex, facilitating ubiquitination of HIFα. Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα. Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα. Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth. Our work thus reveals that RHOBTB3 serves as a scaffold to organize a multi-subunit complex that promotes the hydroxylation, ubiquitination and degradation of HIFα.

  11. Tertiary Structural Rearrangements upon Oxidation of Methionine145 in Calmodulin Promotes Targeted Proteasomal Degradation

    SciTech Connect

    Sacksteder, Colette A.; Whittier, Jennifer E.; Xiong, Yijia; Li, Jinhui; Galeva, Nadezhda A.; Jacoby, Michael E.; Purvine, Samuel O.; Williams, Todd D.; Rechsteiner, Martin C.; Bigelow, Diana J.; Squier, Thomas C.

    2006-08-15

    Mass spectrometry was used to identify possible linkages between the oxidation of specific methionines in calmodulin (CaM) and its degradation by the proteasome. Degradation of oxidized CaM (CaMox) occurs in a multistep process, which involves an initial cleavage that releases a large fragment (Q41-D131) and a carboxyl-terminus peptide (G132-K148) enriched in methionine sulfoxide [Met(O)] prior to rebinding and complete digestion. Rates of CaM degradation correlate with the oxidation of Met145, which is preferentially oxidized by hydrogen peroxide prior to the oxidation of Met144. The specificity of CaMox degradation was confirmed using CaM mutants in which the majority of methionines were replaced by leucines using site-directed mutagenesis, permitting the site-specific oxidation of Met144 or Met145. Oxidation of both Met144 and Met145 results in the selective degradation of CaMox by the proteasome with rates comparable to that observed for fully oxidized CaM. Oxidation of Met144 has little effect on proteolytic degradation; rather, oxidation of Met145 targets CaMox for proteasomal degradation. The selective oxidation of either Met144 or Met145 has little effect on the secondary structure of CaM; rather, oxidation-induced tertiary structural changes mediate the recognition and degradation of CaMox by the proteasome. Thus, oxidation of Met145 can function as a sensor that has the potential to regulate cellular metabolism through the targeted modulation of CaM abundance in response to oxidative stress.

  12. Proteasomal and Autophagic Degradation Systems.

    PubMed

    Dikic, Ivan

    2017-06-20

    Autophagy and the ubiquitin-proteasome system are the two major quality control pathways responsible for cellular homeostasis. As such, they provide protection against age-associated changes and a plethora of human diseases. Ubiquitination is utilized as a degradation signal by both systems, albeit in different ways, to mark cargoes for proteasomal and lysosomal degradation. Both systems intersect and communicate at multiple points to coordinate their actions in proteostasis and organelle homeostasis. This review summarizes molecular details of how proteasome and autophagy pathways are functionally interconnected in cells and indicates common principles and nodes of communication that can be therapeutically exploited.

  13. Kaposi's Sarcoma-Associated Herpesvirus RTA Promotes Degradation of the Hey1 Repressor Protein through the Ubiquitin Proteasome Pathway▿

    PubMed Central

    Gould, Faye; Harrison, Sally M.; Hewitt, Eric W.; Whitehouse, Adrian

    2009-01-01

    The Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) protein regulates the latent-lytic switch by transactivating a variety of KSHV lytic and cellular promoters. RTA is a novel E3 ubiquitin ligase that targets a number of transcriptional repressor proteins for degradation by the ubiquitin proteasome pathway. Herein, we show that RTA interacts with the cellular transcriptional repressor protein Hey1. We demonstrate that Hey1 is a target for RTA-mediated ubiquitination and is subsequently degraded by the proteasome. Moreover, a Cys-plus-His-rich region within RTA is important for RTA-mediated degradation of Hey1. We confirm that Hey1 represses the RTA promoter and, furthermore, show that Hey1 binds to the RTA promoter. An interaction was observed between Hey1 and the corepressor mSin3A, and this interaction was abolished in the presence of RTA. Additionally, mSin3A associated with the RTA promoter in nonreactivated, but not reactivated, BCBL1 cells. Small interfering RNA knockdown of Hey1 in HEK 293T cells latently infected with the recombinant virus rKSHV.219 led to increased levels of RTA expression upon reactivation but was insufficient to induce complete lytic reactivation. These results suggest that other additional transcriptional repressors are also important in maintenance of KSHV latency. Taken together, our results suggest that Hey1 has a contributory role in the maintenance of KSHV latency and that disruption of the Hey1 repressosome by RTA-targeted degradation may be one step in the mechanism to regulate lytic reactivation. PMID:19369342

  14. E6AP inhibits G-CSFR turnover and functions by promoting its ubiquitin-dependent proteasome degradation.

    PubMed

    Chhabra, Stuti; Kumar, Yogesh; Thacker, Gatha; Kapoor, Isha; Lochab, Savita; Sanyal, Sabyasachi; Bhatt, Madan L B; Chattopadhyay, Naibedya; Trivedi, Arun Kumar

    2017-10-01

    Granulocyte colony-stimulating factor receptor (G-CSFR) plays a crucial role in regulating myeloid cell survival, proliferation, and neutrophilic granulocyte precursor cells maturation. Previously, we demonstrated that Fbw7α negatively regulates G-CSFR and its downstream signaling through ubiquitin-proteasome mediated degradation. However, whether additional ubiquitin ligases for G-CSFR exist is not known. Identifying multiple E3 ubiquitin ligases for G-CSFR shall improve our understanding of activation and subsequent attenuation of G-CSFR signaling required for differentiation and proliferation. Here, for the first time we demonstrate that E6 associated protein (E6AP), an E3 ubiquitin ligase physically associates with G-CSFR and targets it for ubiquitin-mediated proteasome degradation and thereby attenuates its functions. We further show that E6AP promoted G-CSFR degradation leads to reduced phosphorylation of signal transducer and activator of transcription 3 (STAT3) which is required for G-CSF dependent granulocytic differentiation. More importantly, our finding shows that E6AP also targets mutant form of G-SCFR (G-CSFR-T718), frequently observed in severe congenital neutropenia (SCN) patients that very often culminate to AML, however, at a quite slower rate than wild type G-CSFR. In addition, our data showed that knockdown of E6AP restores G-CSFR and its signaling thereby promoting granulocytic differentiation. Collectively, our data demonstrates that E6AP facilitates ubiquitination and subsequent degradation of G-CSFR leading to attenuation of its downstream signaling and inhibition of granulocytic differentiation. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Oxidative challenge enhances REGγ-proteasome-dependent protein degradation.

    PubMed

    Zhang, Yuanyuan; Liu, Shuang; Zuo, Qiuhong; Wu, Lin; Ji, Lei; Zhai, Wanli; Xiao, Jianru; Chen, Jiwu; Li, Xiaotao

    2015-05-01

    Elimination of oxidized proteins is important to cells as accumulation of damaged proteins causes cellular dysfunction, disease, and aging. Abundant evidence shows that the 20S proteasome is largely responsible for degradation of oxidative proteins in both ubiquitin-dependent and ubiquitin-independent pathways. However, the role of the REGγ-proteasome in degrading oxidative proteins remains unclear. Here, we focus on two of the well-known REGγ-proteasome substrates, p21(Waf1/Cip1) and hepatitis C virus (HCV) core protein, to analyze the impact of oxidative stress on REGγ-proteasome functions. We demonstrate that REGγ-proteasome is essential for oxidative stress-induced rapid degradation of p21 and HCV proteins. Silencing REGγ abrogated this response in multiple cell lines. Furthermore, pretreatment with proteasome inhibitor MG132 completely blunted oxidant-induced p21 degradation, indicating a proteasome-dependent action. Cellular oxidation promoted REGγ-proteasome-dependent trypsin-like activity by enhancing the interaction between REGγ and 20S proteasome. Antioxidant could counteract oxidation-induced protein degradation, indicating that REGγ-proteasome activity may be regulated by redox state. This study provides further insights into the actions of a unique proteasome pathway in response to an oxidative stress environment, implying a novel molecular basis for REGγ-proteasome functions in antioxidation. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. 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; Gu, Jianxin

    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.

  17. Arctigenin promotes degradation of inducible nitric oxide synthase through CHIP-associated proteasome pathway and suppresses its enzyme activity.

    PubMed

    Yao, Xiangyang; Li, Guilan; Lü, Chaotian; Xu, Hui; Yin, Zhimin

    2012-10-01

    Arctigenin, a natural dibenzylbutyrolactone lignan compound, has been reported to possess anti-inflammatory properties. Previous works showed that arctigenin decreased lipopolysaccharide (LPS)-induced iNOS at transcription level. However, whether arctigenin could regulate iNOS at the post-translational level is still unclear. In the present study, we demonstrated that arctigenin promoted the degradation of iNOS which is expressed under LPS stimulation in murine macrophage-like RAW 264.7 cells. Such degradation of iNOS protein is due to CHIP-associated ubiquitination and proteasome-dependency. Furthermore, arctigenin decreased iNOS phosphorylation through inhibiting ERK and Src activation, subsequently suppressed iNOS enzyme activity. In conclusion, our research displays a new finding that arctigenin can promote the ubiqitination and degradation of iNOS after LPS stimulation. iNOS activity regulated by arctigenin is likely to involve a multitude of crosstalking mechanisms. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Steviol reduces MDCK Cyst formation and growth by inhibiting CFTR channel activity and promoting proteasome-mediated CFTR degradation.

    PubMed

    Yuajit, Chaowalit; Homvisasevongsa, Sureeporn; Chatsudthipong, Lisa; Soodvilai, Sunhapas; Muanprasat, Chatchai; Chatsudthipong, Varanuj

    2013-01-01

    Cyst enlargement in polycystic kidney disease (PKD) involves cAMP-activated proliferation of cyst-lining epithelial cells and transepithelial fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. This study aimed to investigate an inhibitory effect and detailed mechanisms of steviol and its derivatives on cyst growth using a cyst model in Madin-Darby canine kidney (MDCK) cells. Among 4 steviol-related compounds tested, steviol was found to be the most potent at inhibiting MDCK cyst growth. Steviol inhibition of cyst growth was dose-dependent; steviol (100 microM) reversibly inhibited cyst formation and cyst growth by 72.53.6% and 38.2±8.5%, respectively. Steviol at doses up to 200 microM had no effect on MDCK cell viability, proliferation and apoptosis. However, steviol acutely inhibited forskolin-stimulated apical chloride current in MDCK epithelia, measured with the Ussing chamber technique, in a dose-dependent manner. Prolonged treatment (24 h) with steviol (100 microM) also strongly inhibited forskolin-stimulated apical chloride current, in part by reducing CFTR protein expression in MDCK cells. Interestingly, proteasome inhibitor, MG-132, abolished the effect of steviol on CFTR protein expression. Immunofluorescence studies demonstrated that prolonged treatment (24 h) with steviol (100 microM) markedly reduced CFTR expression at the plasma membrane. Taken together, the data suggest that steviol retards MDCK cyst progression in two ways: first by directly inhibiting CFTR chloride channel activity and second by reducing CFTR expression, in part, by promoting proteasomal degradation of CFTR. Steviol and related compounds therefore represent drug candidates for treatment of polycystic kidney disease.

  19. Steviol Reduces MDCK Cyst Formation and Growth by Inhibiting CFTR Channel Activity and Promoting Proteasome-Mediated CFTR Degradation

    PubMed Central

    Yuajit, Chaowalit; Homvisasevongsa, Sureeporn; Chatsudthipong, Lisa; Soodvilai, Sunhapas; Muanprasat, Chatchai; Chatsudthipong, Varanuj

    2013-01-01

    Cyst enlargement in polycystic kidney disease (PKD) involves cAMP-activated proliferation of cyst-lining epithelial cells and transepithelial fluid secretion into the cyst lumen via cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. This study aimed to investigate an inhibitory effect and detailed mechanisms of steviol and its derivatives on cyst growth using a cyst model in Madin-Darby canine kidney (MDCK) cells. Among 4 steviol-related compounds tested, steviol was found to be the most potent at inhibiting MDCK cyst growth. Steviol inhibition of cyst growth was dose-dependent; steviol (100 microM) reversibly inhibited cyst formation and cyst growth by 72.53.6% and 38.2±8.5%, respectively. Steviol at doses up to 200 microM had no effect on MDCK cell viability, proliferation and apoptosis. However, steviol acutely inhibited forskolin-stimulated apical chloride current in MDCK epithelia, measured with the Ussing chamber technique, in a dose-dependent manner. Prolonged treatment (24 h) with steviol (100 microM) also strongly inhibited forskolin-stimulated apical chloride current, in part by reducing CFTR protein expression in MDCK cells. Interestingly, proteasome inhibitor, MG-132, abolished the effect of steviol on CFTR protein expression. Immunofluorescence studies demonstrated that prolonged treatment (24 h) with steviol (100 microM) markedly reduced CFTR expression at the plasma membrane. Taken together, the data suggest that steviol retards MDCK cyst progression in two ways: first by directly inhibiting CFTR chloride channel activity and second by reducing CFTR expression, in part, by promoting proteasomal degradation of CFTR. Steviol and related compounds therefore represent drug candidates for treatment of polycystic kidney disease. PMID:23536832

  20. The ER retention protein RER1 promotes alpha-synuclein degradation via the proteasome.

    PubMed

    Park, Hyo-Jin; Ryu, Daniel; Parmar, Mayur; Giasson, Benoit I; McFarland, Nikolaus R

    2017-01-01

    Abnormal accumulation of α-synuclein (αSyn) has been linked to endoplasmic-reticulum (ER) stress, defective intracellular protein/vesicle trafficking, and cytotoxicity. Targeting factors involved in ER-related protein processing and trafficking may, therefore, be a key to modulating αSyn levels and associated toxicity. Recently retention in endoplasmic reticulum 1 (RER1) has been identified as an important ER retrieval/retention factor for Alzheimer's disease proteins and negatively regulates amyloid-β peptide levels. Here, we hypothesized that RER1 might also play an important role in retention/retrieval of αSyn and mediate levels. We expressed RER1 and a C-terminal mutant RER1Δ25, which lacks the ER retention/retrieval function, in HEK293 and H4 neuroglioma cells. RER1 overexpression significantly decreased levels of both wild type and A30P, A53T, and E46K disease causal mutants of αSyn, whereas the RER1Δ25 mutant had a significantly attenuated effect on αSyn. RER1 effects were specific to αSyn and had little to no effect on either βSyn or the Δ71-82 αSyn mutant, which both lack the NAC domain sequence critical for synuclein fibrillization. Tests with proteasomal and macroautophagy inhibitors further demonstrate that RER1 effects on αSyn are primarily mediated through the ubiquitin-proteasome system. RER1 also appears to interact with the ubiquitin ligase NEDD4. RER1 in human diseased brain tissues co-localizes with αSyn-positive Lewy bodies. Together, these findings provide evidence that RER1 is a novel and potential important mediator of elevated αSyn levels. Further investigation of the mechanism of RER1 and downstream effectors on αSyn may yield novel therapeutic targets for modulation in Parkinson disease and related synucleinopathies.

  1. The E3 ubiquitin ligase TRIM31 attenuates NLRP3 inflammasome activation by promoting proteasomal degradation of NLRP3

    PubMed Central

    Song, Hui; Liu, Bingyu; Huai, Wanwan; Yu, Zhongxia; Wang, Wenwen; Zhao, Jing; Han, Lihui; Jiang, Guosheng; Zhang, Lining; Gao, Chengjiang; Zhao, Wei

    2016-01-01

    The NLRP3 inflammasome has a fundamental role in host defence against microbial pathogens and its deregulation may cause diverse inflammatory diseases. NLRP3 protein expression is a rate-limiting step for inflammasome activation, thus its expression must be tightly controlled to maintain immune homeostasis and avoid detrimental effects. However, how NLRP3 expression is regulated remains largely unknown. In this study, we identify E3 ubiquitin ligase TRIM31 as a feedback suppressor of NLRP3 inflammasome. TRIM31 directly binds to NLRP3, promotes K48-linked polyubiquitination and proteasomal degradation of NLRP3. Consequently, TRIM31 deficiency enhances NLRP3 inflammasome activation and aggravates alum-induced peritonitis in vivo. Furthermore, TRIM31 deficiency attenuates the severity of dextran sodium sulfate (DSS)-induced colitis, an inflammatory bowel diseases model in which NLRP3 possesses protective roles. Thus, our research describes a mechanism by which TRIM31 limits NLRP3 inflammasome activity under physiological conditions and suggests TRIM31 as a potential therapeutic target for the intervention of NLRP3 inflammasome related diseases. PMID:27929086

  2. The F-box Protein FBXO25 Promotes the Proteasome-dependent Degradation of ELK-1 Protein*

    PubMed Central

    Teixeira, Felipe R.; Manfiolli, Adriana O.; Soares, Cláudia S.; Baqui, Munira M. A.; Koide, Tie; Gomes, Marcelo D.

    2013-01-01

    FBXO25 is one of the 69 known human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of SKP1, Rbx1, Cullin1, and F-box protein (SCF1) that are involved in targeting proteins for degradation across the ubiquitin proteasome system. However, the substrates of most SCF E3 ligases remain unknown. Here, we applied an in chip ubiquitination screen using a human protein microarray to uncover putative substrates for the FBXO25 protein. Among several novel putative targets identified, the c-fos protooncogene regulator ELK-1 was characterized as the first endogenous substrate for SCF1(FBXO25) E3 ligase. FBXO25 interacted with and mediated the ubiquitination and proteasomal degradation of ELK-1 in HEK293T cells. In addition, FBXO25 overexpression suppressed induction of two ELK-1 target genes, c-fos and egr-1, in response to phorbol 12-myristate 13-acetate. Together, our findings show that FBXO25 mediates ELK-1 degradation through the ubiquitin proteasome system and thereby plays a role in regulating the activation of ELK-1 pathway in response to mitogens. PMID:23940030

  3. The F-box protein FBXO25 promotes the proteasome-dependent degradation of ELK-1 protein.

    PubMed

    Teixeira, Felipe R; Manfiolli, Adriana O; Soares, Cláudia S; Baqui, Munira M A; Koide, Tie; Gomes, Marcelo D

    2013-09-27

    FBXO25 is one of the 69 known human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of SKP1, Rbx1, Cullin1, and F-box protein (SCF1) that are involved in targeting proteins for degradation across the ubiquitin proteasome system. However, the substrates of most SCF E3 ligases remain unknown. Here, we applied an in chip ubiquitination screen using a human protein microarray to uncover putative substrates for the FBXO25 protein. Among several novel putative targets identified, the c-fos protooncogene regulator ELK-1 was characterized as the first endogenous substrate for SCF1(FBXO25) E3 ligase. FBXO25 interacted with and mediated the ubiquitination and proteasomal degradation of ELK-1 in HEK293T cells. In addition, FBXO25 overexpression suppressed induction of two ELK-1 target genes, c-fos and egr-1, in response to phorbol 12-myristate 13-acetate. Together, our findings show that FBXO25 mediates ELK-1 degradation through the ubiquitin proteasome system and thereby plays a role in regulating the activation of ELK-1 pathway in response to mitogens.

  4. NEDD8 ultimate buster-1 long (NUB1L) protein promotes transfer of NEDD8 to proteasome for degradation through the P97UFD1/NPL4 complex.

    PubMed

    Liu, Shuai; Yang, Hui; Zhao, Jian; Zhang, Yu-Hang; Song, Ai-Xin; Hu, Hong-Yu

    2013-10-25

    The NEDD8 protein and neddylation levels in cells are modulated by NUB1L or NUB1 through proteasomal degradation, but the underlying molecular mechanism is not well understood. Here, we report that NUB1L down-regulated the protein levels of NEDD8 and neddylation through specifically recognizing NEDD8 and P97/VCP. NUB1L directly interacted with NEDD8, but not with ubiquitin, on the key residue Asn-51 of NEDD8 and with P97/VCP on its positively charged VCP binding motif. In coordination with the P97-UFD1-NPL4 complex (P97(UFD1/NPL4)), NUB1L promotes transfer of NEDD8 to proteasome for degradation. This mechanism is also exemplified by the canonical neddylation of cullin 1 for SCF (SKP1-cullin1-F-box) ubiquitin E3 ligases that is exquisitely regulated by the turnover of NEDD8.

  5. HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway (Biochemical and Biophysical Research Communications, v. 444, isse 4)

    SciTech Connect

    Wang, Xiaozhen; Lu, Guang; Li, Li; Yi, Juan; Yan, Kaowen; Wang, Yaqing; Zhu, Baili; Kuang, Jingyu; Lin, Ming; Zhang, Sha; Shao, Genze

    2014-02-21

    Highlights: • The 2000–2634aa region of HUWE1 mediates the interaction with BRCA1 degron. • HUWE1 promotes the degradation of BRCA1 through the ubiquitin–proteasome pathway. • HUWE1 expression is inversely correlated with BRCA1 in breast cancer cells. • RNAi inhibition of HUWE1 confers increased resistance of MCF-10F cells to IR and MMC. - Abstract: The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control of BRCA1 protein level. HUWE1 binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.

  6. HUWE1 interacts with BRCA1 and promotes its degradation in the ubiquitin–proteasome pathway (Biochemical and Biophysical Research Communications, v. 444 issue 3)

    SciTech Connect

    Wang, Xiaozhen; Lu, Guang; Li, Li; Yi, Juan; Yan, Kaowen; Wang, Yaqing; Zhu, Baili; Kuang, Jingyu; Lin, Ming; Zhang, Sha; Shao, Genze

    2014-02-14

    Highlights: • The 2000–2634 aa region of HUWE1 mediates the interaction with BRCA1 degron. • HUWE1 promotes the degradation of BRCA1 through the ubiquitin–proteasome pathway. • HUWE1 expression is inversely correlated with BRCA1 in breast cancer cells. • RNAi inhibition of HUWE1 confers increased resistance of MCF-10F cells to IR and MMC. - Abstract: The cellular BRCA1 protein level is essential for its tumor suppression activity and is tightly regulated through multiple mechanisms including ubiquitn–proteasome system. E3 ligases are involved to promote BRCA1 for ubiquitination and degradation. Here, we identified HUWE1/Mule/ARF-BP1 as a novel BRCA1-interacting protein involved in the control of BRCA1 protein level. HUWE1binds BRCA1 through its N-terminus degron domain. Depletion of HUWE1 by siRNA-mediated interference significantly increases BRCA1 protein levels and prolongs the half-life of BRCA1. Moreover, exogenous expression of HUWE1 promotes BRCA1 degradation through the ubiquitin–proteasome pathway, which could explain an inverse correlation between HUWE1 and BRCA1 levels in MCF10F, MCF7 and MDA-MB-231 breast cancer cells. Consistent with a functional role for HUWE1 in regulating BRCA1-mediated cellular response to DNA damage, depletion of HUWE1 by siRNA confers increased resistance to ionizing radiation and mitomycin. These data indicate that HUWE1 is a critical negative regulator of BRCA1 and suggest a new molecular mechanism for breast cancer pathogenesis.

  7. p53 mutations promote proteasomal activity.

    PubMed

    Oren, Moshe; Kotler, Eran

    2016-07-27

    p53 mutations occur very frequently in human cancer. Besides abrogating the tumour suppressive functions of wild-type p53, many of those mutations also acquire oncogenic gain-of-function activities. Augmentation of proteasome activity is now reported as a common gain-of-function mechanism shared by different p53 mutants, which promotes cancer resistance to proteasome inhibitors.

  8. Proteasome involvement in a complex cascade mediating SigT degradation during differentiation of Streptomyces coelicolor.

    PubMed

    Mao, Xu-Ming; Ren, Ni-Ni; Sun, Ning; Wang, Feng; Zhou, Ri-Cheng; Tang, Yi; Li, Yong-Quan

    2014-02-14

    In Streptomyces coelicolor, the ECF sigma factor SigT negatively regulates cell differentiation, and is degraded by ClpP protease in a dual positive feedback manner. Here we further report that the proteasome is required for degradation of SigT, but not for degradation of its anti-sigma factor RstA, and RstA can protect SigT from degradation independent of the proteasome. Meanwhile, deletion of the proteasome showed reduced production of secondary metabolites, and the fermentation medium from wild type could promote SigT degradation. Furthermore, overexpression of redD or actII-orf4 in the proteasome-deficiency mutant resulted in SigT degradation and over-production of both undecylprodigiosin and actinorhodin. Therefore the proteasome is required for SigT degradation by affecting the production of secondary metabolites during cell differentiation.

  9. 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

  10. Prefoldins Negatively Regulate Cold Acclimation in Arabidopsis thaliana by Promoting Nuclear Proteasome-Mediated HY5 Degradation.

    PubMed

    Perea-Resa, Carlos; Rodríguez-Milla, Miguel A; Iniesto, Elisa; Rubio, Vicente; Salinas, Julio

    2017-06-05

    The process of cold acclimation is an important adaptive response whereby many plants from temperate regions increase their freezing tolerance after being exposed to low non-freezing temperatures. The correct development of this response relies on proper accumulation of a number of transcription factors that regulate expression patterns of cold-responsive genes. Multiple studies have revealed a variety of molecular mechanisms involved in promoting the accumulation of these transcription factors. Interestingly, however, the mechanisms implicated in controlling such accumulation to ensure their adequate levels remain largely unknown. In this work, we demonstrate that prefoldins (PFDs) control the levels of HY5, an Arabidopsis transcription factor with a key role in cold acclimation by activating anthocyanin biosynthesis, in response to low temperature. Our results show that, under cold conditions, PFDs accumulate into the nucleus through a DELLA-dependent mechanism, where they interact with HY5, triggering its ubiquitination and subsequent degradation. The degradation of HY5 would result, in turn, in anthocyanin biosynthesis attenuation, ensuring the accurate development of cold acclimation. These findings uncover an unanticipated nuclear function for PFDs in plant responses to abiotic stresses. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

  11. Parkin mediates proteasome-dependent protein degradation and rupture of the outer mitochondrial membrane.

    PubMed

    Yoshii, Saori R; Kishi, Chieko; Ishihara, Naotada; Mizushima, Noboru

    2011-06-03

    Upon mitochondrial depolarization, Parkin, a Parkinson disease-related E3 ubiquitin ligase, translocates from the cytosol to mitochondria and promotes their degradation by mitophagy, a selective type of autophagy. Here, we report that in addition to mitophagy, Parkin mediates proteasome-dependent degradation of outer membrane proteins such as Tom20, Tom40, Tom70, and Omp25 of depolarized mitochondria. By contrast, degradation of the inner membrane and matrix proteins largely depends on mitophagy. Furthermore, Parkin induces rupture of the outer membrane of depolarized mitochondria, which also depends on proteasomal activity. Upon induction of mitochondrial depolarization, proteasomes are recruited to mitochondria in the perinuclear region. Neither proteasome-dependent degradation of outer membrane proteins nor outer membrane rupture is required for mitophagy. These results suggest that Parkin regulates degradation of outer and inner mitochondrial membrane proteins differently through proteasome- and mitophagy-dependent pathways.

  12. SIAH-1 interacts with the Kaposi's sarcoma-associated herpesvirus-encoded ORF45 protein and promotes its ubiquitylation and proteasomal degradation.

    PubMed

    Abada, Rinat; Dreyfuss-Grossman, Tsofia; Herman-Bachinsky, Yifat; Geva, Haim; Masa, Shiri-Rivka; Sarid, Ronit

    2008-03-01

    Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8, is a potentially tumorigenic virus implicated in the etiology of Kaposi's sarcoma, primary effusion lymphoma, and some forms of multicentric Castleman's disease. The open reading frame 45 (ORF45) protein, encoded by the KSHV genome, is capable of inhibiting virus-dependent interferon induction and appears to be essential for both early and late stages of infection. In the present study, we show, both in yeast two-hybrid assays and in mammalian cells, that the ORF45 protein interacts with the cellular ubiquitin E3 ligase family designated seven in absentia homologue (SIAH). We provide evidence that SIAH-1 promotes the degradation of KSHV ORF45 through a RING domain-dependent mechanism and via the ubiquitin-proteasome system. Furthermore, our data indicate the involvement of SIAH-1 in the regulation of the expression of ORF45 in KSHV-infected cells. Since the availability of KSHV ORF45 is expected to influence the course of KSHV infection, our findings identify a novel biological role for SIAH proteins as modulators of virus infection.

  13. Cysteine String Protein Promotes Proteasomal Degradation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by Increasing Its Interaction with the C Terminus of Hsp70-interacting Protein and Promoting CFTR Ubiquitylation*S⃞

    PubMed Central

    Schmidt, Béla Z.; Watts, Rebecca J.; Aridor, Meir; Frizzell, Raymond A.

    2009-01-01

    Cysteine string protein (Csp) is a J-domain-containing protein whose overexpression blocks the exit of cystic fibrosis transmembrane conductance regulator (CFTR) from the endoplasmic reticulum (ER). Another method of blocking ER exit, the overexpression of Sar1-GTP, however, yielded twice as much immature CFTR compared with Csp overexpression. This finding suggested that Csp not only inhibits CFTR ER exit but also facilitates the degradation of immature CFTR. This was confirmed by treatment with a proteasome inhibitor, which returned the level of immature CFTR to that found in cells expressing Sar1-GTP only. CspH43Q, which does not interact with Hsc70/Hsp70 efficiently, did not promote CFTR degradation, suggesting that the pro-degradative effect of Csp requires Hsc70/Hsp70 binding/activation. In agreement with this, Csp overexpression increased the amount of Hsc70/Hsp70 co-immunoprecipitated with CFTR, whereas overexpression of CspH43Q did not. The Hsc70/Hsp70 binding partner C terminus of Hsp70-interacting protein (CHIP) can target CFTR for proteasome-mediated degradation. Csp overexpression also increased the amount of CHIP co-immunoprecipitated with CFTR. In addition, CHIP interacted directly with Csp, which was confirmed by in vitro binding experiments. Csp overexpression also increased CFTR ubiquitylation and reduced the half-life of immature CFTR. These findings indicate that Csp not only regulates the exit of CFTR from the ER, but that this action is accompanied by Hsc70/Hsp70 and CHIP-mediated CFTR degradation. PMID:19098309

  14. Fos family protein degradation by the proteasome.

    PubMed

    Gomard, Tiphanie; Jariel-Encontre, Isabelle; Basbous, Jihane; Bossis, Guillaume; Moquet-Torcy, Gabriel; Mocquet-Torcy, Gabriel; Piechaczyk, Marc

    2008-10-01

    c-Fos proto-oncoprotein defines a family of closely related transcription factors (Fos proteins) also comprising Fra-1, Fra-2, FosB and DeltaFosB, the latter two proteins being generated by alternative splicing. Through the regulation of many genes, most of them still unidentified, they regulate major functions from the cell level up to the whole organism. Thus they are involved in the control of proliferation, differentiation and apoptosis, as well as in the control of responses to stresses, and they play important roles in organogenesis, immune responses and control of cognitive functions, among others. Fos proteins are intrinsically unstable. We have studied how two of them, c-Fos and Fra-1, are degraded. Departing from the classical scenario where unstable key cell regulators are hydrolysed by the proteasome after polyubiquitination, we showed that the bulk of c-Fos and Fra-1 can be broken down independently of any prior ubiquitination. Certain conserved structural domains suggest that similar mechanisms may also apply to Fra-2 and FosB. Computer search indicates that certain motifs shared by the Fos proteins and putatively responsible for instability are found in no other protein, suggesting the existence of degradation mechanisms specific for this protein family. Under particular signalling conditions, others have shown that a part of cytoplasmic c-Fos requires ubiquitination for fast turnover. This poses the question of the multiplicity of degradation pathways that apply to proteins depending on their intracellular localization.

  15. Hsp90 Enhances Degradation of Oxidized Calmodulin by the 20S Proteasome

    SciTech Connect

    Whittier, Jennifer E.; Xiong, Yijia; Rechsteiner, Martin C.; Squier, Thomas C.

    2004-10-29

    The 20S proteasome has been suggested to play a critical role in mediating the degradation of abnormal proteins under conditions of oxidative stress, and has been found in tight association with the molecular chaperone Hsp90. To elucidate the role of Hsp90 in promoting the degradation of oxidized calmodulin (CaMox), which accumulates in senescent brain during normal biological aging, we have purified the 20S proteasome free of Hsp90 from red blood cells and assessed its ability to recognize and degrade CaMox in the absence and presence of added Hsp90. The purified 20S proteasome does not degrade CaMox to any appreciable extent. However, following association with Hsp90, the 20S proteasome selectively degrades CaMox. This degradation is sensitive to both proteasome and Hsp90-specific inhibitors, and is further enhanced in the presence of 2 mM ATP. Irrespective of the presence of Hsp90 we find that unoxidized CaM is not significantly degraded. Furthermore, the ability of the proteasome to degrade commonly used fluorogenic peptides is not affected by Hsp90, indicating that there is no change in the accessibility of the catalytic core. Direct binding measurements demonstrate that Hsp90 selectively associates with CaMox; essentially no binding is observed between Hsp90 and unoxidized CaM. Since oxidation has previously been shown to induce both global conformational changes and a reduction in helical content of CaM, these results suggest that Hsp90 in association with the 20S proteasome selectively associates with partially unfolded proteins to promote their degradation by the proteasome.

  16. Calreticulin and Arginylated Calreticulin Have Different Susceptibilities to Proteasomal Degradation*

    PubMed Central

    Goitea, Victor E.; Hallak, Marta E.

    2015-01-01

    Post-translational arginylation has been suggested to target proteins for proteasomal degradation. The degradation mechanism for arginylated calreticulin (R-CRT) localized in the cytoplasm is unknown. To evaluate the effect of arginylation on CRT stability, we examined the metabolic fates and degradation mechanisms of cytoplasmic CRT and R-CRT in NIH 3T3 and CHO cells. Both CRT isoforms were found to be proteasomal substrates, but the half-life of R-CRT (2 h) was longer than that of cytoplasmic CRT (0.7 h). Arginylation was not required for proteasomal degradation of CRT, although R-CRT displays ubiquitin modification. A CRT mutant incapable of dimerization showed reduced metabolic stability of R-CRT, indicating that R-CRT dimerization may protect it from proteasomal degradation. Our findings, taken together, demonstrate a novel function of arginylation: increasing the half-life of CRT in cytoplasm. PMID:25969538

  17. Involvement of proteasomal subunits zeta and iota in RNA degradation.

    PubMed Central

    Petit, F; Jarrousse, A S; Dahlmann, B; Sobek, A; Hendil, K B; Buri, J; Briand, Y; Schmid, H P

    1997-01-01

    We have identified two distinct subunits of 20 S proteasomes that are associated with RNase activity. Proteasome subunits zeta and iota, eluted from two-dimensional Western blots, hydrolysed tobacco mosaic virus RNA, whereas none of the other subunits degraded this substrate under the same conditions. Additionally, proteasomes were dissociated by 6 M urea, and subunit zeta, containing the highest RNase activity, was isolated by anion-exchange chromatography and gel filtration. Purified subunit zeta migrated as a single spot on two-dimensional PAGE with a molecular mass of approx. 28 kDa. Addition of anti-(subunit zeta) antibodies led to the co-precipitation of this proteasome subunit and nuclease activity. This is the first evidence that proteasomal alpha-type subunits are associated with an enzymic activity, and our results provide further evidence that proteasomes may be involved in cellular RNA metabolism. PMID:9337855

  18. Sulforaphane Induced Apoptosis via Promotion of Mitochondrial Fusion and ERK1/2-Mediated 26S Proteasome Degradation of Novel Pro-survival Bim and Upregulation of Bax in Human Non-Small Cell Lung Cancer Cells.

    PubMed

    Geng, Yang; Zhou, Yan; Wu, Sai; Hu, Yabin; Lin, Kai; Wang, Yalin; Zheng, Zhongnan; Wu, Wei

    2017-01-01

    Previous studies in our laboratory showed that sulforaphane (SFN) induced apoptosis by sustained activation of extracellular regulated protein kinases 1/2 (ERK1/2). However, the underlying mechanisms associated with SFN-induced apoptosis and downstream cascades which are modulated by ERK1/2 were not elucidated. Herein we demonstrated for the first time that alteration of mitochondrial dynamics contributed to SFN-induced apoptosis in human non-small cell lung cancer (NSCLC) cells. Reports showed that protein Bim not only induced apoptosis but also promoted proliferation under certain circumstances. We found that Bim was related to cell growth in NSCLC cells. Pro-survival Bim downregulation was shown to induce apoptosis in response to SFN. Further, Using the ERK1/2 inhibitor, PD98059, we found that SFN upregulated Bax and downregulated Bim through the ERK1/2-dependent signaling pathway. Furthermore, SFN activated ERK1/2 to increase 26S proteasome activity to degrade Bim, while the proteasome inhibitor MG132 reversed this effect. Therefore, SFN phosphorylated ERK1/2 and activated the proteasome system leading to the degradation of Bim, which contributed to apoptosis in NSCLC cells. These findings provided a novel insight into SFN-related therapeutics in cancer treatment.

  19. Proteasomal and lysosomal protein degradation and heart disease.

    PubMed

    Wang, Xuejun; Robbins, Jeffrey

    2014-06-01

    In the cell, the proteasome and lysosomes represent the most important proteolytic machineries, responsible for the protein degradation in the ubiquitin-proteasome system (UPS) and autophagy, respectively. Both the UPS and autophagy are essential to protein quality and quantity control. Alterations in cardiac proteasomal and lysosomal degradation are remarkably associated with most heart disease in humans and are implicated in the pathogenesis of congestive heart failure. Studies carried out in animal models and in cell culture have begun to establish both sufficiency and, in some cases, the necessity of proteasomal functional insufficiency or lysosomal insufficiency as a major pathogenic factor in the heart. This review article highlights some recent advances in the research into proteasome and lysosome protein degradation in relation to cardiac pathology and examines the emerging evidence for enhancing degradative capacities of the proteasome and/or lysosome as a new therapeutic strategy for heart disease. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy". Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. IRE1 directs proteasomal and lysosomal degradation of misfolded rhodopsin

    PubMed Central

    Chiang, Wei-Chieh; Messah, Carissa; Lin, Jonathan H.

    2012-01-01

    Endoplasmic reticulum (ER) is responsible for folding of secreted and membrane proteins in eukaryotic cells. Disruption of ER protein folding leads to ER stress. Chronic ER stress can cause cell death and is proposed to underlie the pathogenesis of many human diseases. Inositol-requiring enzyme 1 (IRE1) directs a key unfolded protein response signaling pathway that controls the fidelity of ER protein folding. IRE1 signaling may be particularly helpful in preventing chronic ER stress and cell injury by alleviating protein misfolding in the ER. To examine this, we used a chemical-genetic approach to selectively activate IRE1 in mammalian cells and tested how artificial IRE1 signaling affected the fate of misfolded P23H rhodopsin linked to photoreceptor cell death. We found that IRE1 signaling robustly promoted the degradation of misfolded P23H rhodopsin without affecting its wild-type counterpart. We also found that IRE1 used both proteasomal and lysosomal degradation pathways to remove P23H rhodopsin. Surprisingly, when one degradation pathway was compromised, IRE1 signaling could still promote misfolded rhodopsin degradation using the remaining pathway. Last, we showed that IRE1 signaling also reduced levels of several other misfolded rhodopsins with lesser effects on misfolded cystic fibrosis transmembrane conductance regulator. Our findings reveal the diversity of proteolytic mechanisms used by IRE1 to eliminate misfolded rhodopsin. PMID:22219383

  1. Degradation of pro-insulin-receptor proteins by proteasomes.

    PubMed

    Cruz, Miguel; Velasco, Eduardo; Kumate, Jesús

    2004-01-01

    Type-2 diabetes is characterized by hyperinsulinemia, peripheral insulin resistance, and diminished tyrosine phosphorylation activity. It has been recently shown that proteasomes are implicated in the degradation of the insulin receptor substrate-1 (IRS-1) but not in that of the insulin receptor (IR). However, it is unknown whether proteasomes are involved in pro-IR degradation. We used CHO-IR and the 3T3-L1 cells treated with insulin at different concentrations and compared the proteasome activity of IRS-1, IR, and pro-IR degradation either in presence or in absence of lactacystin. A total of 100 nM of insulin allowed degradation of IRS-1 after 6 h of incubation. At 1,000 nM of insulin, pro-IR degradation began at 1 h of incubation, similar to IRS-1 degradation. Surprisingly, at a higher concentration (10 microM) of insulin, a drastic decrease of proteins was observed from the first minute of incubation. This activity was blocked by lactacystin, a specific proteasome inhibitor. According to these results, we propose that pro-IR is degraded by proteasomes.

  2. Emerging Mechanistic Insights into AAA Complexes Regulating Proteasomal Degradation

    PubMed Central

    Förster, Friedrich; Schuller, Jan M.; Unverdorben, Pia; Aufderheide, Antje

    2014-01-01

    The 26S proteasome is an integral element of the ubiquitin-proteasome system (UPS) and, as such, responsible for regulated degradation of proteins in eukaryotic cells. It consists of the core particle, which catalyzes the proteolysis of substrates into small peptides, and the regulatory particle, which ensures specificity for a broad range of substrates. The heart of the regulatory particle is an AAA-ATPase unfoldase, which is surrounded by non-ATPase subunits enabling substrate recognition and processing. Cryo-EM-based studies revealed the molecular architecture of the 26S proteasome and its conformational rearrangements, providing insights into substrate recognition, commitment, deubiquitylation and unfolding. The cytosol proteasomal degradation of polyubiquitylated substrates is tuned by various associating cofactors, including deubiquitylating enzymes, ubiquitin ligases, shuttling ubiquitin receptors and the AAA-ATPase Cdc48/p97. Cdc48/p97 and its cofactors function upstream of the 26S proteasome, and their modular organization exhibits some striking analogies to the regulatory particle. In archaea PAN, the closest regulatory particle homolog and Cdc48 even have overlapping functions, underscoring their intricate relationship. Here, we review recent insights into the structure and dynamics of the 26S proteasome and its associated machinery, as well as our current structural knowledge on the Cdc48/p97 and its cofactors that function in the ubiquitin-proteasome system (UPS). PMID:25102382

  3. Emerging mechanistic insights into AAA complexes regulating proteasomal degradation.

    PubMed

    Förster, Friedrich; Schuller, Jan M; Unverdorben, Pia; Aufderheide, Antje

    2014-08-06

    The 26S proteasome is an integral element of the ubiquitin-proteasome system(UPS) and, as such, responsible for regulated degradation of proteins in eukaryotic cells.It consists of the core particle, which catalyzes the proteolysis of substrates into small peptides, and the regulatory particle, which ensures specificity for a broad range of substrates.The heart of the regulatory particle is an AAA-ATPase unfoldase, which is surrounded by non-ATPase subunits enabling substrate recognition and processing. Cryo-EM-based studies revealed the molecular architecture of the 26S proteasome and its conformational rearrangements, providing insights into substrate recognition, commitment, deubiquitylation and unfolding. The cytosol proteasomal degradation of polyubiquitylated substrates is tuned by various associating cofactors, including deubiquitylating enzymes, ubiquitin ligases,shuttling ubiquitin receptors and the AAA-ATPase Cdc48/p97. Cdc48/p97 and its cofactors function upstream of the 26S proteasome, and their modular organization exhibits some striking analogies to the regulatory particle. In archaea PAN, the closest regulatory particle homolog and Cdc48 even have overlapping functions, underscoring their intricate relationship.Here, we review recent insights into the structure and dynamics of the 26S proteasome and its associated machinery, as well as our current structural knowledge on the Cdc48/p97 and its cofactors that function in the ubiquitin-proteasome system (UPS).

  4. Proteasome activator PA28γ stimulates degradation of GSK3-phosphorylated insulin transcription activator MAFA.

    PubMed

    Kanai, Kenichi; Aramata, Shinsaku; Katakami, Sayo; Yasuda, Kunio; Kataoka, Kohsuke

    2011-08-01

    MAFA is a member of the MAF family of basic leucine zipper transcription factors and is a critical regulator of insulin gene expression and islet β-cell function. To be degraded by the proteasome, MAFA must be phosphorylated by GSK3 and MAP kinases at multiple serine and threonine residues (Ser49, Thr53, Thr57, Ser61, and Ser65) within its amino-terminal domain. In this study, we report that MAFA degradation is stimulated by PA28γ (REGγ and PSME3), a member of a family of proteasome activators that bind and activate the 20S proteasome. To date, only a few PA28γ-proteasome pathway substrates have been identified, including steroid receptor coactivator 3 (SRC3) and the cell cycle inhibitor p21(CIP1). PA28γ binds to MAFA, induces its proteasomal degradation, and thereby attenuates MAFA-driven transcriptional activation of the insulin promoter. Co-expression of GSK3 enhanced the PA28γ-mediated degradation of MAFA, but mutants that contained alanine substitutions at the MAFA phosphorylation sites did not bind PA28γ and were resistant to degradation. We also found that a PA28γ mutant (N151Y) that did not stimulate p21 degradation enhanced MAFA degradation, and another mutant (K188D) that promoted greater p21 degradation did not enhance MAFA degradation.These results suggest that PA28γ stimulates MAFA degradation through a novel molecular mechanism that is distinct from that for the degradation of p21.

  5. Proteasome activator PA28{gamma} stimulates degradation of GSK3-phosphorylated insulin transcription activator MAFA.

    PubMed

    Kanai, Kenichi; Aramata, Shinsaku; Katakami, Sayo; Yasuda, Kunio; Kataoka, Kohsuke

    2011-01-01

    MAFA is a member of the MAF family of basic leucine zipper transcription factors and is a critical regulator of insulin gene expression and islet β-cell function. To be degraded by the proteasome, MAFA must be phosphorylated by GSK3 and MAP kinases at multiple serine and threonine residues (Ser49, Thr53, Thr57, Ser61, and Ser65) within its amino-terminal domain. In this study, we report that MAFA degradation is stimulated by PA28γ (REGγ and PSME3), a member of a family of proteasome activators that bind and activate the 20S proteasome. To date, only a few PA28γ-proteasome pathway substrates have been identified, including steroid receptor coactivator 3 (SRC3) and the cell cycle inhibitor p21 (CIP1). PA28γ binds to MAFA, induces its proteasomal degradation, and thereby attenuates MAFA-driven transcriptional activation of the insulin promoter. Co-expression of GSK3 enhanced the PA28γ-mediated degradation of MAFA, but mutants that contained alanine substitutions at the MAFA phosphorylation sites did not bind PA28γ and were resistant to degradation. We also found that a PA28γ mutant (N151Y) that did not stimulate p21 degradation enhanced MAFA degradation, and another mutant (K188D) that promoted greater p21 degradation did not enhance MAFA degradation. These results suggest that PA28γ stimulates MAFA degradation through a novel molecular mechanism that is distinct from that for the degradation of p21.

  6. 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; Juhnn, Yong-Sung

    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

  7. Acetylation-mediated proteasomal degradation of core histones during DNA repair and spermatogenesis.

    PubMed

    Qian, Min-Xian; Pang, Ye; Liu, Cui Hua; Haratake, Kousuke; Du, Bo-Yu; Ji, Dan-Yang; Wang, Guang-Fei; Zhu, Qian-Qian; Song, Wei; Yu, Yadong; Zhang, Xiao-Xu; Huang, Hai-Tao; Miao, Shiying; Chen, Lian-Bin; Zhang, Zi-Hui; Liang, Ya-Nan; Liu, Shan; Cha, Hwangho; Yang, Dong; Zhai, Yonggong; Komatsu, Takuo; Tsuruta, Fuminori; Li, Haitao; Cao, Cheng; Li, Wei; Li, Guo-Hong; Cheng, Yifan; Chiba, Tomoki; Wang, Linfang; Goldberg, Alfred L; Shen, Yan; Qiu, Xiao-Bo

    2013-05-23

    Histone acetylation plays critical roles in chromatin remodeling, DNA repair, and epigenetic regulation of gene expression, but the underlying mechanisms are unclear. Proteasomes usually catalyze ATP- and polyubiquitin-dependent proteolysis. Here, we show that the proteasomes containing the activator PA200 catalyze the polyubiquitin-independent degradation of histones. Most proteasomes in mammalian testes ("spermatoproteasomes") contain a spermatid/sperm-specific α subunit α4 s/PSMA8 and/or the catalytic β subunits of immunoproteasomes in addition to PA200. Deletion of PA200 in mice abolishes acetylation-dependent degradation of somatic core histones during DNA double-strand breaks and delays core histone disappearance in elongated spermatids. Purified PA200 greatly promotes ATP-independent proteasomal degradation of the acetylated core histones, but not polyubiquitinated proteins. Furthermore, acetylation on histones is required for their binding to the bromodomain-like regions in PA200 and its yeast ortholog, Blm10. Thus, PA200/Blm10 specifically targets the core histones for acetylation-mediated degradation by proteasomes, providing mechanisms by which acetylation regulates histone degradation, DNA repair, and spermatogenesis.

  8. Acetylation-Mediated Proteasomal Degradation of Core Histones during DNA Repair and Spermatogenesis

    PubMed Central

    Qian, Min-Xian; Pang, Ye; Liu, Cui Hua; Haratake, Kousuke; Du, Bo-Yu; Ji, Dan-Yang; Wang, Guang-Fei; Zhu, Qian-Qian; Song, Wei; Yu, Yadong; Zhang, Xiao-Xu; Huang, Hai-Tao; Miao, Shiying; Chen, Lian-Bin; Zhang, Zi-Hui; Liang, Ya-Nan; Liu, Shan; Cha, Hwangho; Yang, Dong; Zhai, Yonggong; Komatsu, Takuo; Tsuruta, Fuminori; Li, Haitao; Cao, Cheng; Li, Wei; Li, Guo-Hong; Cheng, Yifan; Chiba, Tomoki; Wang, Linfang; Goldberg, Alfred L.; Shen, Yan; Qiu, Xiao-Bo

    2013-01-01

    SUMMARY Histone acetylation plays critical roles in chromatin remodeling, DNA repair, and epigenetic regulation of gene expression, but the underlying mechanisms are unclear. Proteasomes usually catalyze ATP- and polyubiquitin-dependent proteolysis. Here we show that the proteasomes containing the activator PA200 catalyze the polyubiquitin-independent degradation of histones. Most proteasomes in mammalian testes (“spermatoproteasomes”) contain a spermatid/sperm-specific α-subunit α4s/PSMA8 and/or the catalytic β-subunits of immunoproteasomes in addition to PA200. Deletion of PA200 in mice abolishes acetylation-dependent degradation of somatic core histones during DNA double-strand breaks, and delays core histone disappearance in elongated spermatids. Purified PA200 greatly promotes ATP-independent proteasomal degradation of the acetylated core histones, but not polyubiquitinated proteins. Furthermore, acetylation on histones is required for their binding to the bromodomain-like regions in PA200 and its yeast ortholog, Blm10. Thus, PA200/Blm10 specifically targets the core histones for acetylation-mediated degradation by proteasomes, providing mechanisms by which acetylation regulates histone degradation, DNA repair, and spermatogenesis. PMID:23706739

  9. E3 ubiquitin ligase CHIP interacts with C-type lectin-like receptor CLEC-2 and promotes its ubiquitin-proteasome degradation.

    PubMed

    Shao, Miaomiao; Li, Lili; Song, Shushu; Wu, Weicheng; Peng, Peike; Yang, Caiting; Zhang, Mingming; Duan, Fangfang; Jia, Dongwei; Zhang, Jie; Wu, Hao; Zhao, Ran; Wang, Lan; Ruan, Yuanyuan; Gu, Jianxin

    2016-10-01

    C-type lectin-like receptor 2 (CLEC-2) was originally identified as a member of non-classical C-type lectin-like receptors in platelets and immune cells. Activation of CLEC-2 is involved in thrombus formation, lymphatic/blood vessel separation, platelet-mediated tumor metastasis and immune response. Nevertheless, the regulation of CLEC-2 expression is little understood. In this study, we identified that the C terminus of Hsc70-interacting protein (CHIP) interacted with CLEC-2 by mass spectrometry analysis, and CHIP decreased the protein expression of CLEC-2 through lysine-48-linked ubiquitination and proteasomal degradation. Deleted and point mutation also revealed that CHIP controlled CLEC-2 protein expression via both tetratricopeptide repeats (TPR) domain and Ubox domain in a HSP70/90-independent manner. Moreover, reduced CHIP expression was associated with decreased CLEC-2 polyubiquitination and increased CLEC-2 protein levels in PMA-induced differentiation of THP-1 monocytes into macrophages. These results indicate that CLEC-2 is the target substrate of E3 ubiquitin ligase CHIP, and suggest that the CHIP/CLEC-2 axis may play an important role in the modulation of immune response. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2016-01-01

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

  11. Proteasomal degradation of beta-carotene metabolite--modified proteins.

    PubMed

    Sommerburg, Olaf; Karius, Nicole; Siems, Werner; Langhans, Claus-Dieter; Leichsenring, Michael; Breusing, Nicolle; Grune, Tilman

    2009-01-01

    Free radical attack on beta-carotene results in the formation of high amounts of carotene breakdown products (CBPs) having biological activities. As several of the CBPs are reactive aldehydes, it has to be considered that these compounds are able to modify proteins. Therefore, the aim of the study was to investigate whether CBP-modification of proteins is leading to damaged proteins recognized and degraded by the proteasomal system. We used the model proteins tau and ferritin to test whether CBPs will modify them and whether such modifications lead to enhanced proteasomal degradation. To modify proteins, we used crude CBPs as a mixture obtained after hypochloric acid derived BC degradation, as well as several single compounds, as apo8'-carotenal, retinal, or beta-ionone. The majority of the CBPs found in our reaction mixture are well known metabolites as described earlier after BC degradation using different oxidants. CBPs are able to modify proteins, and in in vitro studies, we were able to demonstrate that the 20S proteasome is able to recognize and degrade CBP-modified proteins preferentially. In testing the proteolytic response of HT22 cells toward CBPs, we could demonstrate an enhanced protein turnover, which is sensitive to lactacystin. Interestingly, the proteasomal activity is resistant to treatment with CBP. On the other hand, we were able to demonstrate that supraphysiological levels of CBPs might lead to the formation of protein-CBP-adducts that are able to inhibit the proteasome. Therefore, the removal of CBP-modified proteins seems to be catalyzed by the proteasomal system and is effective, if the formation of CBPs is not overwhelming and leading to protein aggregates.

  12. The Ubiquitin Ligase Hul5 Promotes Proteasomal Processivity▿

    PubMed Central

    Aviram, Sharon; Kornitzer, Daniel

    2010-01-01

    The 26S proteasome is a large cytoplasmic protease that degrades polyubiquitinated proteins to short peptides in a processive manner. The proteasome 19S regulatory subcomplex tethers the target protein via its polyubiquitin adduct and unfolds the target polypeptide, which is then threaded into the proteolytic site-containing 20S subcomplex. Hul5 is a 19S subcomplex-associated ubiquitin ligase that elongates ubiquitin chains on proteasome-bound substrates. We isolated hul5Δ as a mutation with which fusions of an unstable cyclin to stable reporter proteins accumulate as partially processed products. These products appear transiently in the wild type but are strongly stabilized in 19S ATPase mutants and in the hul5Δ mutant, supporting a role for the ATPase subunits in the unfolding of proteasome substrates before insertion into the catalytic cavity and suggesting a role for Hul5 in the processive degradation of proteins that are stalled on the proteasome. PMID:20008553

  13. Integration of autophagy, proteasomal degradation, unfolded protein response and apoptosis.

    PubMed

    Benbrook, D M; Long, A

    2012-10-01

    A single cell has the potential to kill an entire human being. Efforts to cure cancer are limited by survival of individual cancer cells despite immune surveillance and toxic therapies. Understanding the intricate network of pathways that maintain cellular homeostasis and mediate stress response or default into cell death is critical to the development of strategies to eradicate cancer. Autophagy, proteasomal degradation and the unfolded protein response (UPR) are cellular pathways that degrade and recycle excess or damaged proteins to maintain cellular homeostasis and survival. This review will discuss autophagy and how it is integrated with proteasomal degradation and UPR to govern cell fate through restoration of cellular homeostasis or default into the apoptotic cell death pathway. The first response of autophagy is macroautophagy, which sequesters cytoplasm including organelles inside double-membraned autophagosome vesicles that fuse with lysosomes to degrade and recycle the contents. Ubiquitination patterns on proteins targeted for degradation determine whether adapter proteins will bring them to developing autophagosomes or to proteasomes. Macroautophagy is followed by chaperone-mediated autophagy (CMA), in which Hsc70 (Heat shock cognate 70) selectively binds proteins with exposed KFERQ motifs and pushes them inside lysosomes through the LAMP-2A (Lysosome-associated membrane protein type 2A) receptor. These two processes and the lesser understood microautophagy, which involves direct engulfment of proteins into lysosomes, occur at basal and induced levels. Insufficient proteasome function or ER stress induction of UPR can induce autophagy, which can mitigate damage and stress. If this network is incapable of repairing the damage or overcoming continued stress, the default pathway of apoptosis is engaged to destroy the cell. Induction of macroautophagy by cancer therapeutics has led to clinical trials investigating combinations of HCQ (hydroxychloriquine

  14. Lysine 419 targets human glucocorticoid receptor for proteasomal degradation.

    PubMed

    Wallace, Andrew D; Cao, Yan; Chandramouleeswaran, Sindhu; Cidlowski, John A

    2010-12-01

    Glucocorticoid receptors (GRs) are members of a highly conserved family of ligand dependent transcription factors which following hormone binding undergo homologous down-regulation reducing the levels of receptor protein. This decline in human GR (hGR) is due in part to a decrease in protein receptor stability that may limit cellular responsiveness to ligand. To examine the role of the proteasome protein degradation pathway in steroid-dependent hGR responsiveness, we utilized the proteasomal inhibitors MG-132, beta-lactone, and epoxomicin. HeLa cells and COS cells were treated with proteasome inhibitors in the presence of the GR agonist dexamethasone (Dex), or were pretreated with proteasomal inhibitor and then Dex. Dexamethasone induced glucocorticoid responsive reporter activity significantly over untreated controls, whereas cells treated with proteasomal inhibitors and Dex together showed 2-3-fold increase in activity. Protein sequence analysis of the hGR protein identified several candidate protein degradation motifs including a PEST element. Mutagenesis of this element at lysine 419 was done and mutant K419A hGR failed to undergo ligand dependent down-regulation. Mutant K419A hGR displayed 2-3-fold greater glucocorticoid responsive reporter activity in the presence of Dex than wild type hGR. These differences in transcriptional activity were not due to altered subcellular localization, since when the mutant K419A hGR was fused with the green fluorescent protein (GFP) it was found to move in and out of the nucleus similarly to wild type hGR. Together these results suggest that the proteasome and the identified PEST degradation motif limit steroid-dependent human glucocorticoid receptor signaling.

  15. 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…

  16. 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…

  17. Morphine Induces Ubiquitin-Proteasome Activity and Glutamate Transporter Degradation*

    PubMed Central

    Yang, Liling; Wang, Shuxing; Sung, Backil; Lim, Grewo; Mao, Jianren

    2008-01-01

    Glutamate transporters play a crucial role in physiological glutamate homeostasis, neurotoxicity, and glutamatergic regulation of opioid tolerance. However, how the glutamate transporter turnover is regulated remains poorly understood. Here we show that chronic morphine exposure induced posttranscriptional down-regulation of the glutamate transporter EAAC1 in C6 glioma cells with a concurrent decrease in glutamate uptake and increase in proteasome activity, which were blocked by the selective proteasome inhibitor MG-132 or lactacystin but not the lysosomal inhibitor chloroquin. At the cellular level, chronic morphine induced the PTEN (phosphatase and tensin homolog deleted on chromosome Ten)-mediated up-regulation of the ubiquitin E3 ligase Nedd4 via cAMP/protein kinase A signaling, leading to EAAC1 ubiquitination and proteasomal degradation. Either Nedd4 or PTEN knockdown with small interfering RNA prevented the morphine-induced EAAC1 degradation and decreased glutamate uptake. These data indicate that cAMP/protein kinase A signaling serves as an intracellular regulator upstream to the activation of the PTEN/Nedd4-mediated ubiquitin-proteasome system activity that is critical for glutamate transporter turnover. Under an in vivo condition, chronic morphine exposure also induced posttranscriptional down-regulation of the glutamate transporter EAAC1, which was prevented by MG-132, and transcriptional up-regulation of PTEN and Nedd4 within the spinal cord dorsal horn. Thus, inhibition of the ubiquitin-proteasome-mediated glutamate transporter degradation may be an important mechanism for preventing glutamate overexcitation and may offer a new strategy for treating certain neurological disorders and improving opioid therapy in chronic pain management. PMID:18539596

  18. Conformational switching of the 26S proteasome enables substrate degradation.

    PubMed

    Matyskiela, Mary E; Lander, Gabriel C; Martin, Andreas

    2013-07-01

    The 26S proteasome is the major eukaryotic ATP-dependent protease, responsible for regulating the proteome through degradation of ubiquitin-tagged substrates. Its regulatory particle, containing the heterohexameric AAA+ ATPase motor and the essential deubiquitinase Rpn11, recognizes substrates, removes their ubiquitin chains and translocates them into the associated peptidase after unfolding, but detailed mechanisms remain unknown. Here we present the 26S proteasome structure from Saccharomyces cerevisiae during substrate degradation, showing that the regulatory particle switches from a preengaged to a translocation-competent conformation. This conformation is characterized by a rearranged ATPase ring with uniform subunit interfaces, a widened central channel coaxially aligned with the peptidase and a spiral orientation of pore loops that suggests a rapid progression of ATP-hydrolysis events around the ring. Notably, Rpn11 moves from an occluded position to directly above the central pore, thus facilitating substrate deubiquitination concomitant with translocation.

  19. The proteasome and the degradation of oxidized proteins: Part III—Redox regulation of the proteasomal system

    PubMed Central

    Höhn, Tobias Jung Annika; Grune, Tilman

    2014-01-01

    Here, we review shortly the current knowledge on the regulation of the proteasomal system during and after oxidative stress. After addressing the components of the proteasomal system and the degradation of oxidatively damaged proteins in part I and II of this series, we address here which changes in activity undergo the proteasome and the ubiquitin-proteasomal system itself under oxidative conditions. While several components of the proteasomal system undergo direct oxidative modification, a number of redox-regulated events are modulating the proteasomal activity in a way it can address the major tasks in an oxidative stress situation: the removal of oxidized proteins and the adaptation of the cellular metabolism to the stress situation. PMID:24563857

  20. Deubiquitinase activity is required for the proteasomal degradation of misfolded cytosolic proteins upon heat-stress

    PubMed Central

    Fang, Nancy N.; Zhu, Mang; Rose, Amalia; Wu, Kuen-Phon; Mayor, Thibault

    2016-01-01

    Elimination of misfolded proteins is crucial for proteostasis and to prevent proteinopathies. Nedd4/Rsp5 emerged as a major E3-ligase involved in multiple quality control pathways that target misfolded plasma membrane proteins, aggregated polypeptides and cytosolic heat-induced misfolded proteins for degradation. It remained unclear how in one case cytosolic heat-induced Rsp5 substrates are destined for proteasomal degradation, whereas other Rsp5 quality control substrates are otherwise directed to lysosomal degradation. Here we find that Ubp2 and Ubp3 deubiquitinases are required for the proteasomal degradation of cytosolic misfolded proteins targeted by Rsp5 after heat-shock (HS). The two deubiquitinases associate more with Rsp5 upon heat-stress to prevent the assembly of K63-linked ubiquitin on Rsp5 heat-induced substrates. This activity was required to promote the K48-mediated proteasomal degradation of Rsp5 HS-induced substrates. Our results indicate that ubiquitin chain editing is key to the cytosolic protein quality control under stress conditions. PMID:27698423

  1. Normoxic destabilization of ATF-4 depends on proteasomal degradation.

    PubMed

    Wottawa, M; Köditz, J; Katschinski, D M

    2010-04-01

    Hypoxia-inducible gene expression is an important physiological adaptive mechanism in response to a decreased oxygen supply. We have recently described an oxygen- and prolyl-4-hydroxylase (PHD)3-dependent stabilization of the activating transcription factor 4 (ATF-4). The aim of the present study was to examine if the normoxic destabilization of ATF-4 is regulated by oxygen-dependent proteasomal degradation. We determined poly-ubiquitination of ATF-4 in normoxia compared to hypoxia by immunoprecipitation and immunoblots. Furthermore, we analysed the expression of the ATF-4 target gene GADD153 as a function of oxygen concentration. ATF-4 protein levels were not detectable in normoxia. Normoxic degradation correlated with an oxygen-dependent poly-ubiquitination of ATF-4, which was hindered by hypoxic incubation of the cells. As a result of hypoxia, GADD153 was expressed. The hypoxic GADD153 expression was attenuated or increased by transfecting the cells with ATF-4 siRNA or PHD3 siRNA respectively. Our results demonstrate the involvement of oxygen-dependent proteasomal degradation of ATF-4 in the hypoxia-induced expression of GADD153. Taken together, hypoxia/PHD3-regulated stabilization of ATF-4 by hindering oxygen-dependent degradation may play a critical role in linking cell fate decisions to oxygen availability.

  2. Proteasome-Mediated Degradation of FRIGIDA Modulates Flowering Time in Arabidopsis during Vernalization[C][W

    PubMed Central

    Kong, Xiangxiang; Wang, Chuntao; Ma, Lan; Zhao, Jinjie; Wei, Jingjing; Zhang, Xiaoming; Loake, Gary J.; Zhang, Ticao; Huang, Jinling; Yang, Yongping

    2014-01-01

    Winter-annual accessions of Arabidopsis thaliana require either exposure to cold stress or vernalization to initiate flowering via FRIGIDA (FRI). FRI acts as a scaffold protein to recruit several chromatin modifiers that epigenetically modify flowering genes. Here, we report that proteasome-mediated FRI degradation regulates flowering during vernalization in Arabidopsis. Our genetic and biochemical experiments demonstrate that FRI directly interacts with the BTB (Bric-a-Brac/Tramtrack/Broad Complex) proteins LIGHT-RESPONSE BTB1 (LRB1) and LRB2 as well as the CULLIN3A (CUL3A) ubiquitin-E3 ligase in vitro and in vivo, leading to proteasomal degradation of FRI during vernalization. The degradation of FRI is accompanied by an increase in the levels of the long noncoding RNA ColdAIR, which reduces the level of histone H3Lys4 trimethylation (H3K4me3) in FLOWERING LOCUS C chromatin to promote flowering. Furthermore, we found that the cold-induced WRKY34 transcription factor binds to the W-box in the promoter region of CUL3A to modulate CUL3A expression. Deficiency of WRKY34 suppressed CUL3A transcription to enhance FRI protein stability and led to late flowering after vernalization. Conversely, overexpression of WRK34 promoted FRI degradation and early flowering through inducing CUL3A accumulation. Together, these data suggest that WRKY34-induced and CUL3A-dependent proteolysis of FRI modulate flowering in response to vernalization. PMID:25538183

  3. Synthetic Uncleavable Ubiquitinated Proteins Dissect Proteasome Deubiquitination and Degradation, and Highlight Distinctive Fate of Tetraubiquitin.

    PubMed

    Singh, Sumeet K; Sahu, Indrajit; Mali, Sachitanand M; Hemantha, Hosahalli P; Kleifeld, Oded; Glickman, Michael H; Brik, Ashraf

    2016-12-14

    Various hypotheses have been proposed regarding how chain length, linkage type, position on substrate, and susceptibility to deubiquitinases (DUBs) affect processing of different substrates by proteasome. Here we report a new strategy for the chemical synthesis of ubiquitinated proteins to generate a set of well-defined conjugates bearing an oxime bond between the chain and the substrate. We confirmed that this isopeptide replacement is resistant to DUBs and to shaving by proteasome. Analyzing products generated by proteasomes ranked how chain length governed degradation outcome. Our results support that (1) the cleavage of the proximal isopeptide bond is not a prerequisite for proteasomal degradation, (2) by overcoming trimming at the proteasome, tetraUb is a fundamentally different signal than shorter chains, and (3) the tetra-ubiquitin chain can be degraded with the substrate. Together these results highlight the usefulness of chemistry to dissect the contribution of proteasome-associated DUBs and the complexity of the degradation process.

  4. Degrasyn activates proteasomal-dependent degradation of c-Myc.

    PubMed

    Bartholomeusz, Geoffrey; Talpaz, Moshe; Bornmann, William; Kong, Ling-Yuan; Donato, Nicholas J

    2007-04-15

    c-Myc is a highly unstable transcription factor whose deregulation and increased expression are associated with cancer. Degrasyn, a small synthetic molecule, induces rapid degradation of c-Myc protein in MM-1 multiple myeloma and other tumor cell lines. Destruction of c-Myc by degrasyn requires the presence of a region of c-Myc between amino acid residues 316 and 378 that has not previously been associated with c-Myc stability. Degrasyn-induced degradation of c-Myc depends on proteasomes but is independent of the degron regions previously shown to be important for ubiquitin-mediated targeting and proteasomal destruction of the protein. Degrasyn-dependent c-Myc proteolysis is not mediated by any previously identified c-Myc regulatory mechanism, does not require new protein synthesis, and does not depend on the nuclear localization of c-Myc. Degrasyn reduced c-Myc levels in A375 melanoma cells and in A375 tumors in nude mice, and this activity correlated with tumor growth inhibition. Together, these results suggest that degrasyn reduces the stability of c-Myc in vitro and in vivo through a unique signaling process that uses c-Myc domains not previously associated with c-Myc regulation.

  5. Open-gate mutants of the mammalian proteasome show enhanced ubiquitin-conjugate degradation.

    PubMed

    Choi, Won Hoon; de Poot, Stefanie A H; Lee, Jung Hoon; Kim, Ji Hyeon; Han, Dong Hoon; Kim, Yun Kyung; Finley, Daniel; Lee, Min Jae

    2016-03-09

    When in the closed form, the substrate translocation channel of the proteasome core particle (CP) is blocked by the convergent N termini of α-subunits. To probe the role of channel gating in mammalian proteasomes, we deleted the N-terminal tail of α3; the resulting α3ΔN proteasomes are intact but hyperactive in the hydrolysis of fluorogenic peptide substrates and the degradation of polyubiquitinated proteins. Cells expressing the hyperactive proteasomes show markedly elevated degradation of many established proteasome substrates and resistance to oxidative stress. Multiplexed quantitative proteomics revealed ∼ 200 proteins with reduced levels in the mutant cells. Potentially toxic proteins such as tau exhibit reduced accumulation and aggregate formation. These data demonstrate that the CP gate is a key negative regulator of proteasome function in mammals, and that opening the CP gate may be an effective strategy to increase proteasome activity and reduce levels of toxic proteins in cells.

  6. Chemical approaches to targeted protein degradation through modulation of the ubiquitin-proteasome pathway.

    PubMed

    Collins, Ian; Wang, Hannah; Caldwell, John J; Chopra, Raj

    2017-03-15

    Manipulation of the ubiquitin-proteasome system to achieve targeted degradation of proteins within cells using chemical tools and drugs has the potential to transform pharmacological and therapeutic approaches in cancer and other diseases. An increased understanding of the molecular mechanism of thalidomide and its analogues following their clinical use has unlocked small-molecule modulation of the substrate specificity of the E3 ligase cereblon (CRBN), which in turn has resulted in the advancement of new immunomodulatory drugs (IMiDs) into the clinic. The degradation of multiple context-specific proteins by these pleiotropic small molecules provides a means to uncover new cell biology and to generate future drug molecules against currently undruggable targets. In parallel, the development of larger bifunctional molecules that bring together highly specific protein targets in complexes with CRBN, von Hippel-Lindau, or other E3 ligases to promote ubiquitin-dependent degradation has progressed to generate selective chemical compounds with potent effects in cells and in vivo models, providing valuable tools for biological target validation and with future potential for therapeutic use. In this review, we survey recent breakthroughs achieved in these two complementary methods and the discovery of new modes of direct and indirect engagement of target proteins with the proteasome. We discuss the experimental characterisation that validates the use of molecules that promote protein degradation as chemical tools, the preclinical and clinical examples disclosed to date, and the future prospects for this exciting area of chemical biology. © 2017 The Author(s).

  7. Heat Shock Proteins Regulate Activation-induced Proteasomal Degradation of the Mature Phosphorylated Form of Protein Kinase C*

    PubMed Central

    Lum, Michelle A.; Balaburski, Gregor M.; Murphy, Maureen E.; Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    Although alterations in stimulus-induced degradation of PKC have been implicated in disease, mechanistic understanding of this process remains limited. Evidence supports the existence of both proteasomal and lysosomal mechanisms of PKC processing. An established pathway involves rate-limiting priming site dephosphorylation of the activated enzyme and proteasomal clearance of the dephosphorylated protein. However, here we show that agonists promote down-regulation of endogenous PKCα with minimal accumulation of a nonphosphorylated species in multiple cell types. Furthermore, proteasome and lysosome inhibitors predominantly protect fully phosphorylated PKCα, pointing to this form as a substrate for degradation. Failure to detect substantive dephosphorylation of activated PKCα was not due to rephosphorylation because inhibition of Hsp70/Hsc70, which is required for re-priming, had only a minor effect on agonist-induced accumulation of nonphosphorylated protein. Thus, PKC degradation can occur in the absence of dephosphorylation. Further analysis revealed novel functions for Hsp70/Hsc70 and Hsp90 in the control of agonist-induced PKCα processing. These chaperones help to maintain phosphorylation of activated PKCα but have opposing effects on degradation of the phosphorylated protein; Hsp90 is protective, whereas Hsp70/Hsc70 activity is required for proteasomal processing of this species. Notably, down-regulation of nonphosphorylated PKCα shows little Hsp70/Hsc70 dependence, arguing that phosphorylated and nonphosphorylated species are differentially targeted for proteasomal degradation. Finally, lysosomal processing of activated PKCα is not regulated by phosphorylation or Hsps. Collectively, these data demonstrate that phosphorylated PKCα is a direct target for agonist-induced proteasomal degradation via an Hsp-regulated mechanism, and highlight the existence of a novel pathway of PKC desensitization in cells. PMID:23900841

  8. Proteasome-Mediated Degradation of the Coactivator p300 Impairs Cardiac Transcription

    PubMed Central

    Poizat, Coralie; Sartorelli, Vittorio; Chung, Gene; Kloner, Robert A.; Kedes, Larry

    2000-01-01

    The transcription of tissue-specific genes is controlled by regulatory factors and cofactors and is suppressed in cardiac cells by the antineoplastic agent doxorubicin. Here we show that exposure of cultured cardiomyocytes to doxorubicin resulted in the rapid depletion of transcripts for MEF2C, dHAND, and NKX2.5, three pivotal regulators of cardiac gene expression. Delivery of exogenous p300, a coactivator of MEF2C and NKX2.5 in cardiomyocytes, restored cardiac transcription despite the presence of doxorubicin. Furthermore, p300 also restored the accumulation of transcripts for MEF2C itself. Importantly, cardiocytes exposed to doxorubicin displayed reduced levels of p300 proteins. This was not due to alterations in the level of p300 transcripts; rather, and surprisingly, doxorubicin promoted selective degradation of p300 mediated by the 26S-proteasome machinery. Doxorubicin had no effect on the general level of ubiquitinated proteins or on the levels of β-catenin, a protein known to be degraded by proteasome-mediated degradation. These results provide evidence for a new mechanism of transcriptional repression caused by doxorubicin in which the selective degradation of p300 results in reduced p300-dependent transcription, including production of MEF2C mRNA. PMID:11073966

  9. p62 links the autophagy pathway and the ubiqutin-proteasome system upon ubiquitinated protein degradation.

    PubMed

    Liu, Wei Jing; Ye, Lin; Huang, Wei Fang; Guo, Lin Jie; Xu, Zi Gan; Wu, Hong Luan; Yang, Chen; Liu, Hua Feng

    2016-01-01

    The ubiquitin-proteasome system (UPS) and autophagy are two distinct and interacting proteolytic systems. They play critical roles in cell survival under normal conditions and during stress. An increasing body of evidence indicates that ubiquitinated cargoes are important markers of degradation. p62, a classical receptor of autophagy, is a multifunctional protein located throughout the cell and involved in many signal transduction pathways, including the Keap1-Nrf2 pathway. It is involved in the proteasomal degradation of ubiquitinated proteins. When the cellular p62 level is manipulated, the quantity and location pattern of ubiquitinated proteins change with a considerable impact on cell survival. Altered p62 levels can even lead to some diseases. The proteotoxic stress imposed by proteasome inhibition can activate autophagy through p62 phosphorylation. A deficiency in autophagy may compromise the ubiquitin-proteasome system, since overabundant p62 delays delivery of the proteasomal substrate to the proteasome despite proteasomal catalytic activity being unchanged. In addition, p62 and the proteasome can modulate the activity of HDAC6 deacetylase, thus influencing the autophagic degradation.

  10. Transport of c-MYC by Kinesin-1 for proteasomal degradation in the cytoplasm.

    PubMed

    Lee, Clement M

    2014-09-01

    c-MYC is an oncogenic transcription factor that is degraded by the proteasome pathway. However, the mechanism that regulates delivery of c-MYC to the proteasome for degradation is not well characterized. Here, the results show that the motor protein complex Kinesin-1 transports c-MYC to the cytoplasm for proteasomal degradation. Inhibition of Kinesin-1 function enhanced ubiquitination of c-MYC and induced aggregation of c-MYC in the cytoplasm. Transport studies showed that the c-MYC aggregates moved from the nucleus to the cytoplasm and KIF5B is responsible for the transport in the cytoplasm. Furthermore, inhibition of the proteasomal degradation process also resulted in an accumulation of c-MYC aggregates in the cytoplasm. Moreover, Kinesin-1 was shown to interact with c-MYC and the proteasome subunit S6a. Inhibition of Kinesin-1 function also reduced c-MYC-dependent transformation activities. Taken together, the results strongly suggest that Kinesin-1 transports c-MYC for proteasomal degradation in the cytoplasm and the proper degradation of c-MYC mediated by Kinesin-1 transport is important for transformation activities of c-MYC. In addition, the results indicate that Kinesin-1 transport mechanism is important for degradation of a number of other proteins as well. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. The Ubiquitination, Disaggregation and Proteasomal Degradation Machineries in Polyglutamine Disease.

    PubMed

    Nath, Samir R; Lieberman, Andrew P

    2017-01-01

    Polyglutamine disorders are chronic, progressive neurodegenerative diseases caused by expansion of a glutamine tract in widely expressed genes. Despite excellent models of disease, a well-documented clinical history and progression, and established genetic causes, there are no FDA approved, disease modifying treatments for these disorders. Downstream of the mutant protein, several divergent pathways of toxicity have been identified over the last several decades, supporting the idea that targeting only one of these pathways of toxicity is unlikely to robustly alleviate disease progression. As a result, a vast body of research has focused on eliminating the mutant protein to broadly prevent downstream toxicity, either by silencing mutant protein expression or leveraging the endogenous protein quality control machinery. In the latter approach, a focus has been placed on four critical components of mutant protein degradation that are active in the nucleus, a key site of toxicity: disaggregation, ubiquitination, deubiquitination, and proteasomal activity. These machineries have unique functional components, but work together as a cellular defense system that can be successfully leveraged to alleviate disease phenotypes in several models of polyglutamine toxicity. This review will highlight recent advances in understanding both the potential and role of these components of the protein quality control machinery in polyglutamine disease pathophysiology.

  12. The Ubiquitination, Disaggregation and Proteasomal Degradation Machineries in Polyglutamine Disease

    PubMed Central

    Nath, Samir R.; Lieberman, Andrew P.

    2017-01-01

    Polyglutamine disorders are chronic, progressive neurodegenerative diseases caused by expansion of a glutamine tract in widely expressed genes. Despite excellent models of disease, a well-documented clinical history and progression, and established genetic causes, there are no FDA approved, disease modifying treatments for these disorders. Downstream of the mutant protein, several divergent pathways of toxicity have been identified over the last several decades, supporting the idea that targeting only one of these pathways of toxicity is unlikely to robustly alleviate disease progression. As a result, a vast body of research has focused on eliminating the mutant protein to broadly prevent downstream toxicity, either by silencing mutant protein expression or leveraging the endogenous protein quality control machinery. In the latter approach, a focus has been placed on four critical components of mutant protein degradation that are active in the nucleus, a key site of toxicity: disaggregation, ubiquitination, deubiquitination, and proteasomal activity. These machineries have unique functional components, but work together as a cellular defense system that can be successfully leveraged to alleviate disease phenotypes in several models of polyglutamine toxicity. This review will highlight recent advances in understanding both the potential and role of these components of the protein quality control machinery in polyglutamine disease pathophysiology. PMID:28381987

  13. Degradation of regulator of calcineurin 1 (RCAN1) is mediated by both chaperone-mediated autophagy and ubiquitin proteasome pathways.

    PubMed

    Liu, Heng; Wang, Pin; Song, Weihong; Sun, Xiulian

    2009-10-01

    Regulator of calcineurin 1 (RCAN1), a gene identified from the critical region of Down syndrome, has been implied in pathogenesis of Alzheimer's disease (AD). RCAN1 expression was shown to be increased in AD brains; however, the mechanism of RCAN1 gene regulation is not well defined. The present study was designed to investigate the molecular mechanism of RCAN1 protein degradation. In addition to being degraded through the ubiquitin proteasome pathway, we found that lysosomal inhibition markedly increased RCAN1 protein expression in a time- and dosage-dependent manner. Inhibition of macroautophagy reduced RCAN1 expression, indicating that RCAN1 degradation is not through a macroautophagy pathway. However, disruption of chaperone-mediated autophagy (CMA) increased RCAN1 expression. Two CMA recognition motifs were identified in RCAN1 protein to mediate its degradation through a CMA-lysosome pathway. A promoter assay further demonstrated that inhibition of RCAN1 degradation in cells reduced calcineurin-NFAT activity. Dysfunctions of ubiquitin-proteasome and autophagy-lysosome pathways have been implicated in neurodegenerative diseases. Therefore, elucidation of RCAN1 degradation by a ubiquitin proteasome pathway and CMA-lysosome pathway in the present study may greatly advance our understanding of AD pathogenesis.

  14. Mutant ubiquitin found in neurodegenerative disorders is a ubiquitin fusion degradation substrate that blocks proteasomal degradation

    PubMed Central

    Lindsten, Kristina; de Vrij, Femke M.S.; Verhoef, Lisette G.G.C.; Fischer, David F.; van Leeuwen, Fred W.; Hol, Elly M.; Masucci, Maria G.; Dantuma, Nico P.

    2002-01-01

    Loss of neurons in neurodegenerative diseases is usually preceded by the accumulation of protein deposits that contain components of the ubiquitin/proteasome system. Affected neurons in Alzheimer's disease often accumulate UBB+1, a mutant ubiquitin carrying a 19–amino acid C-terminal extension generated by a transcriptional dinucleotide deletion. Here we show that UBB+1 is a potent inhibitor of ubiquitin-dependent proteolysis in neuronal cells, and that this inhibitory activity correlates with induction of cell cycle arrest. Surprisingly, UBB+1 is recognized as a ubiquitin fusion degradation (UFD) proteasome substrate and ubiquitinated at Lys29 and Lys48. Full blockade of proteolysis requires both ubiquitination sites. Moreover, the inhibitory effect was enhanced by the introduction of multiple UFD signals. Our findings suggest that the inhibitory activity of UBB+1 may be an important determinant of neurotoxicity and contribute to an environment that favors the accumulation of misfolded proteins. PMID:11980917

  15. Ubiquitin-Proteasome System Inhibition Promotes Long-Term Depression and Synaptic Tagging/Capture.

    PubMed

    Li, Qin; Korte, Martin; Sajikumar, Sreedharan

    2016-06-01

    A balance of protein synthesis and degradation is critical for the dynamic regulation and implementation of long-term memory storage. The role of the ubiquitin-proteasome system (UPS) in regulating the plasticity at potentiated synapses is well studied, but its roles in depressed synaptic populations remain elusive. In this study, we probed the possibility of regulating the UPS by inhibiting the proteasome function during the induction of protein synthesis-independent form of hippocampal long-term depression (early-LTD), an important component of synaptic plasticity. Here, we show that protein degradation is involved in early-LTD induction and interfering with this process facilitates early-LTD to late-LTD. We provide evidence here that under the circumstances of proteasome inhibition brain-derived neurotrophic factor is accumulated as plasticity-related protein and it drives the weakly depressed or potentiated synapses to associativity. Thus, UPS inhibition promotes LTD and establishes associativity between weakly depressed or potentiated synapses through the mechanisms of synaptic tagging/capture or cross-capture.

  16. Degradation of the Neurospora circadian clock protein FREQUENCY through the ubiquitin-proteasome pathway.

    PubMed

    He, Q; Liu, Y

    2005-11-01

    Phosphorylation of the Neurospora circadian clock protein FREQUENCY (FRQ) promotes its degradation through the ubiquitin-proteasome pathway. Ubiquitination of FRQ requires FWD-1 (F-box/WD-40 repeat-containing protein-1), which is the substrate-recruiting subunit of an SCF (SKP/Cullin/F-box)-type ubiquitin ligase. In the fwd-1 mutant strains, FRQ degradation is defective, resulting in the accumulation of hyperphosphorylated FRQ and the loss of the circadian rhythmicities. The CSN (COP9 signalosome) promotes the function of SCF complexes in vivo. But in vitro, deneddylation of cullins by CSN inhibits SCF activity. In Neurospora, the disruption of the csn-2 subunit impairs FRQ degradation and compromises the normal circadian functions. These defects are due to the dramatically reduced levels of FWD-1 in the csn-2 mutant, a result of its rapid degradation. Other components of the SCF(FWD-1) complex, SKP-1 and CUL-1 are also unstable in the mutant. These results establish important roles for SCF(FWD-1) and CSN in the circadian clock of Neurospora and suggest that they are conserved components of the eukaryotic circadian clocks. In addition, these findings resolve the CSN paradox and suggest that the major function of CSN is to maintain the stability of SCF ubiquitin ligases in vivo.

  17. Proteasome Activity Is Affected by Fluctuations in Insulin-Degrading Enzyme Distribution

    PubMed Central

    Sbardella, Diego; Tundo, Grazia Raffaella; Sciandra, Francesca; Bozzi, Manuela; Gioia, Magda; Ciaccio, Chiara; Tarantino, Umberto; Brancaccio, Andrea; Coletta, Massimo; Marini, Stefano

    2015-01-01

    Insulin-Degrading-Enzyme (IDE) is a Zn2+-dependent peptidase highly conserved throughout evolution and ubiquitously distributed in mammalian tissues wherein it displays a prevalent cytosolic localization. We have recently demonstrated a novel Heat Shock Protein-like behaviour of IDE and its association with the 26S proteasome. In the present study, we examine the mechanistic and molecular features of IDE-26S proteasome interaction in a cell experimental model, extending the investigation also to the effect of IDE on the enzymatic activities of the 26S proteasome. Further, kinetic investigations indicate that the 26S proteasome activity undergoes a functional modulation by IDE through an extra-catalytic mechanism. The IDE-26S proteasome interaction was analyzed during the Heat Shock Response and we report novel findings on IDE intracellular distribution that might be of critical relevance for cell metabolism. PMID:26186340

  18. Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators.

    PubMed

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

    2014-05-01

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

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

    SciTech Connect

    Mi, Lixin; Gan, Nanqin; Chung, Fung-Lung

    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.

  20. Atypical ubiquitylation in yeast targets lysine-less Asi2 for proteasomal degradation.

    PubMed

    Boban, Mirta; Ljungdahl, Per O; Foisner, Roland

    2015-01-23

    Proteins are typically targeted for proteasomal degradation by the attachment of a polyubiquitin chain to ϵ-amino groups of lysine residues. Non-lysine ubiquitylation of proteasomal substrates has been considered an atypical and rare event limited to complex eukaryotes. Here we report that a fully functional lysine-less mutant of an inner nuclear membrane protein in yeast, Asi2, is polyubiquitylated and targeted for proteasomal degradation. Efficient degradation of lysine-free Asi2 requires E3-ligase Doa10 and E2 enzymes Ubc6 and Ubc7, components of the endoplasmic reticulum-associated degradation pathway. Together, our data suggest that non-lysine ubiquitylation may be more prevalent than currently considered. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. ELL targets c-Myc for proteasomal degradation and suppresses tumour growth.

    PubMed

    Chen, Yu; Zhou, Chi; Ji, Wei; Mei, Zhichao; Hu, Bo; Zhang, Wei; Zhang, Dawei; Wang, Jing; Liu, Xing; Ouyang, Gang; Zhou, Jiangang; Xiao, Wuhan

    2016-03-24

    Increasing evidence supports that ELL (eleven-nineteen lysine-rich leukaemia) is a key regulator of transcriptional elongation, but the physiological function of Ell in mammals remains elusive. Here we show that ELL functions as an E3 ubiquitin ligase and targets c-Myc for proteasomal degradation. In addition, we identify that UbcH8 serves as a ubiquitin-conjugating enzyme in this pathway. Cysteine 595 of ELL is an active site of the enzyme; its mutation to alanine (C595A) renders the protein unable to promote the ubiquitination and degradation of c-Myc. ELL-mediated c-Myc degradation inhibits c-Myc-dependent transcriptional activity and cell proliferation, and also suppresses c-Myc-dependent xenograft tumour growth. In contrast, the ELL(C595A) mutant not only loses the ability to inhibit cell proliferation and xenograft tumour growth, but also promotes tumour metastasis. Thus, our work reveals a previously unrecognized function for ELL as an E3 ubiquitin ligase for c-Myc and a potential tumour suppressor.

  2. ELL targets c-Myc for proteasomal degradation and suppresses tumour growth

    PubMed Central

    Chen, Yu; Zhou, Chi; Ji, Wei; Mei, Zhichao; Hu, Bo; Zhang, Wei; Zhang, Dawei; Wang, Jing; Liu, Xing; Ouyang, Gang; Zhou, Jiangang; Xiao, Wuhan

    2016-01-01

    Increasing evidence supports that ELL (eleven–nineteen lysine-rich leukaemia) is a key regulator of transcriptional elongation, but the physiological function of Ell in mammals remains elusive. Here we show that ELL functions as an E3 ubiquitin ligase and targets c-Myc for proteasomal degradation. In addition, we identify that UbcH8 serves as a ubiquitin-conjugating enzyme in this pathway. Cysteine 595 of ELL is an active site of the enzyme; its mutation to alanine (C595A) renders the protein unable to promote the ubiquitination and degradation of c-Myc. ELL-mediated c-Myc degradation inhibits c-Myc-dependent transcriptional activity and cell proliferation, and also suppresses c-Myc-dependent xenograft tumour growth. In contrast, the ELL(C595A) mutant not only loses the ability to inhibit cell proliferation and xenograft tumour growth, but also promotes tumour metastasis. Thus, our work reveals a previously unrecognized function for ELL as an E3 ubiquitin ligase for c-Myc and a potential tumour suppressor. PMID:27009366

  3. Redox modulation of cellular metabolism through targeted degradation of signaling proteins by the proteasome

    SciTech Connect

    Squier, Thomas C.

    2006-02-01

    Under conditions of oxidative stress, the 20S proteasome plays a critical role in maintaining cellular homeostasis through the selective degradation of oxidized and damaged proteins. This adaptive stress response is distinct from ubiquitin-dependent pathways in that oxidized proteins are recognized and degraded in an ATP-independent mechanism, which can involve the molecular chaperone Hsp90. Like the regulatory complexes 19S and 11S REG, Hsp90 tightly associates with the 20S proteasome to mediate the recognition of aberrant proteins for degradation. In the case of the calcium signaling protein calmodulin, proteasomal degradation results from the oxidation of a single surface exposed methionine (i.e., Met145); oxidation of the other eight methionines has a minimal effect on the recognition and degradation of calmodulin by the proteasome. Since cellular concentrations of calmodulin are limiting, the targeted degradation of this critical signaling protein under conditions of oxidative stress will result in the downregulation of cellular metabolism, serving as a feedback regulation to diminish the generation of reactive oxygen species. The targeted degradation of critical signaling proteins, such as calmodulin, can function as sensors of oxidative stress to downregulate global rates of metabolism and enhance cellular survival.

  4. Molecular Pathways: Turning Proteasomal Protein Degradation into a Unique Treatment Approach

    PubMed Central

    Stintzing, Sebastian; Lenz, Heinz-Josef

    2015-01-01

    Cancer treatment regimens have evolved from single cytotoxic substances affecting all proliferative tissues towards antibodies and kinase inhibitors targeting tumor specific pathways. Treatment efficacy and cancer survival has overall improved and side effects have become less frequent. The ubiquitin proteasome system (UPS) mediated proteasomal protein degradation is the most critical pathway to regulate the quantity of signal proteins involved in carcinogenesis and tumor progression. These processes are, as well as protein recycling, highly regulated and offer targets for biomarker and drug development. Unspecific proteasome inhibitors such as bortezomib and carfilzomib have shown clinical efficacy and are approved for clinical use. Inhibitors of more substrate specific enzymes of degradation processes are developed and in early clinical trials. The novel compounds focus on the degradation of key regulatory proteins such as p53, p27Kip1 and β-catenin, and inhibitors specific for growth factor receptor kinases turnover are in pre-clinical testing. PMID:24756373

  5. [Proteasome degradation of protein C and plasmin inhibitor mutants: molecular mechanism of congenital protein deficiency].

    PubMed

    Nishio, Miwako; Koyama, Takatoshi; Hirosawa, Shinsaku

    2009-08-01

    In many inherited disorders, protein deficiency is one of the major aetiologies, but the molecular and cellular mechanisms remain unclear. We investigated the intracellular degradation of mutant proteins, using naturally occurring PC and PI mutants that lead to congenital deficiencies. We have shown that proteasomes are very important for the degradation of PC and PI mutants, irrespective of the presence or absence of N-glycosylation moieties. Furthermore, mannose trimming after glucose removal is very important for initiation of the degradation. Inhibition of glucose trimming of the mutant proteins accelerated degradation by the proteasomes, and initiation of the degradation occurs after mannose trimming of the middle chain of N-linked glycosylation by mannosidase I. The binding of molecular chaperons influenced by the presence of N-glycosylation moieties may affect the efficient degradation of the mutant proteins. Cotransfection of endoplasmic reticulum (ER) degradation enhancing alpha-mannosidase like protein (EDEM) accelerated the degradation of N-glycosylated PC. The mutant PC or PI molecules were ubiquitin-independently degraded by proteasomes. Autophagy does not appear to contribute to the degradation of PC and PI mutants. These findings might help to elucidate the molecular mechanisms and potential treatments of congenital deficiencies of proteins in a system of coagulation and fibrinolysis.

  6. FBXO32 Targets c-Myc for Proteasomal Degradation and Inhibits c-Myc Activity.

    PubMed

    Mei, Zhichao; Zhang, Dawei; Hu, Bo; Wang, Jing; Shen, Xian; Xiao, Wuhan

    2015-06-26

    FBXO32 (MAFbx/Atrogin-1) is an E3 ubiquitin ligase that is markedly up-regulated in muscle atrophy. Although some data indicate that FBXO32 may play an important role in tumorigenesis, the molecular mechanism of FBXO32 in tumorigenesis has been poorly understood. Here, we present evidence that FBXO32 targets the oncogenic protein c-Myc for ubiquitination and degradation through the proteasome pathway. Phosphorylation of c-Myc at Thr-58 and Ser-62 is dispensable for FBXO32 to induce c-Myc degradation. Mutation of the lysine 326 in c-Myc reduces c-Myc ubiquitination and prevents the c-Myc degradation induced by FBXO32. Furthermore, overexpression of FBXO32 suppresses c-Myc activity and inhibits cell growth, but knockdown of FBXO32 enhances c-Myc activity and promotes cell growth. Finally, we show that FBXO32 is a direct downstream target of c-Myc, highlighting a negative feedback regulation loop between c-Myc and FBXO32. Thus, FBXO32 may function by targeting c-Myc. This work explains the function of FBXO32 and highlights its mechanisms in tumorigenesis.

  7. FBXO32 Targets c-Myc for Proteasomal Degradation and Inhibits c-Myc Activity*

    PubMed Central

    Mei, Zhichao; Zhang, Dawei; Hu, Bo; Wang, Jing; Shen, Xian; Xiao, Wuhan

    2015-01-01

    FBXO32 (MAFbx/Atrogin-1) is an E3 ubiquitin ligase that is markedly up-regulated in muscle atrophy. Although some data indicate that FBXO32 may play an important role in tumorigenesis, the molecular mechanism of FBXO32 in tumorigenesis has been poorly understood. Here, we present evidence that FBXO32 targets the oncogenic protein c-Myc for ubiquitination and degradation through the proteasome pathway. Phosphorylation of c-Myc at Thr-58 and Ser-62 is dispensable for FBXO32 to induce c-Myc degradation. Mutation of the lysine 326 in c-Myc reduces c-Myc ubiquitination and prevents the c-Myc degradation induced by FBXO32. Furthermore, overexpression of FBXO32 suppresses c-Myc activity and inhibits cell growth, but knockdown of FBXO32 enhances c-Myc activity and promotes cell growth. Finally, we show that FBXO32 is a direct downstream target of c-Myc, highlighting a negative feedback regulation loop between c-Myc and FBXO32. Thus, FBXO32 may function by targeting c-Myc. This work explains the function of FBXO32 and highlights its mechanisms in tumorigenesis. PMID:25944903

  8. Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization.

    PubMed

    Zimmerman, Shawn W; Manandhar, Gaurishankar; Yi, Young-Joo; Gupta, Satish K; Sutovsky, Miriam; Odhiambo, John F; Powell, Michael D; Miller, David J; Sutovsky, Peter

    2011-02-23

    Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced

  9. Proapoptotic fibronectin fragment induces the degradation of ubiquitinated p53 via proteasomes in periodontal ligament cells

    PubMed Central

    Ghosh, Abhijit; Joo, Nam Eok; Chen, Tina Chunyuan; Kapila, Yvonne L.

    2009-01-01

    Background and Objective The extracellular matrix (ECM) plays a key role in signaling necessary for tissue remodeling and cell survival. However, signals from disease-altered ECMs, as that present in inflammatory diseases like periodontitis and arthritis, may lead to apoptosis or programmed cell death of resident cells. Previously, we found that a disease-associated fibronectin fragment triggers apoptosis of primary human periodontal ligament (PDL) cells via a novel apoptotic pathway in which the tumor suppressor, p53, is transcriptionally downregulated. Materials and Methods We used immunofluorescence, transfection assays, western blotting and ELISAs to show that p53 is degraded by a proteasomal pathway in response to a proapoptotic disease-associated fibronectin fragment. Results We now show that under these same apoptotic conditions p53 is further downregulated by post-translational ubiquitination and subsequent targeting to the proteasome for degradation. Pretreatment of cells with the proteasomal inhibitors MG132 and lactacystin rescued the cells from apoptosis. p53 levels in cells transfected with ubiquitin siRNA were resistant to degradation induced by the proapoptotic fibronectin fragment, showing that ubiquitination is important for the proapoptotic fibronectin fragment-induced degradation of p53. Conclusions These data show that a proapoptotic fibronectin matrix induces ubiquitination and degradation of p53 in the proteasome as part of a novel mechanism of apoptosis associated with inflammatory diseases. PMID:20337881

  10. Yeast Deubiquitinase Ubp3 Interacts with the 26 S Proteasome to Facilitate Rad4 Degradation*

    PubMed Central

    Mao, Peng; Smerdon, Michael J.

    2010-01-01

    Deubiquitinating enzymes (DUBs) function in a variety of cellular processes by removing ubiquitin moieties from substrates, but their role in DNA repair has not been elucidated. Yeast Rad4-Rad23 heterodimer is responsible for recognizing DNA damage in nucleotide excision repair (NER). Rad4 binds to UV damage directly while Rad23 stabilizes Rad4 from proteasomal degradation. Here, we show that disruption of yeast deubiquitinase UBP3 leads to enhanced UV resistance, increased repair of UV damage and Rad4 levels in rad23Δ cells, and elevated Rad4 stability. A catalytically inactive Ubp3 (Ubp3-C469A), however, is unable to affect NER or Rad4. Consistent with its role in down-regulating Rad4, Ubp3 physically interacts with Rad4 and the proteasome, both in vivo and in vitro, suggesting that Ubp3 associates with the proteasome to facilitate Rad4 degradation and thus suppresses NER. PMID:20876584

  11. Control of mammalian circadian rhythm by CKIepsilon-regulated proteasome-mediated PER2 degradation.

    PubMed

    Eide, Erik J; Woolf, Margaret F; Kang, Heeseog; Woolf, Peter; Hurst, William; Camacho, Fernando; Vielhaber, Erica L; Giovanni, Andrew; Virshup, David M

    2005-04-01

    The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKIepsilon (casein kinase Iepsilon) has been postulated to prime PER2 for degradation. Supporting this idea, CKIepsilon inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKIepsilon inhibition also slows the degradation of PER2 in cells. CKIepsilon-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein beta-TrCP to a specific site, and dominant negative beta-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.

  12. 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…

  13. Role of Proteasome-Dependent Protein Degradation in Long-Term Operant Memory in "Aplysia"

    ERIC Educational Resources Information Center

    Lyons, Lisa C.; Gardner, Jacob S.; Gandour, Catherine E.; Krishnan, Harini C.

    2017-01-01

    We investigated the in vivo role of protein degradation during intermediate (ITM) and long-term memory (LTM) in "Aplysia" using an operant learning paradigm. The proteasome inhibitor MG-132 inhibited the induction and molecular consolidation of LTM with no effect on ITM. Remarkably, maintenance of steady-state protein levels through…

  14. 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…

  15. Role of Proteasome-Dependent Protein Degradation in Long-Term Operant Memory in "Aplysia"

    ERIC Educational Resources Information Center

    Lyons, Lisa C.; Gardner, Jacob S.; Gandour, Catherine E.; Krishnan, Harini C.

    2017-01-01

    We investigated the in vivo role of protein degradation during intermediate (ITM) and long-term memory (LTM) in "Aplysia" using an operant learning paradigm. The proteasome inhibitor MG-132 inhibited the induction and molecular consolidation of LTM with no effect on ITM. Remarkably, maintenance of steady-state protein levels through…

  16. Direct interaction of menin leads to ubiquitin-proteasomal degradation of β-catenin.

    PubMed

    Kim, Byungho; Song, Tae-Yang; Jung, Kwan Young; Kim, Seul Gi; Cho, Eun-Jung

    2017-10-07

    Menin, encoded by the multiple endocrine neoplasia type 1 (MEN1) gene, is a tumor suppressor and transcription regulator. Menin interacts with various proteins as a scaffold protein and is proposed to play important roles in multiple physiological and pathological processes by controlling gene expression, proliferation, and apoptosis. The mechanisms underlying menin's suppression of tumorigenesis are largely elusive. In this study, we showed that menin was essential for the regulation of canonical Wnt/β-catenin signaling in cultured cells. The C-terminal domain of menin was able to directly interact with and promote ubiquitin-mediated degradation of β-catenin. We further revealed that overexpression of menin down-regulated the transcriptional activity of β-catenin and target gene expression. Moreover, menin efficiently inhibited β-catenin protein levels, transcriptional activity, and proliferation of human renal carcinoma cells with an activated β-catenin pathway. Taken together, our results provide novel molecular insights into the tumor suppressor activity of menin, which is partly mediated by proteasomal degradation of β-catenin and inhibition of Wnt/β-catenin signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. SUMO regulates proteasome-dependent degradation of FLASH/Casp8AP2

    PubMed Central

    Vennemann, Astrid; Hofmann, Thomas G.

    2013-01-01

    FLASH/Casp8AP2 is a huge multifunctional protein involved in multiple cellular processes, reaching from death receptor signaling to regulation of histone gene transcription and histone mRNA processing. Previous work has shown that FLASH localizes to Cajal bodies and promyelocytic leukemia (PML) bodies. However, the function of its nuclear body association remains unclear. Here we demonstrate that murine FLASH is covalently modified by SUMO at Lys residue 1792. Interestingly, ectopic expression of SUMO results in proteasome-dependent degradation of FLASH. A point mutant of FLASH with a mutated SUMO acceptor lysine residue, FLASHK1792R, is resistant to SUMO-induced degradation. Finally, we show that arsenic trioxide, a drug known to potentiate SUMO modification and degradation of PML, triggers recruitment of FLASH to PML bodies and concomitant loss of FLASH protein. Our data suggest that SUMO targets FLASH for proteasome-dependent degradation, which is associated with recruitment of FLASH to PML bodies. PMID:23673342

  18. SUMO regulates proteasome-dependent degradation of FLASH/Casp8AP2.

    PubMed

    Vennemann, Astrid; Hofmann, Thomas G

    2013-06-15

    FLASH/Casp8AP2 is a huge multifunctional protein involved in multiple cellular processes, reaching from death receptor signaling to regulation of histone gene transcription and histone mRNA processing. Previous work has shown that FLASH localizes to Cajal bodies and promyelocytic leukemia (PML) bodies. However, the function of its nuclear body association remains unclear. Here we demonstrate that murine FLASH is covalently modified by SUMO at Lys residue 1792. Interestingly, ectopic expression of SUMO results in proteasome-dependent degradation of FLASH. A point mutant of FLASH with a mutated SUMO acceptor lysine residue, FLASH(K1792R), is resistant to SUMO-induced degradation. Finally, we show that arsenic trioxide, a drug known to potentiate SUMO modification and degradation of PML, triggers recruitment of FLASH to PML bodies and concomitant loss of FLASH protein. Our data suggest that SUMO targets FLASH for proteasome-dependent degradation, which is associated with recruitment of FLASH to PML bodies.

  19. Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination

    PubMed Central

    Braten, Ori; Livneh, Ido; Ziv, Tamar; Admon, Arie; Kehat, Izhak; Caspi, Lilac H.; Gonen, Hedva; Bercovich, Beatrice; Godzik, Adam; Jahandideh, Samad; Jaroszewski, Lukasz; Sommer, Thomas; Kwon, Yong Tae; Guharoy, Mainak; Tompa, Peter; Ciechanover, Aaron

    2016-01-01

    The “canonical” proteasomal degradation signal is a substrate-anchored polyubiquitin chain. However, a handful of proteins were shown to be targeted following monoubiquitination. In this study, we established—in both human and yeast cells—a systematic approach for the identification of monoubiquitination-dependent proteasomal substrates. The cellular wild-type polymerizable ubiquitin was replaced with ubiquitin that cannot form chains. Using proteomic analysis, we screened for substrates that are nevertheless degraded under these conditions compared with those that are stabilized, and therefore require polyubiquitination for their degradation. For randomly sampled representative substrates, we confirmed that their cellular stability is in agreement with our screening prediction. Importantly, the two groups display unique features: monoubiquitinated substrates are smaller than the polyubiquitinated ones, are enriched in specific pathways, and, in humans, are structurally less disordered. We suggest that monoubiquitination-dependent degradation is more widespread than assumed previously, and plays key roles in various cellular processes. PMID:27385826

  20. 20S proteasome activation promotes life span extension and resistance to proteotoxicity in Caenorhabditis elegans

    PubMed Central

    Chondrogianni, Niki; Georgila, Konstantina; Kourtis, Nikos; Tavernarakis, Nektarios; Gonos, Efstathios S.

    2015-01-01

    Protein homeostasis (proteostasis) is one of the nodal points that need to be preserved to retain physiologic cellular/organismal balance. The ubiquitin-proteasome system (UPS) is responsible for the removal of both normal and damaged proteins, with the proteasome being the downstream effector. The proteasome is the major cellular protease with progressive impairment of function during aging and senescence. Despite the documented age-retarding properties of proteasome activation in various cellular models, simultaneous enhancement of the 20S core proteasome content, assembly, and function have never been reported in any multicellular organism. Consequently, the possible effects of the core proteasome modulation on organismal life span are elusive. In this study, we have achieved activation of the 20S proteasome at organismal level. We demonstrate enhancement of proteasome levels, assembly, and activity in the nematode Caenorhabditis elegans, resulting in life span extension and increased resistance to stress. We also provide evidence that the observed life span extension is dependent on the transcriptional activity of Dauer formation abnormal/Forkhead box class O (DAF-16/FOXO), skinhead-1 (SKN-1), and heat shock factor-1 (HSF-1) factors through regulation of downstream longevity genes. We further show that the reported beneficial effects are not ubiquitous but they are dependent on the genetic context. Finally, we provide evidence that proteasome core activation might be a potential strategy to minimize protein homeostasis deficiencies underlying aggregation-related diseases, such as Alzheimer’s disease (AD) or Huntington’s disease (HD). In summary, this is the first report demonstrating that 20S core proteasome up-regulation in terms of both content and activity is feasible in a multicellular eukaryotic organism and that in turn this modulation promotes extension of organismal health span and life span.—Chondrogianni, N., Georgila, K., Kourtis, N

  1. Withaferin A: a proteasomal inhibitor promotes healing after injury and exerts anabolic effect on osteoporotic bone

    PubMed Central

    Khedgikar, V; Kushwaha, P; Gautam, J; Verma, A; Changkija, B; Kumar, A; Sharma, S; Nagar, G K; Singh, D; Trivedi, P K; Sangwan, N S; Mishra, P R; Trivedi, R

    2013-01-01

    Withania somnifera or Ashwagandha is a medicinal herb of Ayurveda. Though the extract and purified molecules, withanolides, from this plant have been shown to have different pharmacological activities, their effect on bone formation has not been studied. Here, we show that one of the withanolide, withaferin A (WFA) acts as a proteasomal inhibitor (PI) and binds to specific catalytic β subunit of the 20S proteasome. It exerts positive effect on osteoblast by increasing osteoblast proliferation and differentiation. WFA increased expression of osteoblast-specific transcription factor and mineralizing genes, promoted osteoblast survival and suppressed inflammatory cytokines. In osteoclast, WFA treatment decreased osteoclast number directly by decreasing expression of tartarate-resistant acid phosphatase and receptor activator of nuclear factor kappa-B (RANK) and indirectly by decreasing osteoprotegrin/RANK ligand ratio. Our data show that in vitro treatment of WFA to calvarial osteoblast cells decreased expression of E3 ubiquitin ligase, Smad ubiquitin regulatory factor 2 (Smurf2), preventing degradation of Runt-related transcription factor 2 (RunX2) and relevant Smad proteins, which are phosphorylated by bone morphogenetic protein 2. Increased Smurf2 expression due to exogenous treatment of tumor necrosis factor α (TNFα) to primary osteoblast cells was decreased by WFA treatment. This was corroborated by using small interfering RNA against Smurf2. Further, WFA also blocked nuclear factor kappa-B (NF-kB) signaling as assessed by tumor necrosis factor stimulated nuclear translocation of p65-subunit of NF-kB. Overall data show that in vitro proteasome inhibition by WFA simultaneously promoted osteoblastogenesis by stabilizing RunX2 and suppressed osteoclast differentiation, by inhibiting osteoclastogenesis. Oral administration of WFA to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased expression of osteogenic genes. WFA

  2. Withaferin A: a proteasomal inhibitor promotes healing after injury and exerts anabolic effect on osteoporotic bone.

    PubMed

    Khedgikar, V; Kushwaha, P; Gautam, J; Verma, A; Changkija, B; Kumar, A; Sharma, S; Nagar, G K; Singh, D; Trivedi, P K; Sangwan, N S; Mishra, P R; Trivedi, R

    2013-08-22

    Withania somnifera or Ashwagandha is a medicinal herb of Ayurveda. Though the extract and purified molecules, withanolides, from this plant have been shown to have different pharmacological activities, their effect on bone formation has not been studied. Here, we show that one of the withanolide, withaferin A (WFA) acts as a proteasomal inhibitor (PI) and binds to specific catalytic β subunit of the 20S proteasome. It exerts positive effect on osteoblast by increasing osteoblast proliferation and differentiation. WFA increased expression of osteoblast-specific transcription factor and mineralizing genes, promoted osteoblast survival and suppressed inflammatory cytokines. In osteoclast, WFA treatment decreased osteoclast number directly by decreasing expression of tartarate-resistant acid phosphatase and receptor activator of nuclear factor kappa-B (RANK) and indirectly by decreasing osteoprotegrin/RANK ligand ratio. Our data show that in vitro treatment of WFA to calvarial osteoblast cells decreased expression of E3 ubiquitin ligase, Smad ubiquitin regulatory factor 2 (Smurf2), preventing degradation of Runt-related transcription factor 2 (RunX2) and relevant Smad proteins, which are phosphorylated by bone morphogenetic protein 2. Increased Smurf2 expression due to exogenous treatment of tumor necrosis factor α (TNFα) to primary osteoblast cells was decreased by WFA treatment. This was corroborated by using small interfering RNA against Smurf2. Further, WFA also blocked nuclear factor kappa-B (NF-kB) signaling as assessed by tumor necrosis factor stimulated nuclear translocation of p65-subunit of NF-kB. Overall data show that in vitro proteasome inhibition by WFA simultaneously promoted osteoblastogenesis by stabilizing RunX2 and suppressed osteoclast differentiation, by inhibiting osteoclastogenesis. Oral administration of WFA to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased expression of osteogenic genes. WFA

  3. Nuclear ubiquitin proteasome degradation affects WRKY45 function in the rice defense program

    PubMed Central

    Matsushita, Akane; Inoue, Haruhiko; Goto, Shingo; Nakayama, Akira; Sugano, Shoji; Hayashi, Nagao; Takatsuji, Hiroshi

    2013-01-01

    The transcriptional activator WRKY45 plays a major role in the salicylic acid/benzothiadiazole-induced defense program in rice. Here, we show that the nuclear ubiquitin–proteasome system (UPS) plays a role in regulating the function of WRKY45. Proteasome inhibitors induced accumulation of polyubiquitinated WRKY45 and transient up-regulation of WRKY45 target genes in rice cells, suggesting that WRKY45 is constantly degraded by the UPS to suppress defense responses in the absence of defense signals. Mutational analysis of the nuclear localization signal indicated that UPS-dependent WRKY45 degradation occurs in the nuclei. Interestingly, the transcriptional activity of WRKY45 after salicylic acid treatment was impaired by proteasome inhibition. The same C-terminal region in WRKY45 was essential for both transcriptional activity and UPS-dependent degradation. These results suggest that UPS regulation also plays a role in the transcriptional activity of WRKY45. It has been reported that AtNPR1, the central regulator of the salicylic acid pathway in Arabidopsis, is regulated by the UPS. We found that OsNPR1/NH1, the rice counterpart of NPR1, was not stabilized by proteasome inhibition under uninfected conditions. We discuss the differences in post-translational regulation of salicylic acid pathway components between rice and Arabidopsis. PMID:23013464

  4. HUWE1 ubiquitinates MyoD and targets it for proteasomal degradation

    SciTech Connect

    Noy, Tahel; Suad, Oded; Taglicht, Daniel; Ciechanover, Aaron

    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.

  5. Nuclear ubiquitin proteasome degradation affects WRKY45 function in the rice defense program.

    PubMed

    Matsushita, Akane; Inoue, Haruhiko; Goto, Shingo; Nakayama, Akira; Sugano, Shoji; Hayashi, Nagao; Takatsuji, Hiroshi

    2013-01-01

    The transcriptional activator WRKY45 plays a major role in the salicylic acid/benzothiadiazole-induced defense program in rice. Here, we show that the nuclear ubiquitin-proteasome system (UPS) plays a role in regulating the function of WRKY45. Proteasome inhibitors induced accumulation of polyubiquitinated WRKY45 and transient up-regulation of WRKY45 target genes in rice cells, suggesting that WRKY45 is constantly degraded by the UPS to suppress defense responses in the absence of defense signals. Mutational analysis of the nuclear localization signal indicated that UPS-dependent WRKY45 degradation occurs in the nuclei. Interestingly, the transcriptional activity of WRKY45 after salicylic acid treatment was impaired by proteasome inhibition. The same C-terminal region in WRKY45 was essential for both transcriptional activity and UPS-dependent degradation. These results suggest that UPS regulation also plays a role in the transcriptional activity of WRKY45. It has been reported that AtNPR1, the central regulator of the salicylic acid pathway in Arabidopsis, is regulated by the UPS. We found that OsNPR1/NH1, the rice counterpart of NPR1, was not stabilized by proteasome inhibition under uninfected conditions. We discuss the differences in post-translational regulation of salicylic acid pathway components between rice and Arabidopsis.

  6. Phosphorylation Regulates the Ubiquitin-independent Degradation of Yeast Pah1 Phosphatidate Phosphatase by the 20S Proteasome*

    PubMed Central

    Hsieh, Lu-Sheng; Su, Wen-Min; Han, Gil-Soo; Carman, George M.

    2015-01-01

    Saccharomyces cerevisiae Pah1 phosphatidate phosphatase, which catalyzes the conversion of phosphatidate to diacylglycerol for triacylglycerol synthesis and simultaneously controls phosphatidate levels for phospholipid synthesis, is subject to the proteasome-mediated degradation in the stationary phase of growth. In this study, we examined the mechanism for its degradation using purified Pah1 and isolated proteasomes. Pah1 expressed in S. cerevisiae or Escherichia coli was not degraded by the 26S proteasome, but by its catalytic 20S core particle, indicating that its degradation is ubiquitin-independent. The degradation of Pah1 by the 20S proteasome was dependent on time and proteasome concentration at the pH optimum of 7.0. The 20S proteasomal degradation was conserved for human lipin 1 phosphatidate phosphatase. The degradation analysis using Pah1 truncations and its fusion with GFP indicated that proteolysis initiates at the N- and C-terminal unfolded regions. The folded region of Pah1, in particular the haloacid dehalogenase-like domain containing the DIDGT catalytic sequence, was resistant to the proteasomal degradation. The structural change of Pah1, as reflected by electrophoretic mobility shift, occurs through its phosphorylation by Pho85-Pho80, and the phosphorylation sites are located within its N- and C-terminal unfolded regions. Phosphorylation of Pah1 by Pho85-Pho80 inhibited its degradation, extending its half-life by ∼2-fold. The dephosphorylation of endogenously phosphorylated Pah1 by the Nem1-Spo7 protein phosphatase, which is highly specific for the sites phosphorylated by Pho85-Pho80, stimulated the 20S proteasomal degradation and reduced its half-life by 2.6-fold. These results indicate that the proteolysis of Pah1 by the 20S proteasome is controlled by its phosphorylation state. PMID:25809482

  7. Phosphorylation regulates the ubiquitin-independent degradation of yeast Pah1 phosphatidate phosphatase by the 20S proteasome.

    PubMed

    Hsieh, Lu-Sheng; Su, Wen-Min; Han, Gil-Soo; Carman, George M

    2015-05-01

    Saccharomyces cerevisiae Pah1 phosphatidate phosphatase, which catalyzes the conversion of phosphatidate to diacylglycerol for triacylglycerol synthesis and simultaneously controls phosphatidate levels for phospholipid synthesis, is subject to the proteasome-mediated degradation in the stationary phase of growth. In this study, we examined the mechanism for its degradation using purified Pah1 and isolated proteasomes. Pah1 expressed in S. cerevisiae or Escherichia coli was not degraded by the 26S proteasome, but by its catalytic 20S core particle, indicating that its degradation is ubiquitin-independent. The degradation of Pah1 by the 20S proteasome was dependent on time and proteasome concentration at the pH optimum of 7.0. The 20S proteasomal degradation was conserved for human lipin 1 phosphatidate phosphatase. The degradation analysis using Pah1 truncations and its fusion with GFP indicated that proteolysis initiates at the N- and C-terminal unfolded regions. The folded region of Pah1, in particular the haloacid dehalogenase-like domain containing the DIDGT catalytic sequence, was resistant to the proteasomal degradation. The structural change of Pah1, as reflected by electrophoretic mobility shift, occurs through its phosphorylation by Pho85-Pho80, and the phosphorylation sites are located within its N- and C-terminal unfolded regions. Phosphorylation of Pah1 by Pho85-Pho80 inhibited its degradation, extending its half-life by ∼2-fold. The dephosphorylation of endogenously phosphorylated Pah1 by the Nem1-Spo7 protein phosphatase, which is highly specific for the sites phosphorylated by Pho85-Pho80, stimulated the 20S proteasomal degradation and reduced its half-life by 2.6-fold. These results indicate that the proteolysis of Pah1 by the 20S proteasome is controlled by its phosphorylation state. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Respiratory syncytial virus infection down-regulates antioxidant enzyme expression by triggering deacetylation-proteasomal degradation of Nrf2.

    PubMed

    Komaravelli, Narayana; Tian, Bing; Ivanciuc, Teodora; Mautemps, Nicholas; Brasier, Allan R; Garofalo, Roberto P; Casola, Antonella

    2015-11-01

    Respiratory syncytial virus (RSV) is the most important cause of viral acute respiratory tract infections and hospitalizations in children, for which no vaccine or treatment is available. RSV infection in cells, mice, and children leads to rapid generation of reactive oxygen species, which are associated with oxidative stress and lung damage, due to a significant decrease in the expression of airway antioxidant enzymes (AOEs). Oxidative stress plays an important role in the pathogenesis of RSV-induced lung disease, as antioxidants ameliorate clinical disease and inflammation in vivo. The aim of this study is to investigate the unknown mechanism(s) of virus-induced inhibition of AOE expression. RSV infection is shown to induce a progressive reduction in nuclear and total cellular levels of the transcription factor NF-E2-related factor 2 (Nrf2), resulting in decreased binding to endogenous AOE gene promoters and decreased AOE expression. RSV induces Nrf2 deacetylation and degradation via the proteasome pathway in vitro and in vivo. Histone deacetylase and proteasome inhibitors block Nrf2 degradation and increase Nrf2 binding to AOE endogenous promoters, resulting in increased AOE expression. Known inducers of Nrf2 are able to increase Nrf2 activation and subsequent AOE expression during RSV infection in vitro and in vivo, with significant amelioration of oxidative stress. This is the first study to investigate the mechanism(s) of virus-induced inhibition of AOE expression. RSV-induced inhibition of Nrf2 activation, due to deacetylation and proteasomal degradation, could be targeted for therapeutic intervention aimed to increase airway antioxidant capacity during infection. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Cytoplasmic Lipid Droplets Are Sites of Convergence of Proteasomal and Autophagic Degradation of Apolipoprotein B

    PubMed Central

    Cheng, Jinglei; Fujita, Akikazu; Tokumoto, Toshinobu

    2006-01-01

    Lipid esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes are used to synthesize very low-density lipoproteins (VLDLs), into which apolipoprotein B (ApoB) is integrated cotranslationally. In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make “ApoB-crescents.” ApoB-crescents were seen in <10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50–100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12–24 h of proteasomal inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two pathways. PMID:16597703

  10. Ubiquitin-proteasome dependent degradation of GABAAα1 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 GABAAα1 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 GABAAα1 protein levels and Lys48-linked polyubiquitination of GABAAα1, but reduced proteasome activity in mouse primary cortical neurons (DIV 14 from E16 embryos). Moreover, treatment with betulinic acid, a proteasome activator significantly decreased GABAAα1 protein levels in cortical neurons indicating the role of polyubiquitination of GABAAα1 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 GABAAα1 in the ER. The siRNA knockdown of SYVN1 significantly increased GABAAα1 protein levels in cortical neurons. Moreover, reduced association between SYVN1 and GABAAα1

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

    PubMed

    Crow, Marni S; Cristea, Ileana M

    2017-04-01

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

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

    PubMed

    Üstün, Suayib; Börnke, Frederik

    2015-05-01

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

  13. The Xanthomonas campestris Type III Effector XopJ Proteolytically Degrades Proteasome Subunit RPT61[OPEN

    PubMed Central

    2015-01-01

    Many animal and plant pathogenic bacteria inject type III effector (T3E) proteins into their eukaryotic host cells to suppress immunity. The Yersinia outer protein J (YopJ) family of T3Es is a widely distributed family of effector proteins found in both animal and plant pathogens, and its members are highly diversified in virulence functions. Some members have been shown to possess acetyltransferase activity; however, whether this is a general feature of YopJ family T3Es is currently unknown. The T3E Xanthomonas outer protein J (XopJ), a YopJ family effector from the plant pathogen Xanthomonas campestris pv vesicatoria, interacts with the proteasomal subunit Regulatory Particle AAA-ATPase6 (RPT6) in planta to suppress proteasome activity, resulting in the inhibition of salicylic acid-related immune responses. Here, we show that XopJ has protease activity to specifically degrade RPT6, leading to reduced proteasome activity in the cytoplasm as well as in the nucleus. Proteolytic degradation of RPT6 was dependent on the localization of XopJ to the plasma membrane as well as on its catalytic triad. Mutation of the Walker B motif of RPT6 prevented XopJ-mediated degradation of the protein but not XopJ interaction. This indicates that the interaction of RPT6 with XopJ is dependent on the ATP-binding activity of RPT6, but proteolytic cleavage additionally requires its ATPase activity. Inhibition of the proteasome impairs the proteasomal turnover of Nonexpressor of Pathogenesis-Related1 (NPR1), the master regulator of salicylic acid responses, leading to the accumulation of ubiquitinated NPR1, which likely interferes with the full induction of NPR1 target genes. Our results show that YopJ family T3Es are not only highly diversified in virulence function but also appear to possess different biochemical activities. PMID:25739698

  14. ARF regulates the stability of p16 protein via REGγ-dependent proteasome degradation.

    PubMed

    Kobayashi, Takashi; Wang, Jingqiang; Al-Ahmadie, Hikmat; Abate-Shen, Cory

    2013-08-01

    The cell-cycle regulatory gene INK4A-ARF (CDKN2A) has two alternative transcripts that produce entirely different proteins, namely p14(ARF) and p16, which have complementary functions as regulators of p53 and pRB tumor suppressor pathways, respectively. The unusual organization of INK4A-ARF has long led to speculation of a need for coordinated regulation of p14(ARF) and p16. We now show that p14(ARF) (ARF) regulates the stability of p16 protein in human cancer cell lines, as well as in mouse embryonic fibroblasts (MEFs). In particular, ARF promotes rapid degradation of p16 protein, which is mediated by the proteasome and, more specifically, by interaction of ARF with one of its subunits, REGγ. Furthermore, this ARF-dependent destabilization of p16 can be abrogated by knockdown of REGγ or by pharmacologic blockade of its nuclear export. Thus, our findings have uncovered a novel crosstalk of 2 key tumor suppressors mediated by a REGγ-dependent mechanism. The ability of ARF to control p16 stability may influence cell-cycle function. The ability of ARF to control p16 stability may influence cell cycle function. Visual Overview: http://mcr.aacrjournals.org/content/current. ©2013 AACR.

  15. Tripartite degrons confer diversity and specificity on regulated protein degradation in the ubiquitin-proteasome system.

    PubMed

    Guharoy, Mainak; Bhowmick, Pallab; Sallam, Mohamed; Tompa, Peter

    2016-01-06

    Specific signals (degrons) regulate protein turnover mediated by the ubiquitin-proteasome system. Here we systematically analyse known degrons and propose a tripartite model comprising the following: (1) a primary degron (peptide motif) that specifies substrate recognition by cognate E3 ubiquitin ligases, (2) secondary site(s) comprising a single or multiple neighbouring ubiquitinated lysine(s) and (3) a structurally disordered segment that initiates substrate unfolding at the 26S proteasome. Primary degron sequences are conserved among orthologues and occur in structurally disordered regions that undergo E3-induced folding-on-binding. Posttranslational modifications can switch primary degrons into E3-binding-competent states, thereby integrating degradation with signalling pathways. Degradation-linked lysines tend to be located within disordered segments that also initiate substrate degradation by effective proteasomal engagement. Many characterized mutations and alternative isoforms with abrogated degron components are implicated in disease. These effects result from increased protein stability and interactome rewiring. The distributed nature of degrons ensures regulation, specificity and combinatorial control of degradation.

  16. Tripartite degrons confer diversity and specificity on regulated protein degradation in the ubiquitin-proteasome system

    PubMed Central

    Guharoy, Mainak; Bhowmick, Pallab; Sallam, Mohamed; Tompa, Peter

    2016-01-01

    Specific signals (degrons) regulate protein turnover mediated by the ubiquitin-proteasome system. Here we systematically analyse known degrons and propose a tripartite model comprising the following: (1) a primary degron (peptide motif) that specifies substrate recognition by cognate E3 ubiquitin ligases, (2) secondary site(s) comprising a single or multiple neighbouring ubiquitinated lysine(s) and (3) a structurally disordered segment that initiates substrate unfolding at the 26S proteasome. Primary degron sequences are conserved among orthologues and occur in structurally disordered regions that undergo E3-induced folding-on-binding. Posttranslational modifications can switch primary degrons into E3-binding-competent states, thereby integrating degradation with signalling pathways. Degradation-linked lysines tend to be located within disordered segments that also initiate substrate degradation by effective proteasomal engagement. Many characterized mutations and alternative isoforms with abrogated degron components are implicated in disease. These effects result from increased protein stability and interactome rewiring. The distributed nature of degrons ensures regulation, specificity and combinatorial control of degradation. PMID:26732515

  17. 20S proteasome activation promotes life span extension and resistance to proteotoxicity in Caenorhabditis elegans.

    PubMed

    Chondrogianni, Niki; Georgila, Konstantina; Kourtis, Nikos; Tavernarakis, Nektarios; Gonos, Efstathios S

    2015-02-01

    Protein homeostasis (proteostasis) is one of the nodal points that need to be preserved to retain physiologic cellular/organismal balance. The ubiquitin-proteasome system (UPS) is responsible for the removal of both normal and damaged proteins, with the proteasome being the downstream effector. The proteasome is the major cellular protease with progressive impairment of function during aging and senescence. Despite the documented age-retarding properties of proteasome activation in various cellular models, simultaneous enhancement of the 20S core proteasome content, assembly, and function have never been reported in any multicellular organism. Consequently, the possible effects of the core proteasome modulation on organismal life span are elusive. In this study, we have achieved activation of the 20S proteasome at organismal level. We demonstrate enhancement of proteasome levels, assembly, and activity in the nematode Caenorhabditis elegans, resulting in life span extension and increased resistance to stress. We also provide evidence that the observed life span extension is dependent on the transcriptional activity of Dauer formation abnormal/Forkhead box class O (DAF-16/FOXO), skinhead-1 (SKN-1), and heat shock factor-1 (HSF-1) factors through regulation of downstream longevity genes. We further show that the reported beneficial effects are not ubiquitous but they are dependent on the genetic context. Finally, we provide evidence that proteasome core activation might be a potential strategy to minimize protein homeostasis deficiencies underlying aggregation-related diseases, such as Alzheimer's disease (AD) or Huntington's disease (HD). In summary, this is the first report demonstrating that 20S core proteasome up-regulation in terms of both content and activity is feasible in a multicellular eukaryotic organism and that in turn this modulation promotes extension of organismal health span and life span.

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

    PubMed Central

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

    2010-01-01

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

  19. APP and APLP1 are degraded through autophagy in response to proteasome inhibition in neuronal cells.

    PubMed

    Zhou, Fangfang; van Laar, Theo; Huang, Huizhe; Zhang, Long

    2011-05-01

    Amyloid beta (Aβ) precursor protein (APP) is a key protein in the pathogenesis of Alzheimer's disease (AD). Both APP and its paralogue APLP1 (amyloid beta precursor-like protein 1) have multiple functions in cell adhesion and proliferation. Previously it was thought that autophagy is a novel beta-amyloid peptide (Aβ)-generating pathway activated in AD. However, the protein proteolysis of APLP1 is still largely unknown. The present study shows that APLP1 is rapidly degraded in neuronal cells in response to stresses, such as proteasome inhibition. Activation of the endoplasmic reticulum (ER) stress by proteasome inhibitors induces autophagy, causing reduction of mature APLP1/APP. Blocking autophagy or JNK stress kinase rescues the protein expression for both APP and APLP1. Therefore, our results suggest that APP/APLP1 is degraded through autophagy and the APLP1 proteolysis is mainly mediated by autophagy-lysosome pathway.

  20. Degradation of mouse NTE-related esterase by macroautophagy and the proteasome.

    PubMed

    Chang, Ping-An; Chen, Yu-Ying; Long, Ding-Xin; Qin, Wen-Zhen; Mou, Xiao-Ling

    2012-06-01

    NTE-related esterase (NRE) is a novel endoplasmic reticulum-anchored lysophospholipase with high homology to neuropathy target esterase (NTE). However, little is known about the regulation of NRE protein. In the current study, we investigated the degradation pathways of mouse NRE (mNRE) in mammalian cells. Based on experiments with inhibitors and inducer of protein degradation pathways, we provide here the first evidence that mNRE is degraded by macroautophagy as well as by the proteasome. Moreover, the contribution of protein domains to the degradation of mNRE was investigated, which showed that the transmembrane and regulatory domain played a role in the degradation of mNRE by macroautophagy and the proteasome respectively. In contrast the C-terminal catalytic domain was not involved in both degradation pathways of mNRE. These findings showed for the first time that the degradation pathways in controlling mNRE quantity and may provide further insight into structure and regulation of mNRE.

  1. Molecular Design, Synthesis, and Evaluation of SNIPER(ER) That Induces Proteasomal Degradation of ERα.

    PubMed

    Okuhira, Keiichiro; Demizu, Yosuke; Hattori, Takayuki; Ohoka, Nobumichi; Shibata, Norihito; Kurihara, Masaaki; Naito, Mikihiko

    2016-01-01

    Manipulation of protein stability using small molecules has a great potential for both basic research and clinical therapy. Based on our protein knockdown technology, we recently developed a novel small molecule SNIPER(ER) that targets the estrogen receptor alpha (ERα) for degradation via the ubiquitin-proteasome system. This chapter describes the design and synthesis of SNIPER(ER) compounds, and methods for the evaluation of their activity in cellular system.

  2. Yeast Pah1p phosphatidate phosphatase is regulated by proteasome-mediated degradation.

    PubMed

    Pascual, Florencia; Hsieh, Lu-Sheng; Soto-Cardalda, Aníbal; Carman, George M

    2014-04-04

    Yeast PAH1-encoded phosphatidate phosphatase is the enzyme responsible for the production of the diacylglycerol used for the synthesis of triacylglycerol that accumulates in the stationary phase of growth. Paradoxically, the growth phase-mediated inductions of PAH1 and phosphatidate phosphatase activity do not correlate with the amount of Pah1p; enzyme abundance declined in a growth phase-dependent manner. Pah1p from exponential phase cells was a relatively stable protein, and its abundance was not affected by incubation with an extract from stationary phase cells. Recombinant Pah1p was degraded upon incubation with the 100,000 × g pellet fraction of stationary phase cells, although the enzyme was stable when incubated with the same fraction of exponential phase cells. MG132, an inhibitor of proteasome function, prevented degradation of the recombinant enzyme. Endogenously expressed and plasmid-mediated overexpressed levels of Pah1p were more abundant in the stationary phase of cells treated with MG132. Pah1p was stabilized in mutants with impaired proteasome (rpn4Δ, blm10Δ, ump1Δ, and pre1 pre2) and ubiquitination (hrd1Δ, ubc4Δ, ubc7Δ, ubc8Δ, and doa4Δ) functions. The pre1 pre2 mutations that eliminate nearly all chymotrypsin-like activity of the 20 S proteasome had the greatest stabilizing effect on enzyme levels. Taken together, these results supported the conclusion that Pah1p is subject to proteasome-mediated degradation in the stationary phase. That Pah1p abundance was stabilized in pah1Δ mutant cells expressing catalytically inactive forms of Pah1p and dgk1Δ mutant cells with induced expression of DGK1-encoded diacylglycerol kinase indicated that alteration in phosphatidate and/or diacylglycerol levels might be the signal that triggers Pah1p degradation.

  3. Yeast Pah1p Phosphatidate Phosphatase Is Regulated by Proteasome-mediated Degradation*

    PubMed Central

    Pascual, Florencia; Hsieh, Lu-Sheng; Soto-Cardalda, Aníbal; Carman, George M.

    2014-01-01

    Yeast PAH1-encoded phosphatidate phosphatase is the enzyme responsible for the production of the diacylglycerol used for the synthesis of triacylglycerol that accumulates in the stationary phase of growth. Paradoxically, the growth phase-mediated inductions of PAH1 and phosphatidate phosphatase activity do not correlate with the amount of Pah1p; enzyme abundance declined in a growth phase-dependent manner. Pah1p from exponential phase cells was a relatively stable protein, and its abundance was not affected by incubation with an extract from stationary phase cells. Recombinant Pah1p was degraded upon incubation with the 100,000 × g pellet fraction of stationary phase cells, although the enzyme was stable when incubated with the same fraction of exponential phase cells. MG132, an inhibitor of proteasome function, prevented degradation of the recombinant enzyme. Endogenously expressed and plasmid-mediated overexpressed levels of Pah1p were more abundant in the stationary phase of cells treated with MG132. Pah1p was stabilized in mutants with impaired proteasome (rpn4Δ, blm10Δ, ump1Δ, and pre1 pre2) and ubiquitination (hrd1Δ, ubc4Δ, ubc7Δ, ubc8Δ, and doa4Δ) functions. The pre1 pre2 mutations that eliminate nearly all chymotrypsin-like activity of the 20 S proteasome had the greatest stabilizing effect on enzyme levels. Taken together, these results supported the conclusion that Pah1p is subject to proteasome-mediated degradation in the stationary phase. That Pah1p abundance was stabilized in pah1Δ mutant cells expressing catalytically inactive forms of Pah1p and dgk1Δ mutant cells with induced expression of DGK1-encoded diacylglycerol kinase indicated that alteration in phosphatidate and/or diacylglycerol levels might be the signal that triggers Pah1p degradation. PMID:24563465

  4. VRK1 regulates Cajal body dynamics and protects coilin from proteasomal degradation in cell cycle

    PubMed Central

    Cantarero, Lara; Sanz-García, Marta; Vinograd-Byk, Hadar; Renbaum, Paul; Levy-Lahad, Ephrat; Lazo, Pedro A.

    2015-01-01

    Cajal bodies (CBs) are nuclear organelles associated with ribonucleoprotein functions and RNA maturation. CBs are assembled on coilin, its main scaffold protein, in a cell cycle dependent manner. The Ser-Thr VRK1 (vaccinia-related kinase 1) kinase, whose activity is also cell cycle regulated, interacts with and phosphorylates coilin regulating assembly of CBs. Coilin phosphorylation is not necessary for its interaction with VRK1, but it occurs in mitosis and regulates coilin stability. Knockdown of VRK1 or VRK1 inactivation by serum deprivation causes a loss of coilin phosphorylation in Ser184 and of CBs formation, which are rescued with an active VRK1, but not by kinase-dead VRK1. The phosphorylation of coilin in Ser184 occurs during mitosis before assembly of CBs. Loss of coilin phosphorylation results in disintegration of CBs, and of coilin degradation that is prevented by proteasome inhibitors. After depletion of VRK1, coilin is ubiquitinated in nuclei, which is partly mediated by mdm2, but its proteasomal degradation occurs in cytosol and is prevented by blocking its nuclear export. We conclude that VRK1 is a novel regulator of CBs dynamics and stability in cell cycle by protecting coilin from ubiquitination and degradation in the proteasome, and propose a model of CB dynamics. PMID:26068304

  5. Geniposide accelerates proteasome degradation of Txnip to inhibit insulin secretion in pancreatic β-cells.

    PubMed

    Liu, C Y; Hao, Y N; Yin, F; Zhang, Y L; Liu, J H

    2017-05-01

    To analyze the role of geniposide in the protein degradation of Txnip and to determine the impact of Txnip on geniposide-regulated GSIS in pancreatic INS-1 cells. The content of Txnip protein was measured by western blot; insulin content and glucose uptake were determined by ELISA; and knockdown of Txnip was the method of RNA interference. Glucose induces a rapid increase in Txnip protein, and geniposide accelerates the degradation of Txnip via proteasome pathway in the presence of high glucose (25 mM) in INS-1 pancreatic β-cells. And MG132, a proteasomal inhibitor, potentiates glucose uptake, metabolism (ATP production) and glucose-stimulated insulin secretion (GSIS) in high-glucose (25 mM)-treated INS-1 cells, but geniposide significantly prevents these effects. Furthermore, the combination of geniposide and Txnip knockdown shows substantial synergistic effects to reduce glucose uptake, metabolism and GSIS in high-glucose (25 mM)-treated INS-1 cells. Txnip protein played an essential role in glucose uptake, metabolism and GSIS, and geniposide could accelerate the degradation via proteasome pathway in high-glucose-treated pancreatic INS-1 cells.

  6. Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers.

    PubMed

    Khmelinskii, Anton; Meurer, Matthias; Ho, Chi-Ting; Besenbeck, Birgit; Füller, Julia; Lemberg, Marius K; Bukau, Bernd; Mogk, Axel; Knop, Michael

    2016-01-15

    Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs.

  7. Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers

    PubMed Central

    Khmelinskii, Anton; Meurer, Matthias; Ho, Chi-Ting; Besenbeck, Birgit; Füller, Julia; Lemberg, Marius K.; Bukau, Bernd; Mogk, Axel; Knop, Michael

    2016-01-01

    Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs. PMID:26609072

  8. VRK1 regulates Cajal body dynamics and protects coilin from proteasomal degradation in cell cycle.

    PubMed

    Cantarero, Lara; Sanz-García, Marta; Vinograd-Byk, Hadar; Renbaum, Paul; Levy-Lahad, Ephrat; Lazo, Pedro A

    2015-06-12

    Cajal bodies (CBs) are nuclear organelles associated with ribonucleoprotein functions and RNA maturation. CBs are assembled on coilin, its main scaffold protein, in a cell cycle dependent manner. The Ser-Thr VRK1 (vaccinia-related kinase 1) kinase, whose activity is also cell cycle regulated, interacts with and phosphorylates coilin regulating assembly of CBs. Coilin phosphorylation is not necessary for its interaction with VRK1, but it occurs in mitosis and regulates coilin stability. Knockdown of VRK1 or VRK1 inactivation by serum deprivation causes a loss of coilin phosphorylation in Ser184 and of CBs formation, which are rescued with an active VRK1, but not by kinase-dead VRK1. The phosphorylation of coilin in Ser184 occurs during mitosis before assembly of CBs. Loss of coilin phosphorylation results in disintegration of CBs, and of coilin degradation that is prevented by proteasome inhibitors. After depletion of VRK1, coilin is ubiquitinated in nuclei, which is partly mediated by mdm2, but its proteasomal degradation occurs in cytosol and is prevented by blocking its nuclear export. We conclude that VRK1 is a novel regulator of CBs dynamics and stability in cell cycle by protecting coilin from ubiquitination and degradation in the proteasome, and propose a model of CB dynamics.

  9. Glucose Deprivation Triggers Protein Kinase C-dependent β-Catenin Proteasomal Degradation*

    PubMed Central

    Choi, Seung-Won; Song, Jun-Kyu; Yim, Ye-Seal; Yun, Ho-Geun; Chun, Kyung-Hee

    2015-01-01

    Autophagy is a conserved process that contributes to cell homeostasis. It is well known that induction mainly occurs in response to nutrient starvation, such as starvation of amino acids and insulin, and its mechanisms have been extensively characterized. However, the mechanisms behind cellular glucose deprivation-induced autophagy are as of now poorly understood. In the present study, we determined a mechanism by which glucose deprivation induced the PKC-dependent proteasomal degradation of β-catenin, leading to autophagy. Glucose deprivation was shown to cause a sub-G1 transition and enhancement of the LC3-II protein levels, whereas β-catenin protein underwent degradation in a proteasome-dependent manner. Moreover, the inhibition of GSK3β was unable to abolish the glucose deprivation-mediated β-catenin degradation or up-regulation of LC3-II protein levels, which suggested GSK3β-independent protein degradation. Intriguingly, the inhibition of PKCα using a pharmacological inhibitor and transfection of siRNA for PKCα was observed to effectively block glucose deprivation-induced β-catenin degradation as well as the increase in LC3-II levels and the accumulation of a sub-G1 population. Together, our results demonstrated a molecular mechanism by which glucose deprivation can induce the GSK3β-independent protein degradation of β-catenin, leading to autophagy. PMID:25691573

  10. Oridonin Triggers Chaperon-mediated Proteasomal Degradation of BCR-ABL in Leukemia

    PubMed Central

    Huang, Huilin; Weng, Hengyou; Dong, Bowen; Zhao, Panpan; Zhou, Hui; Qu, Lianghu

    2017-01-01

    Inducing degradation of oncoproteins by small molecule compounds has the potential to avoid drug resistance and therefore deserves to be exploited for new therapies. Oridonin is a natural compound with promising antitumor efficacy that can trigger the degradation of oncoproteins; however, the direct cellular targets and underlying mechanisms remain unclear. Here we report that oridonin depletes BCR-ABL through chaperon-mediated proteasomal degradation in leukemia. Mechanistically, oridonin poses oxidative stress in cancer cells and directly binds to cysteines of HSF1, leading to the activation of this master regulator of the chaperone system. The resulting induction of HSP70 and ubiquitin proteins and the enhanced binding to CHIP E3 ligase hence target BCR-ABL for ubiquitin-proteasome degradation. Both wild-type and mutant forms of BCR-ABL can be efficiently degraded by oridonin, supporting its efficacy observed in cultured cells as well as mouse tumor xenograft assays with either imatinib-sensitive or -resistant cells. Collectively, our results identify a novel mechanism by which oridonin induces rapid degradation of BCR-ABL as well as a novel pharmaceutical activator of HSF1 that represents a promising treatment for leukemia. PMID:28128329

  11. Chaperone-dependent E3 ligase CHIP ubiquitinates and mediates proteasomal degradation of soluble guanylyl cyclase.

    PubMed

    Xia, Tian; Dimitropoulou, Christiana; Zeng, Jingmin; Antonova, Galina N; Snead, Connie; Venema, Richard C; Fulton, David; Qian, Shuibing; Patterson, Cam; Papapetropoulos, Andreas; Catravas, John D

    2007-11-01

    The nitric oxide receptor soluble guanylyl cyclase (sGC) exists in multimeric protein complexes, including heat shock protein (HSP) 90 and endothelial nitric oxide synthase. Inhibition of HSP90 by geldanamycin causes proteasomal degradation of sGC protein. In this study, we have investigated whether COOH terminus of heat shock protein 70-interacting protein (CHIP), a co-chaperone molecule that is involved in protein folding but is also a chaperone-dependent ubiquitin E3 ligase, could play a role in the process of degradation of sGC. Transient overexpression of CHIP in COS-7 cells degraded heterologous sGC in a concentration-related manner; this downregulation of sGC was abrogated by the proteasome inhibitor MG-132. Transfection of tetratricopeptide repeats and U-box domain CHIP mutants attenuated sGC degradation, suggesting that both domains are indispensable for CHIP function. Results from immunoprecipitation and indirect immunofluorescent microscopy experiments demonstrated that CHIP is associated with sGC, HSP90, and HSP70 in COS-7 cells. Furthermore, CHIP increased the association of HSP70 with sGC. In in vitro ubiquitination assays using purified proteins and ubiquitin enzymes, E3 ligase CHIP directly ubiquitinated sGC; this ubiquitination was potentiated by geldanamycin in COS-7 cells, followed by proteasomal degradation. In rat aortic smooth muscle cells, endogenous sGC was also degraded by adenovirus-infected wild-type CHIP but not by the chaperone interaction-deficient K30A CHIP, whereas CHIP, but not K30A, attenuated sGC expression in, and nitric oxide donor-induced relaxation of, rat aortic rings, suggesting that CHIP plays a regulatory role under physiological conditions. This study reveals a new mechanism for the regulation of sGC, an important mediator of cellular and vascular function.

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

    PubMed Central

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

    2013-01-01

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

  13. Cullin 7 mediates proteasomal and lysosomal degradations of rat Eag1 potassium channels

    PubMed Central

    Hsu, Po-Hao; Ma, Yu-Ting; Fang, Ya-Ching; Huang, Jing-Jia; Gan, Yu-Ling; Chang, Pei-Tzu; Jow, Guey-Mei; Tang, Chih-Yung; Jeng, Chung-Jiuan

    2017-01-01

    Mammalian Eag1 (Kv10.1) potassium (K+) channels are widely expressed in the brain. Several mutations in the gene encoding human Eag1 K+ channel have been associated with congenital neurodevelopmental anomalies. Currently very little is known about the molecules mediating protein synthesis and degradation of Eag1 channels. Herein we aim to ascertain the protein degradation mechanism of rat Eag1 (rEag1). We identified cullin 7 (Cul7), a member of the cullin-based E3 ubiquitin ligase family, as a novel rEag1 binding partner. Immunoprecipitation analyses confirmed the interaction between Cul7 and rEag1 in heterologous cells and neuronal tissues. Cul7 and rEag1 also exhibited significant co-localization at synaptic regions in neurons. Over-expression of Cul7 led to reduced protein level, enhanced ubiquitination, accelerated protein turn-over, and decreased current density of rEag1 channels. We provided further biochemical and morphological evidence suggesting that Cul7 targeted endoplasmic reticulum (ER)- and plasma membrane-localized rEag1 to the proteasome and the lysosome, respectively, for protein degradation. Cul7 also contributed to protein degradation of a disease-associated rEag1 mutant. Together, these results indicate that Cul7 mediates both proteasomal and lysosomal degradations of rEag1. Our findings provide a novel insight to the mechanisms underlying ER and peripheral protein quality controls of Eag1 channels. PMID:28098200

  14. CaMKII regulates proteasome phosphorylation and activity and promotes memory destabilization following retrieval.

    PubMed

    Jarome, Timothy J; Ferrara, Nicole C; Kwapis, Janine L; Helmstetter, Fred J

    2016-02-01

    Numerous studies have suggested that memories "destabilize" and require de novo protein synthesis in order to reconsolidate following retrieval, but very little is known about how this destabilization process is regulated. Recently, ubiquitin-proteasome mediated protein degradation has been identified as a critical regulator of memory trace destabilization following retrieval, though the specific mechanisms controlling retrieval-induced changes in ubiquitin-proteasome activity remain equivocal. Here, we found that proteasome activity is increased in the amygdala in a CaMKII-dependent manner following the retrieval of a contextual fear memory. We show that in vitro inhibition of CaMKII reversed retrieval-induced increases in proteasome activity. Additionally, in vivo pharmacological blockade of CaMKII abolished increases in proteolytic activity and activity related regulatory phosphorylation in the amygdala following retrieval, suggesting that CaMKII was "upstream" of protein degradation during the memory reconsolidation process. Consistent with this, while inhibiting CaMKII in the amygdala did not impair memory following retrieval, it completely attenuated the memory impairments that resulted from post-retrieval protein synthesis blockade. Collectively, these results suggest that CaMKII controls the initiation of the memory reconsolidation process through regulation of the proteasome.

  15. 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; Nishida, Tamotsu; Takahashi, Shigeru Takahashi, Yuji

    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.

  16. Proteasome activator subunit 3 promotes pancreatic cancer growth via c-Myc-glycolysis signaling axis.

    PubMed

    Guo, Jiefang; Hao, Jun; Jiang, Hongxue; Jin, Jing; Wu, Hongyu; Jin, Zhendong; Li, Zhaoshen

    2017-02-01

    Pancreatic cancer has the worst prognosis among all cancers and novel markers and therapeutic targets are desperately needed for this terribly deadly disease. Proteasome activator subunit 3 (PSME3) is highly involved in the initiation and progression of many human cancers. However, the potential effect of PSME3 on pancreatic cancer remains largely unknown. In the present study, we first found that PSME3 was significantly upregulated in pancreatic cancer cells and tissues at both mRNA and protein levels using qRT-PCR, western blot analysis, Oncomine data mining and immunohistochemical analysis. High PSME3 expression was positively correlated with tumor size and pM stage, and was significantly correlated with poor prognosis in pancreatic cancer patients revealed by Kaplan-Meier analysis. Gene set enrichment analysis demonstrated that the gene sets related to cell proliferation and metastasis were positively correlated with elevated PSME3 expression. Consistently, silencing of PSME3 suppressed cell proliferation and invasive capacity of pancreatic cancer. Mechanistically, PSME3 inhibited the degradation of c-Myc and thus enhanced glycolysis, which ultimately led to the oncogenic effects of PSME3 on pancreatic cancer. Collectively, our data suggest that PSME3 plays oncogenic roles in pancreatic cancer by inhibiting c-Myc degradation to promote glycolysis, and could serve as a novel therapeutic target for pancreatic cancer treatment. Copyright © 2016. Published by Elsevier Ireland Ltd.

  17. Retinoblastoma protein co-purifies with proteasomal insulin-degrading enzyme: Implications for cell proliferation control

    SciTech Connect

    Radulescu, Razvan T.; Duckworth, William C.; Levy, Jennifer L.; Fawcett, Janet

    2010-04-30

    Previous investigations on proteasomal preparations containing insulin-degrading enzyme (IDE; EC 3.4.24.56) have invariably yielded a co-purifying protein with a molecular weight of about 110 kDa. We have now found both in MCF-7 breast cancer and HepG2 hepatoma cells that this associated molecule is the retinoblastoma tumor suppressor protein (RB). Interestingly, the amount of RB in this protein complex seemed to be lower in HepG2 vs. MCF-7 cells, indicating a higher (cytoplasmic) protein turnover in the former vs. the latter cells. Moreover, immunofluorescence showed increased nuclear localization of RB in HepG2 vs. MCF-7 cells. Beyond these subtle differences between these distinct tumor cell types, our present study more generally suggests an interplay between RB and IDE within the proteasome that may have important growth-regulatory consequences.

  18. Proteasome inhibitors promote the sequestration of PrPSc into aggresomes within the cytosol of prion-infected CAD neuronal cells.

    PubMed

    Dron, Michel; Dandoy-Dron, Françoise; Farooq Salamat, Muhammad Khalid; Laude, Hubert

    2009-08-01

    Dysfunction of the endoplasmic reticulum associated protein degradation/proteasome system is believed to contribute to the initiation or aggravation of neurodegenerative disorders associated with protein misfolding, and there is some evidence to suggest that proteasome dysfunctions might be implicated in prion disease. This study investigated the effect of proteasome inhibitors on the biogenesis of both the cellular (PrP(C)) and abnormal (PrP(Sc)) forms of prion protein in CAD neuronal cells, a newly introduced prion cell system. In uninfected cells, proteasome impairment altered the intracellular distribution of PrP(C), leading to a strong accumulation in the Golgi apparatus. Moreover, a detergent-insoluble and weakly protease-resistant PrP species of 26 kDa, termed PrP(26K), accumulated in the cells, whether they were prion-infected or not. However, no evidence was found that, in infected cells, this PrP(26K) species converts into the highly proteinase K-resistant PrP(Sc). In the infected cultures, proteasome inhibition caused an increased intracellular aggregation of PrP(Sc) that was deposited into large aggresomes. These findings strengthen the view that, in neuronal cells expressing wild-type PrP(C) from the natural promoter, proteasomal impairment may affect both the process of PrP(C) biosynthesis and the subcellular sites of PrP(Sc) accumulation, despite the fact that these two effects could essentially be disconnected.

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

    SciTech Connect

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

    2014-08-08

    Highlights: • Protein stability of Rbfox3 splice isoforms is differentially regulated. • Rbfox3-d31, an Rbfox3 isoform lacking the RRM, is highly susceptible to degradation. • The protein stability of Rbfox3-d31 is regulated by the ubiquitin–proteasome pathway. • Rbfox3-d31 inhibits the nuclear localization of Rbfox2. • Rbfox3-d31 inhibits the splicing activity of Rbfox2. - Abstract: 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.

  20. Molecular shredders: how proteasomes fulfill their role.

    PubMed

    Groll, Michael; Clausen, Tim

    2003-12-01

    The 20S proteasome is a large, cylinder-shaped protease that is found in all domains of life and plays a crucial role in cellular protein turnover. It has multiple catalytic centers located within the hollow cavity of a molecular cage. This architecture prevents unwanted degradation of endogenous proteins and promotes processive degradation of substrates by restricting the dissociation of partially digested polypeptides. Although this kind of self-compartmentalization is generally conserved, the proteasomes of bacteria, archaea and eukaryotes show many differences in architecture, subunit composition and regulation. The structure of the 20S proteasome and its inherent role in the regulation of proteasome function are gradually being elucidated.

  1. A nuclear ubiquitin-proteasome pathway targets the inner nuclear membrane protein Asi2 for degradation.

    PubMed

    Boban, Mirta; Pantazopoulou, Marina; Schick, Anna; Ljungdahl, Per O; Foisner, Roland

    2014-08-15

    The nuclear envelope consists of inner and outer nuclear membranes. Whereas the outer membrane is an extension of the endoplasmic reticulum, the inner nuclear membrane (INM) represents a unique membranous environment containing specific proteins. The mechanisms of integral INM protein degradation are unknown. Here, we investigated the turnover of Asi2, an integral INM protein in Saccharomyces cerevisiae. We report that Asi2 is degraded by the proteasome independently of the vacuole and that it exhibited a half-life of ∼45 min. Asi2 exhibits enhanced stability in mutants lacking the E2 ubiquitin conjugating enzymes Ubc6 or Ubc7, or the E3 ubiquitin ligase Doa10. Consistent with these data, Asi2 is post-translationally modified by poly-ubiquitylation in a Ubc7- and Doa10-dependent manner. Importantly Asi2 degradation is significantly reduced in a sts1-2 mutant that fails to accumulate proteasomes in the nucleus, indicating that Asi2 is degraded in the nucleus. Our results reveal a molecular pathway that affects the stability of integral proteins of the inner nuclear membrane and indicate that Asi2 is subject to protein quality control in the nucleus. © 2014. Published by The Company of Biologists Ltd.

  2. The Role of the Ubiquitin Proteasome Pathway in Keratin Intermediate Filament Protein Degradation

    PubMed Central

    Rogel, Micah R.; Jaitovich, Ariel; Ridge, Karen M.

    2010-01-01

    Lung injury, whether caused by hypoxic or mechanical stresses, elicits a variety of responses at the cellular level. Alveolar epithelial cells respond and adapt to such injurious stimuli by reorganizing the cellular cytoskeleton, mainly accomplished through modification of the intermediate filament (IF) network. The structural and mechanical integrity in epithelial cells is maintained through this adaptive reorganization response. Keratin, the predominant IF expressed in epithelial cells, displays highly dynamic properties in response to injury, sometimes in the form of degradation of the keratin IF network. Post-translational modification, such as phosphorylation, targets keratin proteins for degradation in these circumstances. As with other structural and regulatory proteins, turnover of keratin is regulated by the ubiquitin (Ub)-proteasome pathway. The degradation process begins with activation of Ub by the Ub-activating enzyme (E1), followed by the exchange of Ub to the Ub-conjugating enzyme (E2). E2 shuttles the Ub molecule to the substrate-specific Ub ligase (E3), which then delivers the Ub to the substrate protein, thereby targeting it for degradation. In some cases of injury and IF-related disease, aggresomes form in epithelial cells. The mechanisms that regulate aggresome formation are currently unknown, although proteasome overload may play a role. Therefore, a more complete understanding of keratin degradation—causes, mechanisms, and consequences—will allow for a greater understanding of epithelial cell biology and lung pathology alike. PMID:20160151

  3. UV Irradiation Triggers Cylindromatosis Translocation, Modification, and Degradation in a Proteasome-Independent Manner.

    PubMed

    Zhou, Ping; Hao, Ziwei; Wang, Xincheng; Gao, Jinmin; Li, Dengwen; Xie, Songbo; Zhang, Tong-Cun

    2016-03-01

    The tumor suppressor, cylindromatosis (CYLD), is a negative regulator of NF-κB signaling by removing lysine 63-linked ubiquitin chains from multiple NF-κB signaling components, including TRAF2, TRAF6, and NEMO. How CYLD itself is regulated, however, remains yet to be characterized. In this study, we present the first evidence that UV irradiation is able to induce CYLD translocation from the cytoplasm to microtubules and that the cytoskeleton-associated CYLD is subject to posttranslational modification and degradation in a proteasome-independent manner. By immunostaining, we found that CYLD displayed microtubule-like filament localization under ultraviolet (UV) irradiation. Further studies revealed that the cytoskeleton-associated CYLD underwent posttranslational modification, which in turn contributed to CYLD degradation in an unknown manner, distinct from proteasome-mediated degradation under normal conditions. Collectively, our data suggest that UV-induced CYLD degradation might serve as an underlying mechanism for UV-induced NF-κB pathway activation.

  4. Ribosomal proteins produced in excess are degraded by the ubiquitin–proteasome system

    PubMed Central

    Sung, Min-Kyung; Reitsma, Justin M.; Sweredoski, Michael J.; Hess, Sonja; Deshaies, Raymond J.

    2016-01-01

    Ribosome assembly is an essential process that consumes prodigious quantities of cellular resources. Ribosomal proteins cannot be overproduced in Saccharomyces cerevisiae because the excess proteins are rapidly degraded. However, the responsible quality control (QC) mechanisms remain poorly characterized. Here we demonstrate that overexpression of multiple proteins of the small and large yeast ribosomal subunits is suppressed. Rpl26 overexpressed from a plasmid can be detected in the nucleolus and nucleoplasm, but it largely fails to assemble into ribosomes and is rapidly degraded. However, if the endogenous RPL26 loci are deleted, plasmid-encoded Rpl26 assembles into ribosomes and localizes to the cytosol. Chemical and genetic perturbation studies indicate that overexpressed ribosomal proteins are degraded by the ubiquitin–proteasome system and not by autophagy. Inhibition of the proteasome led to accumulation of multiple endogenous ribosomal proteins in insoluble aggregates, consistent with the operation of this QC mechanism in the absence of ribosomal protein overexpression. Our studies reveal that ribosomal proteins that fail to assemble into ribosomes are rapidly distinguished from their assembled counterparts and ubiquitinated and degraded within the nuclear compartment. PMID:27385339

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

    DOE PAGES

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

    2015-05-21

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

  6. Lysine 269 is essential for cyclin D1 ubiquitylation by the SCFFbx4/αB-crystallin ligase and subsequent proteasome-dependent degradation

    PubMed Central

    O., Barbash; E., Egan; L.L., Pontano; J., Kosak; Diehl, J. Alan

    2009-01-01

    Protein ubiquitylation is a complex enzymatic process that results in the covalent attachment of ubiquitin, via Gly-76 of ubiquitin, to an ε-NH2-group of an internal lysine residue in a given substrate. While E3 ligases frequently utilize lysines adjacent to the degron within the substrate, many substrates can be targeted to the proteasome via polyubiquitylation of any lysine. We have assessed the role of lysine residues proximal to the cyclin D1 phosphodegron for ubiquitylation by the SCFFbx4/αB-crystallin ubiquitin ligase and subsequent proteasome-dependent degradation of cyclin D1. The work described herein reveals a requisite role for Lys-269 (K269) for the rapid, poly-ubiquitin mediated degradation of cyclin D1. Mutation of lysine 269, which is proximal to the phosphodegron sequence surrounding Thr-286 in cyclin D1, not only stabilizes cyclin D1, but also triggers cyclin D1 accumulation within the nucleus thereby promoting cell transformation. In addition, D1-K269R is resistant to genotoxic stress induced degradation, similar to non-phosphorylatable D1-T286A, supporting the critical role for the post-translational regulation of cyclin D1 in the response to DNA damaging agents. Strikingly, while mutation of lysine 269 to arginine inhibits cyclin D1 degradation, it does not inhibit cyclin D1 ubiquitylation in vivo demonstrating that ubiquitylation of a specific lysine can influence substrate targeting to the 26S proteasome. PMID:19767775

  7. Mouse Mammary Tumor Virus Signal Peptide Uses a Novel p97-Dependent and Derlin-Independent Retrotranslocation Mechanism To Escape Proteasomal Degradation

    PubMed Central

    Byun, Hyewon; Das, Poulami; Yu, Houqing; Aleman, Alejandro; Lozano, Mary M.; Matouschek, Andreas

    2017-01-01

    ABSTRACT Multiple pathogens, including viruses and bacteria, manipulate endoplasmic reticulum-associated degradation (ERAD) to avoid the host immune response and promote their replication. The betaretrovirus mouse mammary tumor virus (MMTV) encodes Rem, which is a precursor protein that is cleaved into a 98-amino-acid signal peptide (SP) and a C-terminal protein (Rem-CT). SP uses retrotranslocation for ER membrane extraction and yet avoids ERAD by an unknown mechanism to enter the nucleus and function as a Rev-like protein. To determine how SP escapes ERAD, we used a ubiquitin-activated interaction trap (UBAIT) screen to trap and identify transient protein interactions with SP, including the ERAD-associated p97 ATPase, but not E3 ligases or Derlin proteins linked to retrotranslocation, polyubiquitylation, and proteasomal degradation of extracted proteins. A dominant negative p97 ATPase inhibited both Rem and SP function. Immunoprecipitation experiments indicated that Rem, but not SP, is polyubiquitylated. Using both yeast and mammalian expression systems, linkage of a ubiquitin-like domain (UbL) to SP or Rem induced degradation by the proteasome, whereas SP was stable in the absence of the UbL. ERAD-associated Derlin proteins were not required for SP activity. Together, these results suggested that Rem uses a novel p97-dependent, Derlin-independent retrotranslocation mechanism distinct from other pathogens to avoid SP ubiquitylation and proteasomal degradation. PMID:28351922

  8. Inhibition of histone deacetylases by chlamydocin induces apoptosis and proteasome-mediated degradation of survivin.

    PubMed

    De Schepper, Stefanie; Bruwiere, Hélène; Verhulst, Tinne; Steller, Ulf; Andries, Luc; Wouters, Walter; Janicot, Michel; Arts, Janine; Van Heusden, Jim

    2003-02-01

    The naturally occurring cyclic tetrapeptide chlamydocin is a very potent inhibitor of cell proliferation. Here we show that chlamydocin is a highly potent histone deacetylase (HDAC) inhibitor, inhibiting HDAC activity in vitro with an IC(50) of 1.3 nM. Like other HDAC inhibitors, chlamydocin induces the accumulation of hyperacetylated histones H3 and H4 in A2780 ovarian cancer cells, increases the expression of p21(cip1/waf1), and causes an accumulation of cells in G(2)/M phase of the cell cycle. In addition, chlamydocin induces apoptosis by activating caspase-3, which in turn leads to the cleavage of p21(cip1/waf1) into a 15-kDa breakdown product and drives cells from growth arrest into apoptosis. Concomitant with the activation of caspase-3 and cleavage of p21(cip1/waf1), chlamydocin decreases the protein level of survivin, a member of the inhibitor of apoptosis protein family that is selectively expressed in tumors. Although our data indicate a potential link between degradation of survivin and activation of the apoptotic pathway induced by HDAC inhibitors, stable overexpression of survivin does not suppress the activation of caspase-3 or cleavage of p21(cip1/waf1) induced by chlamydocin treatment. The decrease of survivin protein level is mediated by degradation via proteasomes since it can be inhibited by specific proteasome inhibitors. Taken together, our results show that induction of apoptosis by chlamydocin involves caspase-dependent cleavage of p21(cip1/waf1), which is strikingly associated with proteasome-mediated degradation of survivin.

  9. cMyBP-C was decreased via KLHL3-mediated proteasomal degradation in congenital heart diseases.

    PubMed

    Wang, Leitong; Lai, Guangrui; Chu, Guoming; Liang, Xiaoyan; Zhao, Yanyan

    2017-03-15

    Cardiac myosin binding protein C (cMyBP-C) is a cardiac structural and regulatory protein; mutations of cMyBP-C are frequently associated with hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Cardiac special transcription factors may regulate the expression of cMyBP-C. However, the role of cMyBP-C in congenital heart diseases (CHD) remains poorly understood. In the current study, western blotting and the MRM approach showed that cMyBP-C expression was significantly reduced in fetuses with CHD compared to those without. Furthermore, we found that cMyBP-C interacted with KLHL3 by immunoprecipitation and immunofluorescence, and the degradation of cMyBP-C was caused by KLHL3-mediated ubiquitination. In addition, homocysteine (Hcy, a risk factor of CHD) treatment caused a decrease in cMyBP-C and an increase in KLHL3 expression, and the proteasome inhibitor MG132 reversed the Hcy-induced reduction of cMyBP-C expression. Finally, we verified that reduced cMyBP-C by Hcy promoted apoptosis in cardiomyocytes. These results demonstrate that Hcy decreases the expression of cMyBP-C through a KLHL3-mediated ubiquitin-proteasome pathway, and thereby influences heart development.

  10. Berberine Suppresses Cyclin D1 Expression through Proteasomal Degradation in Human Hepatoma Cells

    PubMed Central

    Wang, Ning; Wang, Xuanbin; Tan, Hor-Yue; Li, Sha; Tsang, Chi Man; Tsao, Sai-Wah; Feng, Yibin

    2016-01-01

    The aim of this study is to explore the underlying mechanism on berberine-induced Cyclin D1 degradation in human hepatic carcinoma. We observed that berberine could suppress both in vitro and in vivo expression of Cyclin D1 in hepatoma cells. Berberine exhibits dose- and time-dependent inhibition on Cyclin D1 expression in human hepatoma cell HepG2. Berberine increases the phosphorylation of Cyclin D1 at Thr286 site and potentiates Cyclin D1 nuclear export to cytoplasm for proteasomal degradation. In addition, berberine recruits the Skp, Cullin, F-box containing complex-β-Transducin Repeat Containing Protein (SCFβ-TrCP) complex to facilitate Cyclin D1 ubiquitin-proteasome dependent proteolysis. Knockdown of β-TrCP blocks Cyclin D1 turnover induced by berberine; blocking the protein degradation induced by berberine in HepG2 cells increases tumor cell resistance to berberine. Our results shed light on berberine′s potential as an anti-tumor agent for clinical cancer therapy. PMID:27854312

  11. Berberine Suppresses Cyclin D1 Expression through Proteasomal Degradation in Human Hepatoma Cells.

    PubMed

    Wang, Ning; Wang, Xuanbin; Tan, Hor-Yue; Li, Sha; Tsang, Chi Man; Tsao, Sai-Wah; Feng, Yibin

    2016-11-15

    The aim of this study is to explore the underlying mechanism on berberine-induced Cyclin D1 degradation in human hepatic carcinoma. We observed that berberine could suppress both in vitro and in vivo expression of Cyclin D1 in hepatoma cells. Berberine exhibits dose- and time-dependent inhibition on Cyclin D1 expression in human hepatoma cell HepG2. Berberine increases the phosphorylation of Cyclin D1 at Thr286 site and potentiates Cyclin D1 nuclear export to cytoplasm for proteasomal degradation. In addition, berberine recruits the Skp, Cullin, F-box containing complex-β-Transducin Repeat Containing Protein (SCF(β-TrCP)) complex to facilitate Cyclin D1 ubiquitin-proteasome dependent proteolysis. Knockdown of β-TrCP blocks Cyclin D1 turnover induced by berberine; blocking the protein degradation induced by berberine in HepG2 cells increases tumor cell resistance to berberine. Our results shed light on berberine's potential as an anti-tumor agent for clinical cancer therapy.

  12. Heat Shock Protein 70 Regulates Degradation of the Mumps Virus Phosphoprotein via the Ubiquitin-Proteasome Pathway

    PubMed Central

    Kubota, Toru; Kita, Shunsuke; Nakatsu, Yuichiro; Aoki, Natsuko; Mori, Yoshio; Maenaka, Katsumi; Takeda, Makoto; Kidokoro, Minoru

    2014-01-01

    ABSTRACT Mumps virus (MuV) infection induces formation of cytoplasmic inclusion bodies (IBs). Growing evidence indicates that IBs are the sites where RNA viruses synthesize their viral RNA. However, in the case of MuV infection, little is known about the viral and cellular compositions and biological functions of the IBs. In this study, pulldown purification and N-terminal amino acid sequencing revealed that stress-inducible heat shock protein 70 (Hsp72) was a binding partner of MuV phosphoprotein (P protein), which was an essential component of the IB formation. Immunofluorescence and immunoblotting analyses revealed that Hsp72 was colocalized with the P protein in the IBs, and its expression was increased during MuV infection. Knockdown of Hsp72 using small interfering RNAs (siRNAs) had little, if any, effect on viral propagation in cultured cells. Knockdown of Hsp72 caused accumulation of ubiquitinated P protein and delayed P protein degradation. These results show that Hsp72 is recruited to IBs and regulates the degradation of MuV P protein through the ubiquitin-proteasome pathway. IMPORTANCE Formation of cytoplasmic inclusion bodies (IBs) is a common characteristic feature in mononegavirus infections. IBs are considered to be the sites of viral RNA replication and transcription. However, there have been few studies focused on host factors recruited to the IBs and their biological functions. Here, we identified stress-inducible heat shock protein 70 (Hsp72) as the first cellular partner of mumps virus (MuV) phosphoprotein (P protein), which is an essential component of the IBs and is involved in viral RNA replication/transcription. We found that the Hsp72 mobilized to the IBs promoted degradation of the MuV P protein through the ubiquitin-proteasome pathway. Our data provide new insight into the role played by IBs in mononegavirus infection. PMID:25552722

  13. Docosahexaenoic acid-mediated protein aggregates may reduce proteasome activity and delay myotube degradation during muscle atrophy in vitro

    PubMed Central

    Shin, Seung Kyun; Kim, Ji Hyeon; Lee, Jung Hoon; Son, Young Hoon; Lee, Min Wook; Kim, Hak Joong; Noh, Sue Ah; Kim, Kwang Pyo; Kim, In-Gyu; Lee, Min Jae

    2017-01-01

    Proteasomes are the primary degradation machinery for oxidatively damaged proteins that compose a class of misfolded protein substrates. Cellular levels of reactive oxygen species increase with age and this cellular propensity is particularly harmful when combined with the age-associated development of various human disorders including cancer, neurodegenerative disease and muscle atrophy. Proteasome activity is reportedly downregulated in these disease conditions. Herein, we report that docosahexaenoic acid (DHA), a major dietary omega-3 polyunsaturated fatty acid, mediates intermolecular protein cross-linkages through oxidation, and the resulting protein aggregates potently reduce proteasomal activity both in vitro and in cultured cells. Cellular models overexpressing aggregation-prone proteins such as tau showed significantly elevated levels of tau aggregates and total ubiquitin conjugates in the presence of DHA, thereby reflecting suppressed proteasome activity. Strong synergetic cytotoxicity was observed when the cells overexpressing tau were simultaneously treated with DHA. Antioxidant N-acetyl cysteine significantly desensitized the cells to DHA-induced oxidative stress. DHA significantly delayed the proteasomal degradation of muscle proteins in a cellular atrophy model. Thus, the results of our study identified DHA as a potent inducer of cellular protein aggregates that inhibit proteasome activity and potentially delay systemic muscle protein degradation in certain pathologic conditions. PMID:28104914

  14. Enhanced Degradation of Misfolded Proteins Promotes Tumorigenesis.

    PubMed

    Chen, Liang; Brewer, Michael D; Guo, Lili; Wang, Ruoxing; Jiang, Peng; Yang, Xiaolu

    2017-03-28

    An adequate cellular capacity to degrade misfolded proteins is critical for cell survival and organismal health. A diminished capacity is associated with aging and neurodegenerative diseases; however, the consequences of an enhanced capacity remain undefined. Here, we report that the ability to clear misfolded proteins is increased during oncogenic transformation and is reduced upon tumor cell differentiation. The augmented capacity mitigates oxidative stress associated with oncogenic growth and is required for both the initiation and maintenance of malignant phenotypes. We show that tripartite motif-containing (TRIM) proteins select misfolded proteins for proteasomal degradation. The higher degradation power in tumor cells is attributed to the upregulation of the proteasome and especially TRIM proteins, both mediated by the antioxidant transcription factor Nrf2. These findings establish a critical role of TRIMs in protein quality control, connect the clearance of misfolded proteins to antioxidant defense, and suggest an intrinsic characteristic of tumor cells.

  15. CHIP stabilizes amyloid precursor protein via proteasomal degradation and p53-mediated trans-repression of β-secretase.

    PubMed

    Singh, Amir Kumar; Pati, Uttam

    2015-08-01

    In patient with Alzheimer's disease (AD), deposition of amyloid-beta Aβ, a proteolytic cleavage of amyloid precursor protein (APP) by β-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 Aβ 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 Aβ. 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. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  16. CHIP stabilizes amyloid precursor protein via proteasomal degradation and p53-mediated trans-repression of β-secretase

    PubMed Central

    Singh, Amir Kumar; Pati, Uttam

    2015-01-01

    In patient with Alzheimer’s disease (AD), deposition of amyloid-beta Aβ, a proteolytic cleavage of amyloid precursor protein (APP) by β-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 Aβ 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 Aβ. CHIPUbox 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

  17. Dietary apigenin potentiates the inhibitory effect of interferon-α on cancer cell viability through inhibition of 26S proteasome-mediated interferon receptor degradation

    PubMed Central

    Li, Sheng; Yang, Li-juan; Wang, Ping; He, Yu-jiao; Huang, Jun-mei; Liu, Han-wei; Shen, Xiao-fei; Wang, Fei

    2016-01-01

    Background Type I interferons (IFN-α/β) have broad and potent immunoregulatory and antiproliferative activities. However, it is still known whether the dietary flavonoids exhibit their antiviral and anticancer properties by modulating the function of type I IFNs. Objective This study aimed at determining the role of apigenin, a dietary plant flavonoid abundant in common fruits and vegetables, on the type I IFN-mediated inhibition of cancer cell viability. Design Inhibitory effect of apigenin on human 26S proteasome, a known negative regulator of type I IFN signaling, was evaluated in vitro. Molecular docking was conducted to know the interaction between apigenin and subunits of 26S proteasome. Effects of apigenin on JAK/STAT pathway, 26S proteasome-mediated interferon receptor stability, and cancer cells viability were also investigated. Results Apigenin was identified to be a potent inhibitor of human 26S proteasome in a cell-based assay. Apigenin inhibited the chymotrypsin-like, caspase-like, and trypsin-like activities of the human 26S proteasome and increased the ubiquitination of endogenous proteins in cells. Results from computational modeling of the potential interactions of apigenin with the chymotrypsin site (β5 subunit), caspase site (β1 subunit), and trypsin site (β2 subunit) of the proteasome were consistent with the observed proteasome inhibitory activity. Apigenin enhanced the phosphorylation of signal transducer and activator of transcription proteins (STAT1 and STAT2) and promoted the endogenous IFN-α-regulated gene expression. Apigenin inhibited the IFN-α-stimulated ubiquitination and degradation of type I interferon receptor 1 (IFNAR1). Apigenin also sensitized the inhibitory effect of IFN-α on viability of cervical carcinoma HeLa cells. Conclusion These results suggest that apigenin potentiates the inhibitory effect of IFN-α on cancer cell viability by activating JAK/STAT signaling pathway through inhibition of 26S proteasome

  18. A splice variant of the human phosphohistidine phosphatase 1 (PHPT1) is degraded by the proteasome.

    PubMed

    Inturi, Raviteja; Wäneskog, Marcus; Vlachakis, Dimitrios; Ali, Yeasmeen; Ek, Pia; Punga, Tanel; Bjerling, Pernilla

    2014-12-01

    Regulation of protein activity by phosphorylation is central in many cellular processes. Phosphorylation of serine, threonine and tyrosine residues is well documented and studied. In addition, other amino acids, like histidine can be phosphorylated, but neither the mechanism nor the function of this modification is well understood. Nevertheless, there is a 14 kDa enzyme with phosphohistidine phosphatase activity, named PHPT1, found in most animals, but not in bacteria, plant or fungi. There are a few splice variant transcripts formed from the human PHPT1 locus and some of them are predicted to form variant proteins, but studies of these proteins are lacking. In order to get insight into the possible function of the variant transcripts encoded at the PHPT1 locus, ectopic expression of PHPT1 transcript variant 6, predicted to be degraded by the non-sense mediated mRNA decay pathway, in HeLa cells was undertaken. In HeLa cells the splice variant protein was degraded by the proteasome, unlike the wild type protein. Using an in silico modeling approach the variant C-terminal end of the proteins were predicted to form different secondary structures that might explain the different properties of the two proteins. The specific degradation of the PHPT1 splice variant indicates that at least for the PHPT1 protein, the quality control and the self-guarding of the cellular system works at two levels, first at the RNA level, aberrant transcripts are degraded by the non-sense mediated mRNA decay pathway, and the small amount of proteins that are formed will be degraded by the proteasome.

  19. Hepatitis C virus core protein inhibits E6AP expression via DNA methylation to escape from ubiquitin-dependent proteasomal degradation.

    PubMed

    Kwak, Juri; Shim, Joo Hee; Tiwari, Indira; Jang, Kyung Lib

    2016-09-28

    The E6-associated protein (E6AP) is a ubiquitin ligase that mediates ubiquitination and proteasomal degradation of hepatitis C virus (HCV) core protein. Given the role of HCV core protein as a major component of the viral nucleocapsid, as well as a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis, HCV has likely evolved a strategy to counteract the host anti-viral defense mechanism of E6AP and maximize its potential to produce infectious virus particles. In the present study, we found that HCV core protein derived from either ectopic expression or HCV infection inhibits E6AP expression via promoter hypermethylation in human hepatocytes. As a result, the potential of E6AP to ubiquitinate and degrade HCV core protein through the ubiquitin-proteasome system was severely impaired, which in turn led to stimulation of virus propagation. The effects of HCV core protein were almost completely abolished when the E6AP level was restored by ectopic expression of E6AP, treatment with a universal DNA methyltransferase (DNMT) inhibitor, 5-Aza-2'dC, or knock-down of DNMT1. In conclusion, HCV core protein inhibits E6AP expression via DNA methylation to protect itself from ubiquitin-dependent proteasomal degradation and stimulate virus propagation, providing a potential target for the development of anti-viral drugs against HCV. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  20. Two waves of proteasome-dependent protein degradation in the hippocampus are required for recognition memory consolidation.

    PubMed

    Figueiredo, Luciana S; Dornelles, Arethuza S; Petry, Fernanda S; Falavigna, Lucio; Dargél, Vinicius A; Köbe, Luiza M; Aguzzoli, Cristiano; Roesler, Rafael; Schröder, Nadja

    2015-04-01

    Healthy neuronal function and synaptic modification require a concert of synthesis and degradation of proteins. Increasing evidence indicates that protein turnover mediated by proteasome activity is involved in long-term synaptic plasticity and memory. However, its role in different phases of memory remains debated, and previous studies have not examined the possible requirement of protein degradation in recognition memory. Here, we show that the proteasome inhibitor, lactacystin (LAC), infused into the CA1 area of the hippocampus at two specific time points during consolidation, impairs 24-retention of memory for object recognition in rats. Administration of LAC after retrieval did not affect retention. These findings provide the first evidence for a requirement of proteasome activity in recognition memory, indicate that protein degradation in the hippocampus is necessary during selective time windows of memory consolidation, and further our understanding of the role of protein turnover in memory formation. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Geldanamycin-induced degradation of Chk1 is mediated by proteasome

    SciTech Connect

    Nomura, M.; E-mail: nomura413jp@yahoo.co.jp; Nomura, N.; Yamashita, J.

    2005-09-30

    Checkpoint kinase 1 (Chk1) is a cell cycle regulator and a heat shock protein 90 (Hsp90) client. It is essential for cell proliferation and survival. In this report, we analyzed the mechanisms of Chk1 regulation in U87MG glioblastoma cells using Geldanamycin (GA), which interferes with the function of Hsp90. GA reduced Chk1 protein level but not its mRNA level in glioblastoma cells. Co-treatment with GA and cycloheximide (CHX), a protein synthesis inhibitor, induced a decrease of half-life of the Chk1 protein to 3 h and resulted in Chk1 down-regulation. CHX alone induced only 32% reduction of Chk1 protein even after 24 h. These findings indicated that reduction of Chk1 by GA was due to destabilization and degradation of the protein. In addition, GA-induced down-regulation of Chk1 was reversed by MG132, a specific proteasome inhibitor. And it was revealed that Chk1 was ubiquitinated by GA. These results have indicated that degradation of Chk1 by GA was mediated by the ubiquitin-proteasome pathway in U87MG glioblastoma cells.

  2. Ikaros is degraded by proteasome-dependent mechanism in the early phase of apoptosis induction

    SciTech Connect

    He, Li-Cai; Xu, Han-Zhang; Gu, Zhi-Min; Liu, Chuan-Xu; Chen, Guo-Qiang; Wang, Yue-Fei; Wen, Dong-Hua; Wu, Ying-Li

    2011-03-18

    Research highlights: {yields} Chemotherapeutic drugs or UV treatment reduces Ikaros prior to caspase-3 activation. {yields} Etoposide treatment does not alter the mRNA but shortens the half-life of Ikaros. {yields} MG132 or epoxomicin but not calpeptin inhibits etoposide-induced Ikaros degradation. {yields} Overexpression of Ikaros accelerates etoposide-induced apoptosis in NB4 cells. -- Abstract: Ikaros is an important transcription factor involved in the development and differentiation of hematopoietic cells. In this work, we found that chemotherapeutic drugs or ultraviolet radiation (UV) treatment could reduce the expression of full-length Ikaros (IK1) protein in less than 3 h in leukemic NB4, Kasumi-1 and Jurkat cells, prior to the activation of caspase-3. Etoposide treatment could not alter the mRNA level of IK1 but it could shorten the half-life of IK1. Co-treatment with the proteasome inhibitor MG132 or epoxomicin but not calpain inhibitor calpeptin inhibited etoposide-induced Ikaros downregulation. Overexpression of IK1 could accelerate etoposide-induced apoptosis in NB4 cells, as evidenced by the increase of Annexin V positive cells and the more early activation of caspase 3. To our knowledge, this is the first report to show that upon chemotherapy drugs or UV treatment, IK1 could be degraded via the proteasome system in the early phase of apoptosis induction. These data might shed new insight on the role of IK1 in apoptosis and the post-translational regulation of IK1.

  3. Ubiquitin-proteasomal degradation of COX-2 in TGF-β stimulated human endometrial cells is mediated through endoplasmic reticulum mannosidase I.

    PubMed

    Singh, Mohan; Chaudhry, Parvesh; Parent, Sophie; Asselin, Eric

    2012-01-01

    Cyclooxygenase (COX)-2 is a key regulatory enzyme in the production of prostaglandins (PG) during various physiological processes. Mechanisms of COX-2 regulation in human endometrial stromal cells (human endometrial stromal cells) are not fully understood. In this study, we investigate the role of TGF-β in the regulation of COX-2 in human uterine stromal cells. Each TGF-β isoform decreases COX-2 protein level in human uterine stromal cells in Smad2/3-dependent manner. The decrease in COX-2 is accompanied by a decrease in PG synthesis. Knockdown of Smad4 using specific small interfering RNA prevents the decrease in COX-2 protein, confirming that Smad pathway is implicated in the regulation of COX-2 expression in human endometrial stromal cells. Pretreatment with 26S proteasome inhibitor, MG132, significantly restores COX-2 protein and PG synthesis, indicating that COX-2 undergoes proteasomal degradation in the presence of TGF-β. In addition, each TGF-β isoform up-regulates endoplasmic reticulum (ER)-mannosidase I (ERManI) implying that COX-2 degradation is mediated through ER-associated degradation pathway in these cells. Furthermore, inhibition of ERManI activity using the mannosidase inhibitor (kifunensine), or small interfering RNA-mediated knockdown of ERManI, prevents TGF-β-induced COX-2 degradation. Taken together, these studies suggest that TGF-β promotes COX-2 degradation in a Smad-dependent manner by up-regulating the expression of ERManI and thereby enhancing ER-associated degradation and proteasomal degradation pathways.

  4. Valproic Acid Causes Proteasomal Degradation of DICER and Influences miRNA Expression

    PubMed Central

    Zhang, Zhaiyi; Convertini, Paolo; Shen, Manli; Xu, Xiu; Lemoine, Frédéric; de la Grange, Pierre; Andres, Douglas A.; Stamm, Stefan

    2013-01-01

    Valproic acid (VPA) is a commonly used drug to treat epilepsy and bipolar disorders. Known properties of VPA are inhibitions of histone deacetylases and activation of extracellular signal regulated kinases (ERK), which cannot fully explain VPA’s clinical features. We found that VPA induces the proteasomal degradation of DICER, a key protein in the generation of micro RNAs. Unexpectedly, the concentration of several micro RNAs increases after VPA treatment, which is caused by the upregulation of their hosting genes prior to DICER degradation. The data suggest that a loss of DICER protein and changes in micro RNA concentration contributes to the clinical properties of VPA. VPA can be used experimentally to down regulate DICER protein levels, which likely reflects a natural regulation of DICER. PMID:24358235

  5. Inhibition of store-operated calcium entry by sub-lethal levels of proteasome inhibition is associated with STIM1/STIM2 degradation.

    PubMed

    Kuang, Xiu-Li; Liu, Yimei; Chang, Yuhua; Zhou, Jing; Zhang, He; Li, Yiping; Qu, Jia; Wu, Shengzhou

    2016-04-01

    Dysfunction of the ubiquitin-proteasome system (UPS) and calcium homeostasis has been implicated in the neurodegeneration of Alzheimer's and Parkinson's diseases. The cytosolic calcium concentration is maintained by store-operated calcium entry (SOCE), which is repressed by Alzheimer's disease-associated mutants, such as mutant presenilins. We hypothesized that inhibition of UPS impacts SOCE. This study showed that pretreatment with sub-lethal levels of proteasome inhibitors, including MG-132 and clasto-lactacystin-β-lactone (LA), reduced SOCE after depletion of endoplasmic reticulum calcium in rat neurons. With the same treatment, MG-132 and LA reduced the protein levels of stromal interaction molecule 1and 2 (STIM1/2), but not the levels of Orai1 and canonical transient receptor potential channel 1 (TRPC1). STIM1 or STIM2 protein was mobilized to lysosome by MG-132/LA treatment as observed under an immunofluorescence confocal laser microscope. In the neurons, MG-132 and LA degraded p62/SQSTM1, promoted autophagy, converted LC3I to LC3II, and promoted co-localization of LC3 and lysosomes. Rapamycin, which enhances autophagy, reduced STIM1/2 protein levels, whereas bafilomycin, which inhibits autophagy, increased their protein levels. The protein levels of STIM1/2 and the amplitude of SOCE were decreased in SH-SY5Y with decreased protein level of proteasome subunit beta type-5 induced by shRNA. We conclude that sub-lethal levels of proteasome inhibition reduce SOCE and promote autophagy-mediated degradation of STIM1/2. UPS inhibition, a common finding in neurodegenerative diseases, interferes with calcium homeostasis via repression of SOCE. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Histone deacetylase 4 promotes ubiquitin-dependent proteasomal degradation of Sp3 in SH-SY5Y cells treated with di(2-ethylhexyl)phthalate (DEHP), determining neuronal death

    SciTech Connect

    Guida, Natascia; Laudati, Giusy; Galgani, Mario; Santopaolo, Marianna; Montuori, Paolo; Triassi, Maria; Di Renzo, Gianfranco; Canzoniero, Lorella M.T.; Formisano, Luigi

    2014-10-01

    Phthalates, phthalic acid esters, are widely used as plasticizers to produce polymeric materials in industrial production of plastics and daily consumable products. Animal studies have shown that di(2-ethylhexyl)phthalate (DEHP) may cause toxic effects in the rat brain. In the present study, chronic exposure to DEHP (0.1–100 μM) caused dose-dependent cell death via the activation of caspase-3 in neuroblastoma cells. Intriguingly, this harmful effect was prevented by the pan-histone deacetylase (HDAC) inhibitor trichostatin A, by the class II HDAC inhibitor MC-1568, but not by the class I HDAC inhibitor MS-275. Furthermore, DEHP reduced specificity protein 3 (Sp3) gene expression, but not Sp3 mRNA, after 24 and 48 h exposures. However, Sp3 protein reduction was prevented by pre-treatment with MC-1568, suggesting the involvement of class II HDACs in causing this effect. Then, we investigated the possible relationship between DEHP-induced neuronal death and the post-translational mechanisms responsible for the down-regulation of Sp3. Interestingly, DEHP-induced Sp3 reduction was associated to its deacetylation and polyubiquitination. Co-immunoprecipitation studies showed that Sp3 physically interacted with HDAC4 after DEHP exposure, while HDAC4 inhibition by antisense oligodeoxynucleotide reverted the DEHP-induced degradation of Sp3. Notably, Sp3 overexpression was able to counteract the detrimental effect induced by DEHP. Taken together, these results suggest that DEHP exerts its toxic effect by inducing deacetylation of Sp3 via HDAC4, and afterwards, Sp3-polyubiquitination. - Highlights: • Di(2-ethylhexyl)phthalate (DEHP) is cytotoxic to SH-SY5Y cells and cortical neurons. • DEHP-induced cytotoxicity is mediated by apoptosis. • DEHP-induced apoptotic cell death is inhibited by class II HDAC MC-1568. • DEHP neurotoxicity is caused by HDAC4-mediated Sp3 degradation by ubiquitin.

  7. Endoplasmic Reticulum-associated Degradation of Pca1p, a Polytopic Protein, via Interaction with the Proteasome at the Membrane.

    PubMed

    Smith, Nathan; Adle, David J; Zhao, Miaoyun; Qin, Xiaojuan; Kim, Heejeong; Lee, Jaekwon

    2016-07-15

    Endoplasmic reticulum-associated degradation (ERAD) plays a critical role in the destruction of terminally misfolded proteins at the secretory pathway. The system also regulates expression levels of several proteins such as Pca1p, a cadmium exporter in yeast. To gain better insight into the mechanisms underlying ERAD of Pca1p and other polytopic proteins by the proteasome in the cytosol, our study determined the roles for the molecular factors of ERAD in dislodging Pca1p from the endoplasmic reticulum (ER). Inactivation of the 20S proteasome leads to accumulation of ubiquitinated Pca1p in the ER membrane, suggesting a role for the proteasome in extraction of Pca1p from the ER. Pca1p formed a complex with the proteasome at the membrane in a Doa10p E3 ligase-dependent manner. Cdc48p is required for recruiting the proteasome to Pca1p. Although the Ufd2p E4 ubiquitin chain extension enzyme is involved in efficient degradation of Pca1p, Ufd2p-deficient cells did not affect the formation of a complex between Pca1p and the proteasome. Two other polytopic membrane proteins undergoing ERAD, Ste6*p and Hmg2p, also displayed the same outcomes observed for Pca1p. However, poly-ubiquitinated Cpy1*p, a luminal ERAD substrate, was detected in the cytosol independent of proteolytic activities of the proteasome. These results indicate that extraction and degradation of polytopic membrane proteins at the ER is a coupled event. This mechanism would relieve the cost of exposed hydrophobic domains in the cytosol during ERAD. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. PRESERVING MITOCHONDRIAL FUNCTION PREVENTS THE PROTEASOMAL DEGRADATION OF GTP CYCLOHYDROLASE I

    PubMed Central

    SHARMA, SHRUTI; SUN, XUTONG; KUMAR, SANJIV; RAFIKOV, RUSLAN; ARAMBURO, ANGELA; KALKAN, GOKHAN; TIAN, JING; REHMANI, IMRAN; KALLARACKAL, SUPHIN; FINEMAN, JEFFERY R.; BLACK, STEPHEN M.

    2012-01-01

    The development of pulmonary hypertension is a common accompaniment of congenital heart disease (CHD) with increased pulmonary blood flow. Our recent evidence suggests that asymmetric dimethylarginine (ADMA)-induced mitochondrial dysfunction causes endothelial nitric oxide synthase (eNOS) uncoupling secondary to a proteasome-dependent degradation of GTP cyclohydrolase I (GCH1) that results in a decrease in the NOS co-factor, tetrahydrobiopterin (BH4). Decreases in NO signaling are thought to be an early hallmark of endothelial dysfunction. As L-carnitine plays an important role in maintaining mitochondrial function in this study we examined the protective mechanisms and the therapeutic potential of L-carnitine on NO signaling in pulmonary arterial endothelial cells (PAEC) and in a lamb model of CHD and increased pulmonary blood flow (Shunt). Acetyl L-carnitine (ALC) attenuated the ADMA-mediated proteasomal degradation of GCH1. This preservation was associated with a decrease in the association of GCH1 with the Hsp70 and the C-terminus of Hsp70-interacting protein (CHIP) and a decrease in its ubiquitination. This in turn prevented the decrease in BH4 levels induced by ADMA and preserved NO signaling. Treatment of Shunt lambs with L-carnitine also reduced GCH1/CHIP interactions, attenuated the ubiquitination and degradation of GCH1, and increased BH4 levels compared to vehicle treated Shunt lambs. The increases in BH4 were associated with decreased NOS uncoupling and enhanced NO generation. Thus, we conclude that L-carnitine may have a therapeutic potential in the treatment of pulmonary hypertension in children with CHD with increased pulmonary blood flow. PMID:22583703

  9. Natural mutations lead to enhanced proteasomal degradation of human Ncb5or, a novel flavoheme reductase.

    PubMed

    Kálmán, Fanni S; Lizák, Beáta; Nagy, Szilvia K; Mészáros, Tamás; Zámbó, Veronika; Mandl, József; Csala, Miklós; Kereszturi, Eva

    2013-07-01

    NADH cytochrome b5 oxidoreductase (Ncb5or) protects β-cells against oxidative stress and lipotoxicity. The predominant phenotype of lean Ncb5or-null mouse is insulin-dependent diabetes due to β-cell death. This suggests the putative role of NCB5OR polymorphism in human diabetes. Therefore, we aimed to investigate the effect of natural missense mutations on the expression of human NCB5OR. Protein and mRNA levels of five non-synonymous coding variants were analyzed in transfected HEK293 and HepG2 cells. Although the mRNA levels were only slightly affected by the mutations, the amount of Ncb5or protein was largely reduced upon two Glu to Gly replacements in the third exon (p.E87G, p.E93G). These two mutations remarkably and synergistically shortened the half-life of Ncb5or and their effect could be attenuated by proteasome inhibitors. Our results strongly indicate that p.E87G, p.E93G mutations lead to enhanced proteasomal degradation due to manifest conformational alterations in the b5 domain. These data provide first evidence for natural mutations in NCB5OR gene resulting in decreased protein levels and hence having potential implications in human pathology. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  10. REGγ regulates ERα degradation via ubiquitin–proteasome pathway in breast cancer

    SciTech Connect

    Chai, Fan; Liang, Yan; Bi, Jiong; Chen, Li; Zhang, Fan; Cui, Youhong; Jiang, Jun

    2015-01-02

    Highlights: • High expression of REGγ is correlated with ERα status and poor clinical features. • Cell growth, mobility and invasion are significantly impaired by REGγ knockdown. • REGγ indirectly regulates ERα protein expression. - Abstract: REGγ is a proteasome coactivator which regulates proteolytic activity in eukaryotic cells. Abundant lines of evidence have showed that REGγ is over expressed in a number of human carcinomas. However, its precise role in the pathogenesis of cancer is still unclear. In this study, by examining 200 human breast cancer specimens, we demonstrated that REGγ was highly expressed in breast cancers, and the expression of REGγ was positively correlated with breast cancer patient estrogen receptor alpha (ERα) status. Moreover, the expression of REGγ was found positively associated with poor clinical features and low survival rates in ERα positive breast cancer patients. Further cell culture studies using MCF7 and BT474 breast cancer cell lines showed that cell proliferation, motility, and invasion capacities were decreased significantly by REGγ knockdown. Lastly, we demonstrated that REGγ indirectly regulates the degradation of ERα protein via ubiquitin–proteasome pathway. In conclusion, our findings provide the evidence that REGγ expression was positively correlated with ERα status and poor clinical prognosis in ERα positive breast cancer patients. As well, we disclose a new connection between the two molecules that are both highly expressed in most breast cancer cases.

  11. Regulation of mIκBNS stability through PEST-mediated degradation by proteasome

    SciTech Connect

    Park, Koog Chan; Jeong, Jiyeong; Kim, Keun Il

    2014-01-24

    Highlights: • mIκBNS is degraded rapidly by proteasome without ubiquitylation. • N-terminal PEST sequence is responsible for the unstable nature of mIκBNS. • PEST sequence is not critical for nuclear localization of mIκBNS. • There is single bona fide NLS at the C-terminus of mIκBNS. - Abstract: Negative regulatory proteins in a cytokine signaling play a critical role in restricting unwanted excess activation of the signaling pathway. At the same time, negative regulatory proteins need to be removed rapidly from cells to respond properly to the next incoming signal. A nuclear IκB protein called IκBNS is known to inhibit a subset of NF-κB target genes upon its expression by NF-κB activation. Here, we show a mechanism to control the stability of mIκBNS which might be important for cells to prepare the next round signaling. We found that mIκBNS is a short-lived protein of which the stability is controlled by proteasome, independent of ubiquitylation process. We identified that the N-terminal PEST sequence in mIκBNS was critical for the regulation of stability.

  12. Attenuation of glucocorticoid signaling through targeted degradation of p300 via the 26S proteasome pathway.

    PubMed

    Li, Qiao; Su, Anna; Chen, Jihong; Lefebvre, Yvonne A; Haché, Robert J G

    2002-12-01

    The effects of acetylation on gene expression are complex, with changes in chromatin accessibility intermingled with direct effects on transcriptional regulators. For the nuclear receptors, both positive and negative effects of acetylation on specific gene transcription have been observed. We report that p300 and steroid receptor coactivator 1 interact transiently with the glucocorticoid receptor and that the acetyltransferase activity of p300 makes an important contribution to glucocorticoid receptor-mediated transcription. Treatment of cells with the deacetylase inhibitor, sodium butyrate, inhibited steroid-induced transcription and altered the transient association of glucocorticoid receptor with p300 and steroid receptor coactivator 1. Additionally, sustained sodium butyrate treatment induced the degradation of p300 through the 26S proteasome pathway. Treatment with the proteasome inhibitor MG132 restored both the level of p300 protein and the transcriptional response to steroid over 20 h of treatment. These results reveal new levels for the regulatory control of gene expression by acetylation and suggest feedback control on p300 activity.

  13. Quantitative assessment of the degradation of aggregated TDP-43 mediated by the ubiquitin proteasome system and macroautophagy.

    PubMed

    Cascella, Roberta; Fani, Giulia; Capitini, Claudia; Rusmini, Paola; Poletti, Angelo; Cecchi, Cristina; Chiti, Fabrizio

    2017-08-25

    Amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions are neurodegenerative disorders that share the cytosolic deposition of TDP-43 (TAR DNA-binding protein 43) in the CNS. TDP-43 is well known as being actively degraded by both the proteasome and macroautophagy. The well-documented decrease in the efficiency of these clearance systems in aging and neurodegeneration, as well as the genetic evidence that many of the familial forms of TDP-43 proteinopathies involve genes that are associated with them, suggest that a failure of these protein degradation systems is a major factor that contributes to the onset of TDP-43-associated disorders. Here, we inserted preformed human TDP-43 aggregates in the cytosol of murine NSC34 and N2a cells in diffuse form and observed their degradation under conditions in which exogenous TDP-43 is not expressed and endogenous nuclear TDP-43 is not recruited, thereby allowing a time zero to be established in TDP-43 degradation and to observe its disposal kinetically and analytically. TDP-43 degradation was observed in the absence and presence of selective inhibitors and small interfering RNAs against the proteasome and autophagy. We found that cytosolic diffuse aggregates of TDP-43 can be distinguished in 3 different classes on the basis of their vulnerability to degradation, which contributed to the definition-with previous reports-of a total of 6 distinct classes of misfolded TDP-43 species that range from soluble monomer to undegradable macroaggregates. We also found that the proteasome and macroautophagy-degradable pools of TDP-43 are fully distinguishable, rather than in equilibrium between them on the time scale required for degradation, and that a significant crosstalk exists between the 2 degradation processes.-Cascella, R., Fani, G., Capitini, C., Rusmini, P., Poletti, A., Cecchi, C., Chiti, F. Quantitative assessment of the degradation of aggregated TDP-43 mediated by the ubiquitin

  14. Involvement of protein degradation by the ubiquitin proteasome system in opiate addictive behaviors.

    PubMed

    Massaly, Nicolas; Dahan, Lionel; Baudonnat, Mathieu; Hovnanian, Caroline; Rekik, Khaoula; Solinas, Marcello; David, Vincent; Pech, Stéphane; Zajac, Jean-Marie; Roullet, Pascal; Mouledous, Lionel; Frances, Bernard

    2013-03-01

    Plastic changes in the nucleus accumbens (NAcc), a structure occupying a key position in the neural circuitry related to motivation, are among the critical cellular processes responsible for drug addiction. During the last decade, it has been shown that memory formation and related neuronal plasticity may rely not only on protein synthesis but also on protein degradation by the ubiquitin proteasome system (UPS). In this study, we assess the role of protein degradation in the NAcc in opiate-related behaviors. For this purpose, we coupled behavioral experiments to intra-accumbens injections of lactacystin, an inhibitor of the UPS. We show that protein degradation in the NAcc is mandatory for a full range of animal models of opiate addiction including morphine locomotor sensitization, morphine conditioned place preference, intra-ventral tegmental area morphine self-administration and intra-venous heroin self-administration but not for discrimination learning rewarded by highly palatable food. This study provides the first evidence of a specific role of protein degradation by the UPS in addiction.

  15. Degradation Signals for Ubiquitin-Proteasome Dependent Cytosolic Protein Quality Control (CytoQC) in Yeast.

    PubMed

    Maurer, Matthew J; Spear, Eric D; Yu, Allen T; Lee, Evan J; Shahzad, Saba; Michaelis, Susan

    2016-07-07

    Cellular protein quality control (PQC) systems selectively target misfolded or otherwise aberrant proteins for degradation by the ubiquitin-proteasome system (UPS). How cells discern abnormal from normal proteins remains incompletely understood, but involves in part the recognition between ubiquitin E3 ligases and degradation signals (degrons) that are exposed in misfolded proteins. PQC is compartmentalized in the cell, and a great deal has been learned in recent years about ER-associated degradation (ERAD) and nuclear quality control. In contrast, a comprehensive view of cytosolic quality control (CytoQC) has yet to emerge, and will benefit from the development of a well-defined set of model substrates. In this study, we generated an isogenic "degron library" in Saccharomyces cerevisiae consisting of short sequences appended to the C-terminus of a reporter protein, Ura3 About half of these degron-containing proteins are substrates of the integral membrane E3 ligase Doa10, which also plays a pivotal role in ERAD and some nuclear protein degradation. Notably, some of our degron fusion proteins exhibit dependence on the E3 ligase Ltn1/Rkr1 for degradation, apparently by a mechanism distinct from its known role in ribosomal quality control of translationally paused proteins. Ubr1 and San1, E3 ligases involved in the recognition of some misfolded CytoQC substrates, are largely dispensable for the degradation of our degron-containing proteins. Interestingly, the Hsp70/Hsp40 chaperone/cochaperones Ssa1,2 and Ydj1, are required for the degradation of all constructs tested. Taken together, the comprehensive degron library presented here provides an important resource of isogenic substrates for testing candidate PQC components and identifying new ones. Copyright © 2016 Maurer et al.

  16. Degradation Signals for Ubiquitin-Proteasome Dependent Cytosolic Protein Quality Control (CytoQC) in Yeast

    PubMed Central

    Maurer, Matthew J.; Spear, Eric D.; Yu, Allen T.; Lee, Evan J.; Shahzad, Saba; Michaelis, Susan

    2016-01-01

    Cellular protein quality control (PQC) systems selectively target misfolded or otherwise aberrant proteins for degradation by the ubiquitin-proteasome system (UPS). How cells discern abnormal from normal proteins remains incompletely understood, but involves in part the recognition between ubiquitin E3 ligases and degradation signals (degrons) that are exposed in misfolded proteins. PQC is compartmentalized in the cell, and a great deal has been learned in recent years about ER-associated degradation (ERAD) and nuclear quality control. In contrast, a comprehensive view of cytosolic quality control (CytoQC) has yet to emerge, and will benefit from the development of a well-defined set of model substrates. In this study, we generated an isogenic “degron library” in Saccharomyces cerevisiae consisting of short sequences appended to the C-terminus of a reporter protein, Ura3. About half of these degron-containing proteins are substrates of the integral membrane E3 ligase Doa10, which also plays a pivotal role in ERAD and some nuclear protein degradation. Notably, some of our degron fusion proteins exhibit dependence on the E3 ligase Ltn1/Rkr1 for degradation, apparently by a mechanism distinct from its known role in ribosomal quality control of translationally paused proteins. Ubr1 and San1, E3 ligases involved in the recognition of some misfolded CytoQC substrates, are largely dispensable for the degradation of our degron-containing proteins. Interestingly, the Hsp70/Hsp40 chaperone/cochaperones Ssa1,2 and Ydj1, are required for the degradation of all constructs tested. Taken together, the comprehensive degron library presented here provides an important resource of isogenic substrates for testing candidate PQC components and identifying new ones. PMID:27172186

  17. Cables1 protects p63 from proteasomal degradation to ensure deletion of cells after genotoxic stress.

    PubMed

    Wang, Ning; Guo, Lankai; Rueda, Bo R; Tilly, Jonathan L

    2010-08-01

    The p63 gene product regulates epithelial morphogenesis and female germline integrity. In this study, we show that cyclin-dependent kinase 5 and Abl enzyme substrate 1 (Cables1) interacts with the trans-activating (TA) p63alpha isoform to protect it from proteasomal degradation. Using the female germline of Cables1-null mice as an in vivo model, we demonstrate further that oocytes lacking Cables1 exhibit lower basal levels of TAp63alpha and reduced accumulation of phosphorylated TAp63alpha in response to genotoxic stress. This in turn enhances the survival of these cells after ionizing radiation exposure. Thus, Cables1 modulates p63 protein stability and function during genotoxic stress.

  18. Cytokinin inhibits the proteasome-mediated degradation of carbonylated proteins in Arabidopsis leaves

    USDA-ARS?s Scientific Manuscript database

    Under normal conditions, plants contain numerous carbonylated proteins, which are thought to be indicative of oxidative stress damage. Conditions that promote formation of reactive oxygen species (ROS) enhance protein carbonylation, and protein degradation is required to reverse the damage. However,...

  19. BIM(EL), an intrinsically disordered protein, is degraded by 20S proteasomes in the absence of poly-ubiquitylation.

    PubMed

    Wiggins, Ceri M; Tsvetkov, Peter; Johnson, Mark; Joyce, Claire L; Lamb, Christopher A; Bryant, Nia J; Komander, David; Shaul, Yosef; Cook, Simon J

    2011-03-15

    BIM-extra long (BIM(EL)), a pro-apoptotic BH3-only protein and part of the BCL-2 family, is degraded by the proteasome following activation of the ERK1/2 signalling pathway. Although studies have demonstrated poly-ubiquitylation of BIM(EL) in cells, the nature of the ubiquitin chain linkage has not been defined. Using ubiquitin-binding domains (UBDs) specific for defined ubiquitin chain linkages, we show that BIM(EL) undergoes K48-linked poly-ubiquitylation at either of two lysine residues. Surprisingly, BIM(EL)ΔKK, which lacks both lysine residues, was not poly-ubiquitylated but still underwent ERK1/2-driven, proteasome-dependent turnover. BIM has been proposed to be an intrinsically disordered protein (IDP) and some IDPs can be degraded by uncapped 20S proteasomes in the absence of poly-ubiquitylation. We show that BIM(EL) is degraded by isolated 20S proteasomes but that this is prevented when BIM(EL) is bound to its pro-survival target protein MCL-1. Furthermore, knockdown of the proteasome cap component Rpn2 does not prevent BIM(EL) turnover in cells, and inhibition of the E3 ubiquitin ligase β-TrCP, which catalyses poly-Ub of BIM(EL), causes Cdc25A accumulation but does not inhibit BIM(EL) turnover. These results provide new insights into the regulation of BIM(EL) by defining a novel ubiquitin-independent pathway for the proteasome-dependent destruction of this highly toxic protein.

  20. Proteasome-dependent degradation of replisome components regulates faithful DNA replication.

    PubMed

    Roseaulin, Laura C; Noguchi, Chiaki; Noguchi, Eishi

    2013-08-15

    The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCF(Pof3) (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.

  1. It's all about talking: two-way communication between proteasomal and lysosomal degradation pathways via ubiquitin

    PubMed Central

    Liebl, Martina P.

    2016-01-01

    Selective degradation of proteins requires a fine-tuned coordination of the two major proteolytic pathways, the ubiquitin-proteasome system (UPS) and autophagy. Substrate selection and proteolytic activity are defined by a plethora of regulatory cofactors influencing each other. Both proteolytic pathways are initiated by ubiquitylation to mark substrate proteins for degradation, although the size and/or topology of the modification are different. In this context E3 ubiquitin ligases, ensuring the covalent attachment of activated ubiquitin to the substrate, are of special importance. The regulation of E3 ligase activity, competition between different E3 ligases for binding E2 conjugation enzymes and substrates, as well as their interplay with deubiquitylating enzymes (DUBs) represent key events in the cross talk between the UPS and autophagy. The coordination between both degradation routes is further influenced by heat shock factors and ubiquitin-binding proteins (UBPs) such as p97, p62, or optineurin. Mutations in enzymes and ubiquitin-binding proteins or a general decline of both proteolytic systems during aging result in accumulation of damaged and aggregated proteins. Thus further mechanistic understanding of how UPS and autophagy communicate might allow therapeutic intervention especially against age-related diseases. PMID:27225656

  2. Mouse Mammary Tumor Virus Signal Peptide Uses a Novel p97-Dependent and Derlin-Independent Retrotranslocation Mechanism To Escape Proteasomal Degradation.

    PubMed

    Byun, Hyewon; Das, Poulami; Yu, Houqing; Aleman, Alejandro; Lozano, Mary M; Matouschek, Andreas; Dudley, Jaquelin P

    2017-03-28

    Multiple pathogens, including viruses and bacteria, manipulate endoplasmic reticulum-associated degradation (ERAD) to avoid the host immune response and promote their replication. The betaretrovirus mouse mammary tumor virus (MMTV) encodes Rem, which is a precursor protein that is cleaved into a 98-amino-acid signal peptide (SP) and a C-terminal protein (Rem-CT). SP uses retrotranslocation for ER membrane extraction and yet avoids ERAD by an unknown mechanism to enter the nucleus and function as a Rev-like protein. To determine how SP escapes ERAD, we used a ubiquitin-activated interaction trap (UBAIT) screen to trap and identify transient protein interactions with SP, including the ERAD-associated p97 ATPase, but not E3 ligases or Derlin proteins linked to retrotranslocation, polyubiquitylation, and proteasomal degradation of extracted proteins. A dominant negative p97 ATPase inhibited both Rem and SP function. Immunoprecipitation experiments indicated that Rem, but not SP, is polyubiquitylated. Using both yeast and mammalian expression systems, linkage of a ubiquitin-like domain (UbL) to SP or Rem induced degradation by the proteasome, whereas SP was stable in the absence of the UbL. ERAD-associated Derlin proteins were not required for SP activity. Together, these results suggested that Rem uses a novel p97-dependent, Derlin-independent retrotranslocation mechanism distinct from other pathogens to avoid SP ubiquitylation and proteasomal degradation.IMPORTANCE Bacterial and viral infections produce pathogen-specific proteins that interfere with host functions, including the immune response. Mouse mammary tumor virus (MMTV) is a model system for studies of human complex retroviruses, such as HIV-1, as well as cancer induction. We have shown that MMTV encodes a regulatory protein, Rem, which is cleaved into an N-terminal signal peptide (SP) and a C-terminal protein (Rem-CT) within the endoplasmic reticulum (ER) membrane. SP function requires ER membrane extraction

  3. E11/Podoplanin Protein Stabilization Through Inhibition of the Proteasome Promotes Osteocyte Differentiation in Murine in Vitro Models

    PubMed Central

    Prideaux, Matt; Allen, Steve; Buttle, David J.; Pitsillides, Andrew A.; Farquharson, Colin

    2015-01-01

    The transmembrane glycoprotein E11 is considered critical in early osteoblast–osteocyte transitions (osteocytogenesis), however its function and regulatory mechanisms are still unknown. Using the late osteoblast MLO‐A5 cell line we reveal increased E11 protein/mRNA expression (P < 0.001) concomitant with extensive osteocyte dendrite formation and matrix mineralization (P < 0.001). Transfection with E11 significantly increased mRNA levels (P < 0.001), but immunoblotting failed to detect any correlative increases in E11 protein levels, suggestive of post‐translational degradation. We found that exogenous treatment of MLO‐A5 and osteocytic IDG‐SW3 cells with 10 μM ALLN (calpain and proteasome inhibitor) stabilized E11 protein levels and induced a profound increase in osteocytic dendrite formation (P < 0.001). Treatment with other calpain inhibitors failed to promote similar osteocytogenic changes, suggesting that these effects of ALLN rely upon its proteasome inhibitor actions. Accordingly we found that proteasome‐selective inhibitors (MG132/lactacystin/ Bortezomib/Withaferin‐A) produced similar dose‐dependent increases in E11 protein levels in MLO‐A5 and primary osteoblast cells. This proteasomal targeting was confirmed by immunoprecipitation of ubiquitinylated proteins, which included E11, and by increased levels of ubiquitinylated E11 protein upon addition of the proteasome inhibitors MG132/Bortezomib. Activation of RhoA, the small GTPase, was found to be increased concomitant with the peak in E11 levels and its downstream signaling was also observed to promote MLO‐A5 cell dendrite formation. Our data indicate that a mechanism reliant upon blockade of proteasome‐mediated E11 destabilization contributes to osteocytogenesis and that this may involve downstream targeting of RhoA. This work adds to our mechanistic understanding of the factors regulating bone homeostasis, which may lead to future therapeutic approaches. J. Cell

  4. Toxoplasma gondii infection of activated J774-A1 macrophages causes inducible nitric oxide synthase degradation by the proteasome pathway.

    PubMed

    Padrão, Juliana da Cruz; Cabral, Gabriel Rabello de Abreu; da Silva, Maria de Fátima Sarro; Seabra, Sergio Henrique; DaMatta, Renato Augusto

    2014-10-01

    Classically activated macrophages produce nitric oxide (NO), which is a potent microbicidal agent. NO production is catalyzed by inducible nitric oxide synthase (iNOS), which uses arginine as substrate producing NO and citruline. However, it has been demonstrated that NO production is inhibited after macrophage infection of Toxoplasma gondii, the agent of toxoplasmosis, due to iNOS degradation. Three possible iNOS degradation pathways have been described in activated macrophages: proteasome, calpain and lysosomal. To identify the iNOS degradation pathway after T. gondii infection, J774-A1 macrophage cell line was activated with lipopolysaccharide and interferon-gamma for 24 h, treated with the following inhibitors, lactacystin (proteasome), calpeptin (calpain), or concanamycin A (lysosomal), and infected with the parasite. NO production and iNOS expression were evaluated after 2 and 6 h of infection. iNOS was degraded in J774-A1 macrophages infected with T. gondii. However, treatment with lactacystin maintained iNOS expression in J774-A1 macrophages infected for 2 h by T. gondii, and after 6 h iNOS was localized in aggresomes. iNOS was degraded after parasite infection of J774-A1 macrophages treated with calpeptin or concanamycin A. NO production confirmed iNOS expression profiles. These results indicate that T. gondii infection of J774-A1 macrophages caused iNOS degradation by the proteasome pathway.

  5. Apelin promotes diabetic nephropathy by inducing podocyte dysfunction via inhibiting proteasome activities

    PubMed Central

    Guo, Caixia; Liu, Yu; Zhao, Wenjie; Wei, Shengnan; Zhang, Xiaoli; Wang, Wenying; Zeng, Xiangjun

    2015-01-01

    Podocyte injuries are associated with progression of diabetic nephropathy (DN). Apelin, an adipocyte-derived peptide, has been reported to be a promoting factor for DN. In this study, we aim to determine whether apelin promotes progression of DN by inducing podocyte dysfunction. kk-Ay mice were used as models for DN. Apelin and its antagonist, F13A were intraperitoneally administered for 4 weeks, respectively. Renal function and foot process proteins were analysed to evaluate the effects of apelin on kk-Ay mice and podocytes. Apelin increased albuminuria and decreased podocyte foot process proteins expression in kk-Ay mice, which is consistent with the results that apelin receptor (APLNR) levels increased in glomeruli of patients or mice with DN. In cultured podocytes, high glucose increased APLNR expression and apelin administration was associated with increased permeability and decreased foot process proteins levels. All these dysfunctions were associated with decreased 26S proteasome activities and increased polyubiquitinated proteins in both kk-Ay mice and cultured podocytes, as demonstrated by 26S proteasome activation with cyclic adenosine monophosphate (cAMP) or oleuropein. These effects seemed to be related to endoplasmic reticulum (ER) stress, as apelin increased C/EBP homologous protein (CHOP) and peiFα levels while cAMP or oleuropein reduced it in high glucose and apelin treated podocytes. These results suggest that apelin induces podocyte dysfunction in DN through ER stress which was induced by decreased proteasome activities in podocytes. PMID:26103809

  6. Rice ROOT ARCHITECTURE ASSOCIATED1 Binds the Proteasome Subunit RPT4 and Is Degraded in a D-Box and Proteasome-Dependent Manner1[W][OA

    PubMed Central

    Han, Ye; Cao, Hong; Jiang, Jiafu; Xu, Yunyuan; Du, Jizhou; Wang, Xin; Yuan, Ming; Wang, Zhiyong; Xu, Zhihong; Chong, Kang

    2008-01-01

    Root growth is mainly determined by cell division and subsequent elongation in the root apical area. Components regulating cell division in root meristematic cells are largely unknown. Previous studies have identified rice (Oryza sativa) ROOT ARCHITECTURE ASSOCIATED1 (OsRAA1) as a regulator in root development. Yet, the function of OsRAA1 at the cellular and molecular levels is unclear. Here, we show that OsRAA1-overexpressed transgenic rice showed reduced primary root growth, increased numbers of cells in metaphase, and reduced numbers of cells in anaphase, which suggests that OsRAA1 is responsible for limiting root growth by inhibiting the onset of anaphase. The expression of OsRAA1 in fission yeast also induced metaphase arrest, which is consistent with the fact that OsRAA1 functions through a conserved mechanism of cell cycle regulation. Moreover, a colocalization assay has shown that OsRAA1 is expressed predominantly at spindles during cell division. Yeast two-hybrid and pull-down assays, as well as a bimolecular fluorescence complementation assay, all have revealed that OsRAA1 interacts with a rice homolog of REGULATORY PARTICLE TRIPLE-A ATPASE4, a component that is involved in the ubiquitin pathway. Treating transgenic rice with specific inhibitors of the 26S proteasome blocked the degradation of OsRAA1 and increased the number of cells in metaphase. Mutation of a putative ubiquitination-targeting D-box (RGSLDLISL) in OsRAA1 interrupted the destruction of OsRAA1 in transgenic yeast. These results suggest that ubiquitination and proteasomic proteolysis are involved in OsRAA1 degradation, which is essential for the onset of anaphase, and that OsRAA1 may modulate root development mediated by the ubiquitin-proteasome pathway as a novel regulatory factor of the cell cycle. PMID:18701670

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

    PubMed

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

    2011-06-03

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

  8. Apoptosis inducer NGFI-B is degraded by the proteasome and stabilized by treatment with EGF

    SciTech Connect

    Strom, Bjorn O.; Paulsen, Ragnhild E.

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer NGFI-B is a molecular target for some anti-cancer drugs. Black-Right-Pointing-Pointer NGFI-B turnover may be important for their anti-cancer action. Black-Right-Pointing-Pointer NGFI-B is degraded by the proteasome. Black-Right-Pointing-Pointer NGFI-B is stabilized by treatment with EGF. Black-Right-Pointing-Pointer Mimicking the EGF-induced phosphorylation also stabilizes the protein. -- Abstract: NGFI-B is a nuclear receptor and immediate early gene that is upregulated in many different tumour cell lines. As it is involved in cell death and survival, it has been suggested as a target for anti-cancer drugs. The protein level of NGFI-B is important for its functions and may be regulated through induction or stabilization. NGFI-B protein stability was studied using the protein synthesis inhibitor cycloheximide in CV1 cells transiently transfected with NGFI-B. Inhibiting the proteasome with MG132 stabilized NGFI-B, indicating that the proteasome is responsible for break-down of NGFI-B, as it is for many nuclear receptors. In order to determine regions responsible for the break-down of NGFI-B two N-terminal regions with high PEST-scores were deleted. Deletion of amino acids 122-195 containing a PEST-sequence which includes an ERK2 phosphorylation target, gave a more stable protein. In addition, treatment of the cells with the ERK2 activator EGF increased the stability of wild type NGFI-B. We then tested whether a mutation at threonine 142 influenced the stability of NGFI-B. We found that the phosphorylation-mimicking mutant NGFI-B T142E had an increased stability, while the non-phosphorylable mutant (T142A) showed similar stability to the wild type. Thus, EGF-stimulation of cells may be a mechanism for priming the cells for effects of NGFI-B by increasing its stability.

  9. Homogeneous, bioluminescent proteasome assays.

    PubMed

    O'Brien, Martha A; Moravec, Richard A; Riss, Terry L; Bulleit, Robert F

    2015-01-01

    Protein degradation is mediated predominantly through the ubiquitin-proteasome pathway. The importance of the proteasome in regulating degradation of proteins involved in cell-cycle control, apoptosis, and angiogenesis led to the recognition of the proteasome as a therapeutic target for cancer. The proteasome is also essential for degrading misfolded and aberrant proteins, and impaired proteasome function has been implicated in neurodegerative and cardiovascular diseases. Robust, sensitive assays are essential for monitoring proteasome activity and for developing inhibitors of the proteasome. Peptide-conjugated fluorophores are widely used as substrates for monitoring proteasome activity, but fluorogenic substrates can exhibit significant background and can be problematic for screening because of cellular autofluorescence or interference from fluorescent library compounds. Furthermore, fluorescent proteasome assays require column-purified 20S or 26S proteasome (typically obtained from erythrocytes), or proteasome extracts from whole cells, as their samples. To provide assays more amenable to high-throughput screening, we developed a homogeneous, bioluminescent method that combines peptide-conjugated aminoluciferin substrates and a stabilized luciferase. Using substrates for the chymotrypsin-like, trypsin-like, and caspase-like proteasome activities in combination with a selective membrane permeabilization step, we developed single-step, cell-based assays to measure each of the proteasome catalytic activities. The homogeneous method eliminates the need to prepare individual cell extracts as samples and has adequate sensitivity for 96- and 384-well plates. The simple "add and read" format enables sensitive and rapid proteasome assays ideal for inhibitor screening.

  10. Ubiquitin degradation with its substrate, or as a monomer in a ubiquitination-independent mode, provides clues to proteasome regulation

    PubMed Central

    Shabek, Nitzan; Herman-Bachinsky, Yifat; Ciechanover, Aaron

    2009-01-01

    The mechanisms that regulate the ubiquitin (Ub)-proteasome system's own components, although critically important, are largely unknown. Ub, a principal component of the system, must be maintained at adequate levels to support cellular homeostasis under basal and stressed conditions. It was suggested that Ub is degraded as part of the polyubiquitin chain along with its substrate. Here, we demonstrate in a direct manner that Ub is indeed degraded in a “piggyback” mechanism. Also, it has been shown that monomeric Ub can be rapidly degraded when a C-terminal tail of a minimal length is fused to it. The tail, which may represent the substrate or part of it, or a naturally occurring extended form of Ub, probably allows entry of the protein into the 20S catalytic chamber, while Ub serves as an anchor to the 19S complex. Here, we show that shorter-tailed Ubs, such as UBB+1, bind to the proteasome but because they cannot be efficiently degraded, they inhibit the degradation of other Ub system's substrates such as Myc, p21, Mdm2, and MyoD. The inhibition depends on the ability of the tailed Ubs to be ubiquitinated: their mere binding to the proteasome is not sufficient. Interestingly, the inhibition affects only substrates that must undergo ubiquitination for their degradation: ornithine decarboxylase that is targeted by the proteasome in a Ub-independent manner, is not affected by the short-tailed ubiquitinated Ubs, suggesting it binds to the 19S complex in a site different from that to which ubiquitinated substrates bind. PMID:19581590

  11. Proteasomes regulate hepatitis B virus replication by degradation of viral core-related proteins in a two-step manner.

    PubMed

    Zheng, Zi-Hua; Yang, Hui-Ying; Gu, Lin; Peng, Xiao-Mou

    2016-10-01

    The cellular proteasomes presumably inhibit the replication of hepatitis B virus (HBV) due to degradation of the viral core protein (HBcAg). Common proteasome inhibitors, however, either enhance or inhibit HBV replication. In this study, the exact degradation process of HBcAg and its influences on HBV replication were further studied using bioinformatic analysis, protease digestion assays of recombinant HBcAg, and proteasome inhibitor treatments of HBV-producing cell line HepG2.2.15. Besides HBcAg and hepatitis B e antigen precursor, common hepatitis B core-related antigens (HBcrAgs), the small and the large degradation intermediates of these HBcrAgs (HBcrDIs), were regularly found in cytosol of HepG2.2.15 cells. Further, the results of investigation reveal that the degradation process of cytosolic HBcrAgs in proteasomes consists of two steps: the limited proteolysis into HBcrDIs by the trypsin-like (TL) activity and the complete degradation of HBcrDIs by the chymotrypsin-like (chTL) activity. Concordantly, HBcrAgs and the large HBcrDI or HBcrDIs (including the small HBcrDI) were accumulated when the TL or chTL activity was inhibited, which generally correlated with enhancement and inhibition of HBV replication, respectively. The small HBcrDI inhibited HBV replication by assembling into the nucleocapsids and preventing the victim particles from being mature enough for envelopment. The two-step degradation manner may highlight some new anti-HBV strategies.

  12. Co-chaperone HSJ1a dually regulates the proteasomal degradation of ataxin-3.

    PubMed

    Gao, Xue-Chao; Zhou, Chen-Jie; Zhou, Zi-Ren; Zhang, Yu-Hang; Zheng, Xue-Ming; Song, Ai-Xin; Hu, Hong-Yu

    2011-01-01

    Homo sapiens J domain protein (HSJ1) is a J-domain containing co-chaperone that is known to stimulate ATPase activity of HSP70 chaperone, while it also harbors two ubiquitin (Ub)-interacting motifs (UIMs) that may bind with ubiquitinated substrates and potentially function in protein degradation. We studied the effects of HSJ1a on the protein levels of both normal and the disease--related polyQ-expanded forms of ataxin-3 (Atx3) in cells. The results demonstrate that the N-terminal J-domain and the C-terminal UIM domain of HSJ1a exert opposite functions in regulating the protein level of cellular overexpressed Atx3. This dual regulation is dependent on the binding of the J-domain with HSP70, and the UIM domain with polyUb chains. The J-domain down-regulates the protein level of Atx3 through HSP70 mediated proteasomal degradation, while the UIM domain may alleviate this process via maintaining the ubiquitinated Atx3. We propose that co-chaperone HSJ1a orchestrates the balance of substrates in stressed cells in a Yin-Yang manner.

  13. Rootletin prevents Cep68 from VHL-mediated proteasomal degradation to maintain centrosome cohesion.

    PubMed

    Yin, Huilong; Zheng, Lu; Liu, Weixiao; Zhang, Dachuan; Li, Wei; Yuan, Li

    2017-04-01

    Centrosome cohesion, mostly regarded as a proteinaceous linker between parental centrioles, ensures the interphase centrosome(s) to function as a single microtubule-organizing center. Maintenance of centrosome cohesion counts on a number of centrosomal linker proteins because depletion of any of those leads to premature centrosome separation in interphase, termed centrosome splitting. However, the underlying mechanisms of the dependence are unknown. Here, we show that absence of Rootletin triggers the von Hippel-Lindau tumour suppressor protein (VHL)-mediated proteasomal degradation of Cep68 and, in turn, results in centrosome splitting. The VHL E3 ligase complex ubiquitinates Cep68 in vitro and in vivo. Co-silencing of Rootletin and VHL reverts Cep68 loss and centrosome splitting. Expression of a stable mutant of Cep68, either diminishing its polyubiquitylation or eliminating binding to β-domain of VHL, also suppresses centrosome splitting provoked by Rootletin depletion. We propose that the archetypal linker protein Rootletin maintains centrosome cohesion in part through inhibition of VHL-mediated Cep68 degradation.

  14. Celastrol induces proteasomal degradation of FANCD2 to sensitize lung cancer cells to DNA crosslinking agents

    PubMed Central

    Wang, Gui-Zhen; Liu, Yong-Qiang; Cheng, Xin; Zhou, Guang-Biao

    2015-01-01

    The Fanconi anemia (FA) pathway plays a key role in interstrand crosslink (ICL) repair and maintenance of the genomic stability, while inhibition of this pathway may sensitize cancer cells to DNA ICL agents and ionizing radiation (IR). The active FA core complex acts as an E3 ligase to monoubiquitinate FANCD2, which is a functional readout of an activated FA pathway. In the present study, we aimed to identify FANCD2-targeting agents, and found that the natural compound celastrol induced degradation of FANCD2 through the ubiquitin–proteasome pathway. We demonstrated that celastrol downregulated the basal and DNA damaging agent-induced monoubiquitination of FANCD2, followed by proteolytic degradation of the substrate. Furthermore, celastrol treatment abrogated the G2 checkpoint induced by IR, and enhanced the ICL agent-induced DNA damage and inhibitory effects on lung cancer cells through depletion of FANCD2. These results indicate that celastrol is a FANCD2 inhibitor that could interfere with the monoubiquitination and protein stability of FANCD2, providing a novel opportunity to develop FA pathway inhibitor and combinational therapy for malignant neoplasms. PMID:25891850

  15. TRIM27 Negatively Regulates NOD2 by Ubiquitination and Proteasomal Degradation

    PubMed Central

    Zurek, Birte; Schoultz, Ida; Neerincx, Andreas; Napolitano, Luisa M.; Birkner, Katharina; Bennek, Eveline; Sellge, Gernot; Lerm, Maria; Meroni, Germana; Söderholm, Johan D.; Kufer, Thomas A.

    2012-01-01

    NOD2, the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR) member 2 is involved in mediating antimicrobial responses. Dysfunctional NOD2 activity can lead to severe inflammatory disorders, but the regulation of NOD2 is still poorly understood. Recently, proteins of the tripartite motif (TRIM) protein family have emerged as regulators of innate immune responses by acting as E3 ubiquitin ligases. We identified TRIM27 as a new specific binding partner for NOD2. We show that NOD2 physically interacts with TRIM27 via the nucleotide-binding domain, and that NOD2 activation enhances this interaction. Dependent on functional TRIM27, ectopically expressed NOD2 is ubiquitinated with K48-linked ubiquitin chains followed by proteasomal degradation. Accordingly, TRIM27 affects NOD2-mediated pro-inflammatory responses. NOD2 mutations are linked to susceptibility to Crohn's disease. We found that TRIM27 expression is increased in Crohn's disease patients, underscoring a physiological role of TRIM27 in regulating NOD2 signaling. In HeLa cells, TRIM27 is partially localized in the nucleus. We revealed that ectopically expressed NOD2 can shuttle to the nucleus in a Walker A dependent manner, suggesting that NOD2 and TRIM27 might functionally cooperate in the nucleus. We conclude that TRIM27 negatively regulates NOD2-mediated signaling by degradation of NOD2 and suggest that TRIM27 could be a new target for therapeutic intervention in NOD2-associated diseases. PMID:22829933

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

    PubMed

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

    2017-03-15

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

  17. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2.

    PubMed

    Kobayashi, Akira; Kang, Moon-Il; Okawa, Hiromi; Ohtsuji, Makiko; Zenke, Yukari; Chiba, Tomoki; Igarashi, Kazuhiko; Yamamoto, Masayuki

    2004-08-01

    Transcription factor Nrf2 is a major regulator of genes encoding phase 2 detoxifying enzymes and antioxidant stress proteins in response to electrophilic agents and oxidative stress. In the absence of such stimuli, Nrf2 is inactive owing to its cytoplasmic retention by Keap1 and rapid degradation through the proteasome system. We examined the contribution of Keap1 to the rapid turnover of Nrf2 (half-life of less than 20 min) and found that a direct association between Keap1 and Nrf2 is required for Nrf2 degradation. In a series of domain function analyses of Keap1, we found that both the BTB and intervening-region (IVR) domains are crucial for Nrf2 degradation, implying that these two domains act to recruit ubiquitin-proteasome factors. Indeed, Cullin 3 (Cul3), a subunit of the E3 ligase complex, was found to interact specifically with Keap1 in vivo. Keap1 associates with the N-terminal region of Cul3 through the IVR domain and promotes the ubiquitination of Nrf2 in cooperation with the Cul3-Roc1 complex. These results thus provide solid evidence that Keap1 functions as an adaptor of Cul3-based E3 ligase. To our knowledge, Nrf2 and Keap1 are the first reported mammalian substrate and adaptor, respectively, of the Cul3-based E3 ligase system.

  18. A conserved serine residue regulates the stability of Drosophila Salvador and human WW domain-containing adaptor 45 through proteasomal degradation

    SciTech Connect

    Wu, Di Wu, Shian

    2013-04-19

    Highlights: •Ser-17 is key for the stability of Drosophila Sav. •Ala mutation of Ser-17 promotes the proteasomal degradation of Sav. •Ser-17 residue is not the main target of Hpo-induced Sav stabilization. •Hpo-dependent and -independent mechanisms regulate Sav stability. •This mechanism is conserved in the homologue of Sav, human WW45. -- Abstract: The Hippo (Hpo) pathway is a conserved tumor suppressor pathway that controls organ size through the coordinated regulation of apoptosis and proliferation. Drosophila Salvador (Sav), which limits organ size, is a core component of the Hpo pathway. In this study, Ser-17 was shown to be important for the stability of Sav. Alanine mutation of Ser-17 promoted the proteasomal degradation of Sav. Destabilization and stabilization of the Sav protein mediated by alanine mutation of Ser-17 and by Hpo, respectively, were independent of each other. This implies that the stability of Sav is controlled by two mechanisms, one that is Ser-17-dependent and Hpo-independent, and another that is Ser-17-independent and Hpo-dependent. These dual mechanisms also regulated the human counterpart of Drosophila Sav, WW domain-containing adaptor 45 (WW45). The conservation of this regulation adds to its significance in normal physiology and tumorigenesis.

  19. Carnosic acid induces proteasomal degradation of Cyclin B1, RB and SOX2 along with cell growth arrest and apoptosis in GBM cells.

    PubMed

    Cortese, Katia; Daga, Antonio; Monticone, Massimiliano; Tavella, Sara; Stefanelli, Alessia; Aiello, Cinzia; Bisio, Angela; Bellese, Grazia; Castagnola, Patrizio

    2016-06-15

    Carnosic acid (CA) is a diterpenoid found in Rosmarinus officinalis L. and Salvia officinalis L. as well as in many other Lamiaceae. This compound is reported to have antioxidant and antimicrobial properties. In addition, a number of reports showed that CA has a cytotoxic activity toward several cancer cell lines. The aim of this study was to establish whether CA has any specific antiproliferative effect toward human glioblastoma (GBM) cells and to analyze the molecular mechanisms involved. We evaluated cell survival by MTT assay, apoptosis and DNA content by flow cytometry, protein expression and phosphorylation by immunoblot analyses. Our results showed that CA inhibited cell survival on both normal astrocytes and GBM cells. In GBM cells, in particular, CA caused an early G2 block, a reduction in the percentage of cells expressing Ki67, an enhanced expression of p21(WAF) and induced apoptosis. Furthermore, we showed that CA promoted proteasomal degradation of several substrate proteins, including Cyclin B1, retinoblastoma (RB), SOX2, and glial fibrillary acid protein (GFAP), whereas MYC levels were not modified. In addition, CA dramatically reduced the activity of CDKs. In conclusion, our findings strongly suggest that CA promotes a profound deregulation of cell cycle control and reduces the survival of GBM cells via proteasome-mediated degradation of Cyclin B1, RB and SOX2. Copyright © 2016 Elsevier GmbH. All rights reserved.

  20. HIV-1 Tat Recruits HDM2 E3 Ligase To Target IRF-1 for Ubiquitination and Proteasomal Degradation

    PubMed Central

    Remoli, Anna Lisa; Marsili, Giulia; Perrotti, Edvige; Acchioni, Chiara; Sgarbanti, Marco; Borsetti, Alessandra; Hiscott, John

    2016-01-01

    ABSTRACT In addition to its ability to regulate HIV-1 promoter activation, the viral transactivator Tat also functions as a determinant of pathogenesis and disease progression by directly and indirectly modulating the host anti-HIV response, largely through the capacity of Tat to interact with and modulate the activities of multiple host proteins. We previously demonstrated that Tat modulated both viral and host transcriptional machinery by interacting with the cellular transcription factor interferon regulatory factor 1 (IRF-1). In the present study, we investigated the mechanistic basis and functional significance of Tat−IRF-1 interaction and demonstrate that Tat dramatically decreased IRF-1 protein stability. To accomplish this, Tat exploited the cellular HDM2 (human double minute 2 protein) ubiquitin ligase to accelerate IRF-1 proteasome-mediated degradation, resulting in a quenching of IRF-1 transcriptional activity during HIV-1 infection. These data identify IRF-1 as a new target of Tat-induced modulation of the cellular protein machinery and reveal a new strategy developed by HIV-1 to evade host immune responses. PMID:27795392

  1. FBXO32 suppresses breast cancer tumorigenesis through targeting KLF4 to proteasomal degradation.

    PubMed

    Zhou, H; Liu, Y; Zhu, R; Ding, F; Wan, Y; Li, Y; Liu, Z

    2017-01-09

    Krüppel-like factor 4 (KLF4, GKLF) is a zinc-finger transcription factor involved in a large variety of cellular processes, including apoptosis, cell cycle progression, as well as stem cell renewal. KLF4 is critical for cell fate decision and has an ambivalent role in tumorigenesis. Emerging data keep reminding us that KLF4 dysregulation either facilitates or impedes tumor progression, making it important to clarify the regulating network of KLF4. Like most transcription factors, KLF4 has a rather short half-life within the cell and its turnover must be carefully orchestrated by ubiquitination and ubiquitin-proteasome system. To better understand the mechanism of KLF4 ubiquitination, we performed a genome-wide screen of E3 ligase small interfering RNA library based on western blot and identified SCF-FBXO32 to be a new E3 ligase, which is responsible for KLF4 ubiquitination and degradation. The F-box domain is critical for FBXO32-dependent KLF4 ubiquitination and degradation. Furthermore, we demonstrated that FBXO32 physically interacts with the N-terminus (1-60 aa) of KLF4 via its C-terminus (228-355 aa) and directly targets KLF4 for ubiquitination and degradation. We also found out that p38 mitogen-activated protein kinase pathway may be implicated in FBXO32-mediated ubiquitination of KLF4, as p38 kinase inhibitor coincidently abrogates endogenous KLF4 ubiquitination and degradation, as well as FBXO32-dependent exogenous KLF4 ubiquitination and degradation. Finally, FBXO32 inhibits colony formation in vitro and primary tumor initiation and growth in vivo through targeting KLF4 into degradation. Our findings thus further elucidate the tumor-suppressive function of FBXO32 in breast cancer. These results expand our understanding of the posttranslational modification of KLF4 and of its role in breast cancer development and provide a potential target for diagnosis and therapeutic treatment of breast cancer.Oncogene advance online publication, 9 January 2017; doi:10

  2. Cigarette smoke induces Akt protein degradation by the ubiquitin-proteasome system.

    PubMed

    Kim, Sun-Yong; Lee, Ji-Hyun; Huh, Jin Won; Ro, Jai Youl; Oh, Yeon-Mock; Lee, Sang-Do; An, Sungkwan; Lee, Yun-Song

    2011-09-16

    Emphysema is one of the characteristic features of chronic obstructive pulmonary disease, which is caused mainly by cigarette smoking. Recent data have suggested that apoptosis and cell cycle arrest may contribute to the development of emphysema. In this study, we addressed the question of whether and how cigarette smoke affected Akt, which plays a critical role in cell survival and proliferation. In normal human lung fibroblasts, cigarette smoke extract (CSE) caused cell death, accompanying degradation of total and phosphorylated Akt (p-Akt), which was inhibited by MG132. CSE exposure resulted in preferential ubiquitination of the active Akt (myristoylated), rather than the inactive (T308A/S473A double mutant) Akt. Consistent with cytotoxicity, CSE induced a progressive decrease of phosphorylated human homolog of mouse double minute homolog 2 (p-HDM2) and phosphorylated apoptosis signal regulating kinase 1 (p-ASK1) with concomitant elevation of p53, p21, and phosphorylated p38 MAPK. Forced expression of the active Akt reduced both CSE-induced cytotoxicity and alteration in HDM2/p53/p21 and ASK1/p38 MAPK, compared with the inactive Akt. Of note, CSE induced expression of the tetratrico-peptide repeat domain 3 (TTC3), known as a ubiquitin ligase for active Akt. TTC3 siRNAs suppressed not only CSE-induced Akt degradation but also CSE-induced cytotoxicity. Accordingly, rat lungs exposed to cigarette smoke for 3 months showed elevated TTC3 expression and reduced Akt and p-Akt. Taken together, these data suggest that cigarette smoke induces cytotoxicity, partly through Akt degradation via the ubiquitin-proteasome system, in which TTC3 acts as a ubiquitin ligase for active Akt.

  3. The Kaposi's Sarcoma-Associated Herpesvirus ORF34 Protein Binds to HIF-1α and Causes Its Degradation via the Proteasome Pathway

    PubMed Central

    Kousoulas, Konstantin G.

    2013-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent for Kaposi's sarcoma (KS) and two other lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). Kaposi's sarcoma is a highly vascular tumor, and recently both hypoxia-inducible factor 1α (HIF-1α) and HIF-2α were detected in KS samples, indicating a role of HIFs in the KSHV life cycle. Previously, we showed that ORF34, a lytic gene of unassigned function, was activated by hypoxia and that ORF34 transcription was upregulated by both HIFs (M. Haque, D. A. Davis, V. Wang, I. Widmer, and R. Yarchoan, J Virol. 77:6761–6768, 2003). In the present study, we show that coexpression of ORF34 with HIF-1αm (degradation-resistant HIF-1α) caused substantial reduction in HIF-1α-dependent transcription, as evidenced by reporter assays. Two-way immunoprecipitation experiments revealed that ORF34 physically interacted with HIF-1αm in transient expression experiments. Deletion analysis revealed that three different ORF34 domains interacted with the amino-terminal domain of HIF-1α. Also, purified HIF-1α and ORF34 proteins interacted with each other. The observed transcriptional inhibition of HIF-1α-dependent promoters was attributed to degradation of HIF-1α after binding with ORF34, since the overall amount of wild-type HIF-1α but not the degradation-resistant one (HIF-1αm) was reduced in the presence of ORF34. Moreover, ORF34 caused degradation of HIF-1α in a dose-dependent manner. Inhibition of the ubiquitin-dependent pathway by the chemical proteasome inhibitor MG132 prevented HIF-1α degradation in the presence of ORF34. These results show that ORF34 binds to HIF-1α, leading to its degradation via the proteasome-dependent pathway. PMID:23221556

  4. Excess free histone H3 localizes to centrosomes for proteasome-mediated degradation during mitosis in metazoans.

    PubMed

    Wike, Candice L; Graves, Hillary K; Wason, Arpit; Hawkins, Reva; Gopalakrishnan, Jay; Schumacher, Jill; Tyler, Jessica K

    2016-08-17

    The cell tightly controls histone protein levels in order to achieve proper packaging of the genome into chromatin, while avoiding the deleterious consequences of excess free histones. Our accompanying study has shown that a histone modification that loosens the intrinsic structure of the nucleosome, phosphorylation of histone H3 on threonine 118 (H3 T118ph), exists on centromeres and chromosome arms during mitosis. Here, we show that H3 T118ph localizes to centrosomes in humans, flies, and worms during all stages of mitosis. H3 abundance at the centrosome increased upon proteasome inhibition, suggesting that excess free histone H3 localizes to centrosomes for degradation during mitosis. In agreement, we find ubiquitinated H3 specifically during mitosis and within purified centrosomes. These results suggest that targeting of histone H3 to the centrosome for proteasome-mediated degradation is a novel pathway for controlling histone supply, specifically during mitosis.

  5. Phosphorylation regulates mycobacterial proteasome.

    PubMed

    Anandan, Tripti; Han, Jaeil; Baun, Heather; Nyayapathy, Seeta; Brown, Jacob T; Dial, Rebekah L; Moltalvo, Juan A; Kim, Min-Seon; Yang, Seung Hwan; Ronning, Donald R; Husson, Robert N; Suh, Joowon; Kang, Choong-Min

    2014-09-01

    Mycobacterium tuberculosis possesses a proteasome system that is required for the microbe to resist elimination by the host immune system. Despite the importance of the proteasome in the pathogenesis of tuberculosis, the molecular mechanisms by which proteasome activity is controlled remain largely unknown. Here, we demonstrate that the α-subunit (PrcA) of the M. tuberculosis proteasome is phosphorylated by the PknB kinase at three threonine residues (T84, T202, and T178) in a sequential manner. Furthermore, the proteasome with phosphorylated PrcA enhances the degradation of Ino1, a known proteasomal substrate, suggesting that PknB regulates the proteolytic activity of the proteasome. Previous studies showed that depletion of the proteasome and the proteasome-associated proteins decreases resistance to reactive nitrogen intermediates (RNIs) but increases resistance to hydrogen peroxide (H2O2). Here we show that PknA phosphorylation of unprocessed proteasome β-subunit (pre-PrcB) and α-subunit reduces the assembly of the proteasome complex and thereby enhances the mycobacterial resistance to H2O2 and that H2O2 stress diminishes the formation of the proteasome complex in a PknA-dependent manner. These findings indicate that phosphorylation of the M. tuberculosis proteasome not only modulates proteolytic activity of the proteasome, but also affects the proteasome complex formation contributing to the survival of M. tuberculosis under oxidative stress conditions.

  6. Proteasomal degradation of ubiquitinated proteins in oocyte meiosis and fertilization in mammals.

    PubMed

    Karabinova, Pavla; Kubelka, Michal; Susor, Andrej

    2011-10-01

    Gametogenesis and fertilization are the key events in sexual reproduction. In the female, meiosis results in a large oocyte that is competent for fertilization and fundamental for the success of early embryonic development. Progression through meiosis is monitored by fine regulatory mechanisms. In this review, we focus on one of the most well-known regulatory elements, the E3 ligase APC/C, which mediates proteolytic degradation of a number of important substrates via the ubiquitin proteasome pathway (UPP). The UPP also indirectly regulates protein synthesis by affecting proteins involved in RNA metabolism, a process that is paramount for the transcriptionally silent oocyte. During the past few years, more evidence has accumulated to suggest that the UPP has an important role in zona pellucida penetration and gamete fusion in mammals. This review focuses on the function of the UPP in regulating oocyte meiotic maturation in mammals, with special attention to its role in chromosome segregation and polar body extrusion, its role in the acquisition of meiotic/developmental competence and recent advances in our understanding of the UPP role in fertilization.

  7. NQO1 inhibits proteasome-mediated degradation of HIF-1α

    PubMed Central

    Oh, Eun-Taex; Kim, Jung-whan; Kim, Joon Mee; Kim, Soo Jung; Lee, Jae-Seon; Hong, Soon-Sun; Goodwin, Justin; Ruthenborg, Robin J.; Jung, Myung Gu; Lee, Hae-June; Lee, Chul-Ho; Park, Eun Sung; Kim, Chulhee; Park, Heon Joo

    2016-01-01

    Overexpression of NQO1 is associated with poor prognosis in human cancers including breast, colon, cervix, lung and pancreas. Yet, the molecular mechanisms underlying the pro-tumorigenic capacities of NQO1 have not been fully elucidated. Here we show a previously undescribed function for NQO1 in stabilizing HIF-1α, a master transcription factor of oxygen homeostasis that has been implicated in the survival, proliferation and malignant progression of cancers. We demonstrate that NQO1 directly binds to the oxygen-dependent domain of HIF-1α and inhibits the proteasome-mediated degradation of HIF-1α by preventing PHDs from interacting with HIF-1α. NQO1 knockdown in human colorectal and breast cancer cell lines suppresses HIF-1 signalling and tumour growth. Consistent with this pro-tumorigenic function for NQO1, high NQO1 expression levels correlate with increased HIF-1α expression and poor colorectal cancer patient survival. These results collectively reveal a function of NQO1 in the oxygen-sensing mechanism that regulates HIF-1α stability in cancers. PMID:27966538

  8. The hydrophobic patch of ubiquitin is required to protect transactivator–promoter complexes from destabilization by the proteasomal ATPases

    PubMed Central

    Archer, Chase T.; Kodadek, Thomas

    2010-01-01

    Mono-ubiquitylation of a transactivator is known to promote transcriptional activation of certain transactivator proteins. For the Sacchromyces cerevisiae transactivator, GAL4, attachment of mono-ubiquitin prevents destabilization of the DNA–transactivator complex by the ATPases of the 26S proteasome. This inhibition of destabilization depends on the arrangement of ubiquitin; a chain of ubiquitin tetramers linked through lysine 48 did not display the same protective effect as mono-ubiquitin. This led to an investigation into the properties of ubiquitin that may be responsible for this difference in activity between the different forms. We demonstrate the ubiquitin tetramers linked through lysine 63 do protect from proteasomal-mediated destabilization. In addition, we show that the mutating the isoleucine residue at position 44 interferes with proteasomal interaction in vitro and will abolish the protective activity in vivo. Together, these data implicate the hydrophobic patch of ubiquitin as required to protect transactivators from destabilization by the proteasomal ATPases. PMID:19939937

  9. Autophagy maintains ubiquitination-proteasomal degradation of Sirt3 to limit oxidative stress in K562 leukemia cells

    PubMed Central

    Xu, Li; Cao, Yan; Xu, Fei; Yan, Lili; Nie, Meilan; Yuan, Na; Zhang, Suping; Zhao, Ruijin; Wang, Hongbin; Wu, Mengyin; Zhang, Xiaoying; Wang, Jianrong

    2016-01-01

    Sirtuin protein family member 3 (Sirt3) has been suggested as a positive regulator in alleviating oxidative stress by acting on the mitochondrial antioxidant machinery in solid tumors; however, its role and regulation in hematological malignancies has been poorly understood. Here, we show that contrary to what has been reported in solid tumors, in K562 leukemia cells elevated Sirt3 was associated with mitochondrial stress, and depletion of Sirt3 decreased reactive oxygen species (ROS) generation and lipid oxidation, but increased the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), suggesting an opposite role of Sirt3 in regulating oxidative stress in the leukemia cells. Notably, loss of autophagy by deletion of autophagy essential gene or by pharmacological inhibition on autophagic degradation caused a significant accumulation of Sirt3. However, induced activation of autophagy did not cause autophagic degradation of Sirt3. Furthermore, inhibiting proteasome activity accumulated Sirt3 in autophagy-intact but not autophagy-defective cells, and disrupting functional autophagy either genetically or pharmacologically caused significantly less ubiquitination of Sirt3. Therefore, our data suggest that basal but not enhanced autophagy activity maintains ubiquitination-proteasomal degradation of Sirt3 to limit lipid oxidative stress, representing an adaptive mechanism by which autophagy, in collaboration with the ubiquitination-proteasomal system, controls oxidative stress by controlling the levels of certain proteins in K562 leukemia cells. PMID:27232755

  10. The E3 ubiquitin ligase CHIP mediates ubiquitination and proteasomal degradation of PRMT5.

    PubMed

    Zhang, Huan-Tian; Zeng, Ling-Fei; He, Qing-Yu; Tao, W Andy; Zha, Zhen-Gang; Hu, Chang-Deng

    2016-02-01

    Protein arginine methyltransferase 5 (PRMT5) is an important member of the protein arginine methyltransferase family that regulates many cellular processes through epigenetic control of target gene expression. Because of its overexpression in a number of human cancers and its essential role in cell proliferation, transformation, and cell cycle progression, PRMT5 has been recently proposed to function as an oncoprotein in cancer cells. However, how its expression is regulated in cancer cells remains largely unknown. We have previously demonstrated that the transcription of PRMT5 can be negatively regulated by the PKC/c-Fos signaling pathway through modulating the transcription factor NF-Y in prostate cancer cells. In the present study, we demonstrated that PRMT5 undergoes polyubiquitination, possibly through multiple lysine residues. We also identified carboxyl terminus of heat shock cognate 70-interacting protein (CHIP), an important chaperone-dependent E3 ubiquitin ligase that couples protein folding/refolding to protein degradation, as an interacting protein of PRMT5 via mass spectrometry. Their interaction was further verified by co-immuoprecipitation, GST pull-down, and bimolecular fluorescence complementation (BiFC) assay. In addition, we provided evidence that the CHIP/chaperone system is essential for the negative regulation of PRMT5 expression via K48-linked ubiquitin-dependent proteasomal degradation. Given that down-regulation of CHIP and overexpression of PRMT5 have been observed in several human cancers, our finding suggests that down-regulation of CHIP may be one of the mechanisms underlying PRMT5 overexpression in these cancers. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Protein degradation by ubiquitin–proteasome system in formation and labilization of contextual conditioning memory

    PubMed Central

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

    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 case, the inhibition of the UPS during consolidation impairs memory. Similar results were reported for memory reconsolidation. However, in other cases, the inhibition of UPS had no effect on memory consolidation and reconsolidation but impedes the amnesic action of protein synthesis inhibition after retrieval. The last finding suggests a specific action of the UPS inhibitor on memory labilization. However, another interpretation is possible in terms of the synthesis/degradation balance of positive and negative elements in neural plasticity, as was found in the case of long-term potentiation. To evaluate these alternative interpretations, other reconsolidation-interfering drugs than translation inhibitors should be tested. Here we analyzed initially the UPS inhibitor effect in contextual conditioning in crabs. We found that UPS inhibition during consolidation impaired long-term memory. In contrast, UPS inhibition did not affect memory reconsolidation after contextual retrieval but, in fact, impeded memory labilization, blocking the action of drugs that does not affect directly the protein synthesis. To extend these finding to vertebrates, we performed similar experiments in contextual fear memory in mice. We found that the UPS inhibitor in hippocampus affected memory consolidation and blocked memory labilization after retrieval. These findings exclude alternative interpretations to the requirement of UPS in memory labilization and give evidence of this mechanism in both vertebrates and invertebrates. PMID:25135196

  12. Protein degradation by ubiquitin-proteasome system in formation and labilization of contextual conditioning memory.

    PubMed

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

    2014-09-01

    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 case, the inhibition of the UPS during consolidation impairs memory. Similar results were reported for memory reconsolidation. However, in other cases, the inhibition of UPS had no effect on memory consolidation and reconsolidation but impedes the amnesic action of protein synthesis inhibition after retrieval. The last finding suggests a specific action of the UPS inhibitor on memory labilization. However, another interpretation is possible in terms of the synthesis/degradation balance of positive and negative elements in neural plasticity, as was found in the case of long-term potentiation. To evaluate these alternative interpretations, other reconsolidation-interfering drugs than translation inhibitors should be tested. Here we analyzed initially the UPS inhibitor effect in contextual conditioning in crabs. We found that UPS inhibition during consolidation impaired long-term memory. In contrast, UPS inhibition did not affect memory reconsolidation after contextual retrieval but, in fact, impeded memory labilization, blocking the action of drugs that does not affect directly the protein synthesis. To extend these finding to vertebrates, we performed similar experiments in contextual fear memory in mice. We found that the UPS inhibitor in hippocampus affected memory consolidation and blocked memory labilization after retrieval. These findings exclude alternative interpretations to the requirement of UPS in memory labilization and give evidence of this mechanism in both vertebrates and invertebrates.

  13. Cystein-specific ubiquitination protects the peroxisomal import receptor Pex5p against proteasomal degradation.

    PubMed

    Schwartzkopff, Benjamin; Platta, Harald W; Girzalsky, Wolfgang; Erdmann, Ralf

    2015-05-14

    Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type 1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p. These mechanoenzymes recognize ubiquitinated Pex5p-species as substrates for membrane extraction. The PTS1-receptor is either polyubiquitinated via peptide-bonds at two certain lysines and results in proteasomal degradation, or monoubiquitinated via a thioester-bond at a conserved cysteine, which enables the recycling of Pex5p and further rounds of matrix protein import. To investigate the physiological relevance of the conserved N-terminal cysteine of Pex5p, the known target amino acids for ubiquitination were substituted by site-directed mutagenesis. In contrast to Pex5pC6A, Pex5pC6K turned out to be functional in PTS1 import and utilization of oleic acid, independent of the lysines at position 18 and 24. In contrast to wild-type Pex5p, Pex5pC6K displays an ubiquitination pattern, similar to the polyubiquitination pattern of Pex4p or Pex22p mutant strains. Moreover, Pex5pC6K displays a significantly reduced steady-state level when the deubiquitination enzyme Ubp15p is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for Pex5p function. The presence of the cysteine prevents polyubiquitination and rapid degradation of Pex5p.

  14. Cysteine-specific ubiquitination protects the peroxisomal import receptor Pex5p against proteasomal degradation

    PubMed Central

    Schwartzkopff, Benjamin; Platta, Harald W.; Hasan, Sohel; Girzalsky, Wolfgang; Erdmann, Ralf

    2015-01-01

    Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type 1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p. These mechanoenzymes recognize ubiquitinated Pex5p-species as substrates for membrane extraction. The PTS1-receptor is either polyubiquitinated via peptide bonds at two certain lysines and results in proteasomal degradation or monoubiquitinated via a thioester-bond at a conserved cysteine, which enables the recycling of Pex5p and further rounds of matrix protein import. To investigate the physiological relevance of the conserved N-terminal cysteine of Pex5p, the known target amino acids for ubiquitination were substituted by site-directed mutagenesis. In contrast with Pex5pC6A, Pex5pC6K turned out to be functional in PTS1 import and utilization of oleic acid, independent of the lysines at position 18 and 24. In contrast with wild-type Pex5p, Pex5pC6K displays an ubiquitination pattern, similar to the polyubiquitination pattern of Pex4p or Pex22p mutant strains. Moreover, Pex5pC6K displays a significantly reduced steady-state level when the deubiquitinating enzyme Ubp15p is missing. Thus, our results indicate that not the cysteine residue but the position of ubiquitination is important for Pex5p function. The presence of the cysteine prevents polyubiquitination and rapid degradation of Pex5p. PMID:26182377

  15. Gastrin Induces Nuclear Export and Proteasome Degradation of Menin in Enteric Glial Cells.

    PubMed

    Sundaresan, Sinju; Meininger, Cameron A; Kang, Anthony J; Photenhauer, Amanda L; Hayes, Michael M; Sahoo, Nirakar; Grembecka, Jolanta; Cierpicki, Tomasz; Ding, Lin; Giordano, Thomas J; Else, Tobias; Madrigal, David J; Low, Malcolm J; Campbell, Fiona; Baker, Ann-Marie; Xu, Haoxing; Wright, Nicholas A; Merchant, Juanita L

    2017-08-28

    The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors (NETs) such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner's glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia. Primary enteric glial cultures were generated from the VillinCre:Men(1FL/FL):Sst(-/-) mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from VillinCre:Men1FL/FL:Sst(+/+) mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom. Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells, e.g., p75 and S100B, colocalized with gastrin in human duodenal

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

    PubMed

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

    2014-06-24

    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.

  17. DNA Damage Is a Prerequisite for p53-Mediated Proteasomal Degradation of HIF-1α in Hypoxic Cells and Downregulation of the Hypoxia Marker Carbonic Anhydrase IX

    PubMed Central

    Kaluzová, Milota; Kaluz, Stefan; Lerman, Michael I.; Stanbridge, Eric J.

    2004-01-01

    We investigated the relationship between the tumor suppressor p53 and the hypoxia-inducible factor-1 (HIF-1)-dependent expression of the hypoxia marker, carbonic anhydrase IX (CAIX). MCF-7 (wt p53) and Saos-2 (p53-null) cells displayed similar induction of CAIX expression and CA9 promoter activity under hypoxic conditions. Activation of p53 by the DNA damaging agent mitomycin C (MC) was accompanied by a potent repression of CAIX expression and the CA9 promoter in MCF-7 but not in Saos-2 cells. The activated p53 mediated increased proteasomal degradation of HIF-1α protein, resulting in considerably lower steady-state levels of HIF-1α protein in hypoxic MCF-7 cells but not in Saos-2 cells. Overexpression of HIF-1α relieved the MC-induced repression in MCF-7 cells, confirming regulation at the HIF-1α level. Similarly, CA9 promoter activity was downregulated by MC in HCT 116 p53+/+ but not the isogenic p53−/− cells. Activated p53 decreased HIF-1α protein levels by accelerated proteasome-dependent degradation without affecting significantly HIF-1α transcription. In summary, our results demonstrate that the presence of wtp53 under hypoxic conditions has an insignificant effect on the stabilization of HIF-1α protein and HIF-1-dependent expression of CAIX. However, upon activation by DNA damage, wt p53 mediates an accelerated degradation of HIF-1α protein, resulting in reduced activation of CA9 transcription and, correspondingly, decreased levels of CAIX protein. A model outlining the quantitative relationship between p53, HIF-1α, and CAIX is presented. PMID:15199132

  18. Proteasome-Dependent Degradation of Daxx by the Viral E1B-55K Protein in Human Adenovirus-Infected Cells ▿

    PubMed Central

    Schreiner, Sabrina; Wimmer, Peter; Sirma, Hüseyin; Everett, Roger D.; Blanchette, Paola; Groitl, Peter; Dobner, Thomas

    2010-01-01

    The death-associated protein Daxx found in PML (promyelocytic leukemia protein) nuclear bodies (PML-NBs) is involved in transcriptional regulation and cellular intrinsic antiviral resistence against incoming viruses. We found that knockdown of Daxx in a nontransformed human hepatocyte cell line using RNA interference (RNAi) techniques results in significantly increased adenoviral (Ad) replication, including enhanced viral mRNA synthesis and viral protein expression. This Daxx restriction imposed upon adenovirus growth is counteracted by early protein E1B-55K (early region 1B 55-kDa protein), a multifunctional regulator of cell-cycle-independent Ad5 replication. The viral protein binds to Daxx and induces its degradation through a proteasome-dependent pathway. We show that this process is independent of Ad E4orf6 (early region 4 open reading frame 6), known to promote the proteasomal degradation of cellular p53, Mre11, DNA ligase IV, and integrin α3 in combination with E1B-55K. These results illustrate the importance of the PML-NB-associated factor Daxx in virus growth restriction and suggest that E1B-55K antagonizes innate antiviral activities of Daxx and PML-NBs to stimulate viral replication at a posttranslational level. PMID:20484509

  19. BP-1T, an antiangiogenic benzophenone-thiazole pharmacophore, counteracts HIF-1 signalling through p53/MDM2-mediated HIF-1α proteasomal degradation.

    PubMed

    Thirusangu, Prabhu; Vigneshwaran, V; Prashanth, T; Vijay Avin, B R; Malojirao, Vikas H; Rakesh, H; Khanum, Shaukath Ara; Mahmood, Riaz; Prabhakar, B T

    2017-02-01

    Hypoxia is a feature of all solid tumours, contributing to tumour progression. Activation of HIF-1α plays a critical role in promoting tumour angiogenesis and metastasis. Since its expression is positively correlated with poor prognosis for cancer patients, HIF-1α is one of the most convincing anticancer targets. BP-1T is a novel antiproliferative agent with promising antiangiogenic effects. In the present study, the molecular mechanism underlying cytotoxic/antiangiogenic effects of BP-1T on tumour/non-tumour angiogenesis was evaluated. Evidences show that BP-1T exhibits potent cytotoxicity with prolonged activity and effectively regressed neovessel formation both in reliable non-tumour and tumour angiogenic models. The expression of CoCl2-induced HIF-1α was inhibited by BP-1T in various p53 (WT)-expressing cancer cells, including A549, MCF-7 and DLA, but not in mutant p53-expressing SCC-9 cells. Mechanistically, BP-1T mediates the HIF-1α proteasomal degradation by activating p53/MDM2 pathway and thereby downregulated HIF-1α-dependent angiogenic genes such as VEGF-A, Flt-1, MMP-2 and MMP-9 under hypoxic condition of in vitro and in vivo solid tumour, eventually leading to abolition of migration and invasion. Based on these observations, we conclude that BP-1T acts on HIF-1α degradation through p53/MDM2 proteasome pathway.

  20. Design Principles Involving Protein Disorder Facilitate Specific Substrate Selection and Degradation by the Ubiquitin-Proteasome System*

    PubMed Central

    Guharoy, Mainak; Bhowmick, Pallab; Tompa, Peter

    2016-01-01

    The ubiquitin-proteasome system (UPS) regulates diverse cellular pathways by the timely removal (or processing) of proteins. Here we review the role of structural disorder and conformational flexibility in the different aspects of degradation. First, we discuss post-translational modifications within disordered regions that regulate E3 ligase localization, conformation, and enzymatic activity, and also the role of flexible linkers in mediating ubiquitin transfer and reaction processivity. Next we review well studied substrates and discuss that substrate elements (degrons) recognized by E3 ligases are highly disordered: short linear motifs recognized by many E3s constitute an important class of degrons, and these are almost always present in disordered regions. Substrate lysines targeted for ubiquitination are also often located in neighboring regions of the E3 docking motifs and are therefore part of the disordered segment. Finally, biochemical experiments and predictions show that initiation of degradation at the 26S proteasome requires a partially unfolded region to facilitate substrate entry into the proteasomal core. PMID:26851277

  1. Regulation of the Arabidopsis GSK3-like Kinase BRASSINOSTEROID-INSENSITIVE 2 through Proteasome-Mediated Protein Degradation

    PubMed Central

    Peng, Peng; Yan, Zhenyan; Zhu, Yongyou; Li, Jianming

    2008-01-01

    Glycogen synthase kinase 3 (GSK3) is a unique serine/threonine kinase that is implicated in a variety of cellular processes and is regulated by phosphorylation or protein–protein interaction in animal cells. BIN2 is an Arabidopsis GSK3-like kinase that negatively regulates brassinosteroid (BR) signaling. Genetic studies suggested that BIN2 is inhibited in response to BR perception at the cell surface to relieve its inhibitory effects on downstream targets; however, little is known about biochemical mechanisms of its inhibition. Here, we show that BIN2 is regulated by proteasome-mediated protein degradation. Exogenous application of a BR biosynthesis inhibitor and an active BR increased and decreased the amount of BIN2 proteins, respectively. Interestingly, the gain-of-function bin2-1 mutation significantly stabilizes BIN2, making it unresponsive to BR-induced BIN2 depletion. Exogenous application of different plant growth hormones revealed that BIN2 depletion is specifically induced by BR through a functional BR receptor, while treatment of a proteasome inhibitor, MG132, not only prevented the BR-induced BIN2 depletion but also nullified the inhibitory effect of BR on the BIN2 kinase activity. Taken together, our results strongly suggest that proteasome-mediated protein degradation constitutes an important regulatory mechanism for restricting the BIN2 activity. PMID:18726001

  2. MM-1 facilitates degradation of c-Myc by recruiting proteasome and a novel ubiquitin E3 ligase.

    PubMed

    Kimura, Yumiko; Nagao, Arisa; Fujioka, Yuko; Satou, Akiko; Taira, Takahiro; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2007-10-01

    We have reported that a novel c-Myc-binding protein, MM-1, repressed the E-box-dependent transcription activity of c-Myc by recruiting the HDAC1 complex via TIF1beta/KAP1, a transcriptional corepressor. We have also reported that a mutation of A157R in MM-1, which is often observed in patients with leukemia or lymphoma, abrogated all of the repressive activities of MM-1 toward c-Myc, indicating that MM-1 is a novel tumor suppressor. In this study, we found that MM-1 was bound to a component of proteasome and stimulated degradation of c-Myc in human cells. Knockdown of endogenous MM-1 in human HeLa cells by introduction of siRNA against MM-1 stabilized the endogenous c-Myc. To identify proteins that participate in c-Myc degradation by MM-1, in vivo and in vitro binding assays were carried out. The results showed that MM-1 directly bound to Rpt3, a subunit of 26S proteasome, and that c-Myc directly bound to Skp2, which recruited ElonginC, ElonginB and Cullin2, thereby forming a novel ubiquitin E3 ligase. Knockdown of endogenous Cullin2 stabilized the endogenous c-Myc. Thus, MM-1 is a factor that connects c-Myc to the ubiquitin E3 ligase and the proteasome.

  3. A Novel Role for ATM in Regulating Proteasome-Mediated Protein Degradation through Suppression of the ISG15 Conjugation Pathway

    PubMed Central

    Wood, Laurence M.; Sankar, Surendran; Reed, Ryan E.; Haas, Arthur L.; Liu, Leroy F.; McKinnon, Peter; Desai, Shyamal D.

    2011-01-01

    Ataxia Telangiectasia (A-T) is an inherited immunodeficiency disorder wherein mutation of the ATM kinase is responsible for the A-T pathogenesis. Although the precise role of ATM in A-T pathogenesis is still unclear, its function in responding to DNA damage has been well established. Here we demonstrate that in addition to its role in DNA repair, ATM also regulates proteasome-mediated protein turnover through suppression of the ISG15 pathway. This conclusion is based on three major pieces of evidence: First, we demonstrate that proteasome-mediated protein degradation is impaired in A-T cells. Second, we show that the reduced protein turnover is causally linked to the elevated expression of the ubiquitin-like protein ISG15 in A-T cells. Third, we show that expression of the ISG15 is elevated in A-T cells derived from various A-T patients, as well as in brain tissues derived from the ATM knockout mice and A-T patients, suggesting that ATM negatively regulates the ISG15 pathway. Our current findings suggest for the first time that proteasome-mediated protein degradation is impaired in A-T cells due to elevated expression of the ISG15 conjugation pathway, which could contribute to progressive neurodegeneration in A-T patients. PMID:21298066

  4. Induced degradation of Tat by nucleocapsid (NC) via the proteasome pathway and its effect on HIV transcription.

    PubMed

    Hong, Hye-Won; Lee, Seong-Wook; Myung, Heejoon

    2013-04-23

    Human Immunodeficiency Virus type 1 (HIV-1) is a retrovirus that causes acquired immunodeficiency syndrome (AIDS). HIV-1 Tat protein upregulates transcriptional transactivation. The nucleocapsid protein NC of HIV-1 is a component of virion and plays a key role in genome packaging. Herein, we have demonstrated the interaction between NC and Tat by means of a yeast two-hybrid assay, GST pull-down analysis, co-immunoprecipitation and subcellular colocalization analysis. We observed that the level of Tat was significantly reduced in the presence of NC. But NC did not affect mRNA expression level of Tat. The level of Tat in the presence of NC was increased by treating cells with a proteasome inhibitor, MG132. The ubiquitination state of Tat was not seen to increase in the presence of NC, suggesting the proteasomal degradation was independent of ubiquitination. Lowered level of Tat in the presence of NC led to a decrease in Tat-mediated transcriptional transactivation.

  5. Retinoic acid induces proteasome-dependent degradation of retinoic acid receptor α (RARα) and oncogenic RARα fusion proteins

    PubMed Central

    Zhu, Jun; Gianni, Maurizio; Kopf, Eliezer; Honoré, Nicole; Chelbi-Alix, Mounira; Koken, Marcel; Quignon, Frédérique; Rochette-Egly, Cécile; de Thé, Hugues

    1999-01-01

    Analyzing the pathways by which retinoic acid (RA) induces promyelocytic leukemia/retinoic acid receptor α (PML/RARα) catabolism in acute promyelocytic leukemia (APL), we found that, in addition to caspase-mediated PML/RARα cleavage, RA triggers degradation of both PML/RARα and RARα. Similarly, in non-APL cells, RA directly targeted RARα and RARα fusions to the proteasome degradation pathway. Activation of either RARα or RXRα by specific agonists induced degradation of both proteins. Conversely, a mutation in RARα that abolishes heterodimer formation and DNA binding, blocked both RARα and RXRα degradation. Mutations in the RARα DNA-binding domain or AF-2 transcriptional activation region also impaired RARα catabolism. Hence, our results link transcriptional activation to receptor catabolism and suggest that transcriptional up-regulation of nuclear receptors by their ligands may be a feedback mechanism allowing sustained target-gene activation. PMID:10611294

  6. Ubiquitin C-terminal hydrolase-L1 protects cystic fibrosis transmembrane conductance regulator from early stages of proteasomal degradation.

    PubMed

    Henderson, Mark J; Vij, Neeraj; Zeitlin, Pamela L

    2010-04-09

    DeltaF508 cystic fibrosis transmembrane conductance regulator (CFTR) degradation involves ubiquitin modification and efficient proteasomal targeting of the nascent misfolded protein. We show that a deubiquitinating enzyme, ubiquitin C-terminal hydrolase-L1 (UCH-L1), is highly expressed in cystic fibrosis (CF) airway epithelial cells in vitro and in vivo. We hypothesized that the elevation in UCH-L1 in CF cells represents a cellular adaptation to counterbalance excessive proteasomal degradation. The bronchial epithelial cell lines IB3-1 (CF, high UCH-L1 expression) and S9 (non-CF, low UCH-L1 expression) were transiently transfected with wild type (WT) or DeltaF508 CFTR, WT UCH-L1 or small interfering RNA-UCH-L1, and a variety of ubiquitin mutants. We observed a positive correlation between UCH-L1 expression and steady state levels of WT- or DeltaF508-CFTR, and this stabilizing effect was confined to the early stages of CFTR synthesis. Immunolocalization of UCH-L1 by confocal microscopy revealed a partial co-localization with a ribosomal subunit and the endoplasmic reticulum. The UCH-L1-associated increase in CFTR levels was correlated with an increase in ubiquitinated CFTR (CFTR-Ub). Co-transfection with mutant ubiquitins and treatment with proteasome inhibitors suggested that UCH-L1 was reducing the proteasomal targeting of CFTR during synthesis by shortening conjugated polyubiquitin chains. Although not sufficient by itself to rescue mutant CFTR therapeutically, the elevation of UCH-L1 and its effect on CFTR processing provides insight into its potential roles in CF and other diseases.

  7. CD4 Glycoprotein Degradation Induced by Human Immunodeficiency Virus Type 1 Vpu Protein Requires the Function of Proteasomes and the Ubiquitin-Conjugating Pathway

    PubMed Central

    Schubert, Ulrich; Antón, Luis C.; Bačík, Igor; Cox, Josephine H.; Bour, Stéphane; Bennink, Jack R.; Orlowski, Marian; Strebel, Klaus; Yewdell, Jonathan W.

    1998-01-01

    The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic β subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E1 or (ii) following expression of a mutant form of ubiquitin (Lys48 mutated to Arg48) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic

  8. Rapid degradation of mutant SLC25A46 by the ubiquitin-proteasome system results in MFN1/2-mediated hyperfusion of mitochondria.

    PubMed

    Steffen, Janos; Vashisht, Ajay A; Wan, Jijun; Jen, Joanna C; Claypool, Steven M; Wohlschlegel, James A; Koehler, Carla M

    2017-03-01

    SCL25A46 is a mitochondrial carrier protein that surprisingly localizes to the outer membrane and is distantly related to Ugo1. Here we show that a subset of SLC25A46 interacts with mitochondrial dynamics components and the MICOS complex. Decreased expression of SLC25A46 results in increased stability and oligomerization of MFN1 and MFN2 on mitochondria, promoting mitochondrial hyperfusion. A mutation at L341P causes rapid degradation of SLC25A46, which manifests as a rare disease, pontocerebellar hypoplasia. The E3 ubiquitin ligases MULAN and MARCH5 coordinate ubiquitylation of SLC25A46 L341P, leading to degradation by organized activities of P97 and the proteasome. Whereas outer mitochondrial membrane-associated degradation is typically associated with apoptosis or a specialized type of autophagy termed mitophagy, SLC25A46 degradation operates independently of activation of outer membrane stress pathways. Thus SLC25A46 is a new component in mitochondrial dynamics that serves as a regulator for MFN1/2 oligomerization. Moreover, SLC25A46 is selectively degraded from the outer membrane independently of mitophagy and apoptosis, providing a framework for mechanistic studies in the proteolysis of outer membrane proteins.

  9. A non-muscle myosin II motor links NR1 to retrograde trafficking and proteasomal degradation in PC12 cells.

    PubMed

    Vazhappilly, Rema; Wee, Karen Siaw-Ling; Sucher, Nikolaus J; Low, Chian-Ming

    2010-03-01

    Rat pheochromocytoma (PC12) cells have been shown to lack functional NMDA receptors; yet, these cells express NR1 subunits of the NMDA receptor. The reason for the lack of functional receptors has been attributed to the absence of significant levels of NR2 subunits to co-assemble with NR1. It is known that PC12 expresses very low levels of NR2C, with complete absence of other types of NR2 subunits. The purpose of the present study is to describe the molecular mechanism of trafficking and degradation of unassembled NR1 subunits in PC12 cells. The localization of NR1 subunits in PC12 cells were evaluated by immunofluorescence and co-immunoprecipitation, which showed that NR1 was present in the endoplasmic reticulum and cis-middle compartments of the Golgi apparatus. Upon treatment with a proteasome inhibitor, MG132, the ubiquitinylated species of NR1 subunit were detected, suggesting that NR1 is being targeted for endoplasmic reticulum-associated proteasomal degradation. Our previous studies suggest that NR1 subunits from the Golgi do not proceed to trans-Golgi, hence they will require re-routing to the endoplasmic reticulum for degradation. Further investigations on the factors involved in the trafficking of NR1 from Golgi to endoplasmic reticulum were performed using co-immunoprecipitation and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. These revealed the co-association of NR1 with non-muscle myosin heavy chain II isoforms A and B. We also demonstrate the functional significance of this interaction through the use of a myosin inhibitor, blebbistatin, to disrupt brefeldin A-induced Golgi-to-endoplasmic reticulum trafficking of NR1. In conclusion, our results suggest that non-muscle myosin II is involved in the retrograde trafficking of NR1 subunits from the cis/middle-Golgi to the endoplasmic reticulum for proteasomal degradation in PC12.

  10. 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

  11. Androgens upregulate Cdc25C protein by inhibiting its proteasomal and lysosomal degradation pathways.

    PubMed

    Chou, Yu-Wei; Zhang, Li; 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.

  12. Cancer cell death induced by novel small molecules degrading the TACC3 protein via the ubiquitin-proteasome pathway.

    PubMed

    Ohoka, N; Nagai, K; Hattori, T; Okuhira, K; Shibata, N; Cho, N; Naito, M

    2014-11-06

    The selective degradation of target proteins with small molecules is a novel approach to the treatment of various diseases, including cancer. We have developed a protein knockdown system with a series of hybrid small compounds that induce the selective degradation of target proteins via the ubiquitin-proteasome pathway. In this study, we designed and synthesized novel small molecules called SNIPER(TACC3)s, which target the spindle regulatory protein transforming acidic coiled-coil-3 (TACC3). SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3 and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/C(CDH1) mediates the SNIPER(TACC3)-induced degradation of TACC3. Intriguingly, SNIPER(TACC3) selectively induced cell death in cancer cells expressing a larger amount of TACC3 protein than normal cells. These results suggest that protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy for treating cancers overexpressing the TACC3 protein.

  13. Cystic Fibrosis Transmembrane Conductance Regulator Controls Lung Proteasomal Degradation and Nuclear Factor-κB Activity in Conditions of Oxidative Stress

    PubMed Central

    Boncoeur, Emilie; Roque, Telma; Bonvin, Elise; Saint-Criq, Vinciane; Bonora, Monique; Clement, Annick; Tabary, Olivier; Henrion-Caude, Alexandra; Jacquot, Jacky

    2008-01-01

    Cystic fibrosis is a lethal inherited disorder caused by mutations in a single gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein, resulting in progressive oxidative lung damage. In this study, we evaluated the role of CFTR in the control of ubiquitin-proteasome activity and nuclear factor (NF)-κB/IκB-α signaling after lung oxidative stress. After a 64-hour exposure to hyperoxia-mediated oxidative stress, CFTR-deficient (cftr−/−) mice exhibited significantly elevated lung proteasomal activity compared with wild-type (cftr+/+) animals. This was accompanied by reduced lung caspase-3 activity and defective degradation of NF-κB inhibitor IκB-α. In vitro, human CFTR-deficient lung cells exposed to oxidative stress exhibited increased proteasomal activity and decreased NF-κB-dependent transcriptional activity compared with CFTR-sufficient lung cells. Inhibition of the CFTR Cl− channel by CFTRinh-172 in the normal bronchial immortalized cell line 16HBE14o− increased proteasomal degradation after exposure to oxidative stress. Caspase-3 inhibition by Z-DQMD in CFTR-sufficient lung cells mimicked the response profile of increased proteasomal degradation and reduced NF-κB activity observed in CFTR-deficient lung cells exposed to oxidative stress. Taken together, these results suggest that functional CFTR Cl− channel activity is crucial for regulation of lung proteasomal degradation and NF-κB activity in conditions of oxidative stress. PMID:18372427

  14. Bifunctional Anti-Huntingtin Proteasome-Directed Intrabodies Mediate Efficient Degradation of Mutant Huntingtin Exon 1 Protein Fragments

    PubMed Central

    Butler, David C.; Messer, Anne

    2011-01-01

    Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide (CAG)n repeat expansion in the coding sequence of the huntingtin gene, and an expanded polyglutamine (>37Q) tract in the protein. This results in misfolding and accumulation of huntingtin protein (htt), formation of neuronal intranuclear and cytoplasmic inclusions, and neuronal dysfunction/degeneration. Single-chain Fv antibodies (scFvs), expressed as intrabodies that bind htt and prevent aggregation, show promise as immunotherapeutics for HD. Intrastriatal delivery of anti-N-terminal htt scFv-C4 using an adeno-associated virus vector (AAV2/1) significantly reduces the size and number of aggregates in HDR6/1 transgenic mice; however, this protective effect diminishes with age and time after injection. We therefore explored enhancing intrabody efficacy via fusions to heterologous functional domains. Proteins containing a PEST motif are often targeted for proteasomal degradation and generally have a short half life. In ST14A cells, fusion of the C-terminal PEST region of mouse ornithine decarboxylase (mODC) to scFv-C4 reduces htt exon 1 protein fragments with 72 glutamine repeats (httex1-72Q) by ∼80–90% when compared to scFv-C4 alone. Proteasomal targeting was verified by either scrambling the mODC-PEST motif, or via proteasomal inhibition with epoxomicin. For these constructs, the proteasomal degradation of the scFv intrabody proteins themselves was reduced<25% by the addition of the mODC-PEST motif, with or without antigens. The remaining intrabody levels were amply sufficient to target N-terminal httex1-72Q protein fragment turnover. Critically, scFv-C4-PEST prevents aggregation and toxicity of httex1-72Q fragments at significantly lower doses than scFv-C4. Fusion of the mODC-PEST motif to intrabodies is a valuable general approach to specifically target toxic antigens to the proteasome for degradation. PMID:22216210

  15. Sperm proteasome degrades egg envelope glycoprotein ZP1 during fertilization of Japanese quail (Coturnix japonica).

    PubMed

    Sasanami, Tomohiro; Sugiura, Kenichi; Tokumoto, Toshinobu; Yoshizaki, Norio; Dohra, Hideo; Nishio, Shunsuke; Mizushima, Shusei; Hiyama, Gen; Matsuda, Tsukasa

    2012-10-01

    At the time of fertilization, the extracellular matrix surrounding avian oocytes, termed the perivitelline membrane (pvm), is hydrolyzed by a sperm-borne protease, although the actual protease that is responsible for the digestion of the pvm remains to be identified. Here, we show evidence that the ubiquitin-proteasome system is functional in the fertilization of Japanese quail. The activities for the induction of the acrosome reaction and binding to ZP3 as revealed by ligand blotting of purified serum ZP1 are similar to those of pvm ZP1. Western blot analysis of purified ZP1 and ZP3 by the use of the anti-ubiquitin antibody showed that only pvm ZP1 was reactive to the antibody. In vitro penetration assay of the sperm on the pvm indicated that fragments of ZP1 and intact ZP3 were released from the pvm. Western blot analysis using the anti-20S proteasome antibody and ultrastructural analysis showed that immunoreactive proteasome was localized in the acrosomal region of the sperm. Inclusion of specific proteasome inhibitor MG132 in the incubation mixture, or depletion of extracellular ATP by the addition of apyrase, efficiently suppressed the sperm perforation of the pvm. These results demonstrate for the first time that the sperm proteasome is important for fertilization in birds and that the extracellular ubiquitination of ZP1 might occur during its transport via blood circulation.

  16. Fast axonal transport of the proteasome complex depends on membrane interaction and molecular motor function.

    PubMed

    Otero, Maria G; Alloatti, Matías; Cromberg, Lucas E; Almenar-Queralt, Angels; Encalada, Sandra E; Pozo Devoto, Victorio M; Bruno, Luciana; Goldstein, Lawrence S B; Falzone, Tomás L

    2014-04-01

    Protein degradation by the ubiquitin-proteasome system in neurons depends on the correct delivery of the proteasome complex. In neurodegenerative diseases, aggregation and accumulation of proteins in axons link transport defects with degradation impairments; however, the transport properties of proteasomes remain unknown. Here, using in vivo experiments, we reveal the fast anterograde transport of assembled and functional 26S proteasome complexes. A high-resolution tracking system to follow fluorescent proteasomes revealed three types of motion: actively driven proteasome axonal transport, diffusive behavior in a viscoelastic axonema and proteasome-confined motion. We show that active proteasome transport depends on motor function because knockdown of the KIF5B motor subunit resulted in impairment of the anterograde proteasome flux and the density of segmental velocities. Finally, we reveal that neuronal proteasomes interact with intracellular membranes and identify the coordinated transport of fluorescent proteasomes with synaptic precursor vesicles, Golgi-derived vesicles, lysosomes and mitochondria. Taken together, our results reveal fast axonal transport as a new mechanism of proteasome delivery that depends on membrane cargo 'hitch-hiking' and the function of molecular motors. We further hypothesize that defects in proteasome transport could promote abnormal protein clearance in neurodegenerative diseases.

  17. C-terminus of HSC70-Interacting Protein (CHIP) Inhibits Adipocyte Differentiation via Ubiquitin- and Proteasome-Mediated Degradation of PPARγ.

    PubMed

    Kim, Jung-Hoon; Shin, Soyeon; Seo, Jinho; Lee, Eun-Woo; Jeong, Manhyung; Lee, Min-Sik; Han, Hyun-Ji; Song, Jaewhan

    2017-01-06

    PPARγ (Peroxisome proliferator-activated receptor γ) is a nuclear receptor involved in lipid homeostasis and related metabolic diseases. Acting as a transcription factor, PPARγ is a master regulator for adipocyte differentiation. Here, we reveal that CHIP (C-terminus of HSC70-interacting protein) suppresses adipocyte differentiation by functioning as an E3 ligase of PPARγ. CHIP directly binds to and induces ubiquitylation of the PPARγ protein, leading to proteasome-dependent degradation. Stable overexpression or knockdown of CHIP inhibited or promoted adipogenesis, respectively, in 3T3-L1 cells. On the other hand, a CHIP mutant defective in E3 ligase could neither regulate PPARγ protein levels nor suppress adipogenesis, indicating the importance of CHIP-mediated ubiquitylation of PPARγ in adipocyte differentiation. Lastly, a CHIP null embryo fibroblast exhibited augmented adipocyte differentiation with increases in PPARγ and its target protein levels. In conclusion, CHIP acts as an E3 ligase of PPARγ, suppressing PPARγ-mediated adipogenesis.

  18. A conserved serine residue regulates the stability of Drosophila Salvador and human WW domain-containing adaptor 45 through proteasomal degradation.

    PubMed

    Wu, Di; Wu, Shian

    2013-04-19

    The Hippo (Hpo) pathway is a conserved tumor suppressor pathway that controls organ size through the coordinated regulation of apoptosis and proliferation. Drosophila Salvador (Sav), which limits organ size, is a core component of the Hpo pathway. In this study, Ser-17 was shown to be important for the stability of Sav. Alanine mutation of Ser-17 promoted the proteasomal degradation of Sav. Destabilization and stabilization of the Sav protein mediated by alanine mutation of Ser-17 and by Hpo, respectively, were independent of each other. This implies that the stability of Sav is controlled by two mechanisms, one that is Ser-17-dependent and Hpo-independent, and another that is Ser-17-independent and Hpo-dependent. These dual mechanisms also regulated the human counterpart of Drosophila Sav, WW domain-containing adaptor 45 (WW45). The conservation of this regulation adds to its significance in normal physiology and tumorigenesis.

  19. C-terminus of HSC70-Interacting Protein (CHIP) Inhibits Adipocyte Differentiation via Ubiquitin- and Proteasome-Mediated Degradation of PPARγ

    PubMed Central

    Kim, Jung-Hoon; Shin, Soyeon; Seo, Jinho; Lee, Eun-Woo; Jeong, Manhyung; Lee, Min-sik; Han, Hyun-Ji; Song, Jaewhan

    2017-01-01

    PPARγ (Peroxisome proliferator-activated receptor γ) is a nuclear receptor involved in lipid homeostasis and related metabolic diseases. Acting as a transcription factor, PPARγ is a master regulator for adipocyte differentiation. Here, we reveal that CHIP (C-terminus of HSC70-interacting protein) suppresses adipocyte differentiation by functioning as an E3 ligase of PPARγ. CHIP directly binds to and induces ubiquitylation of the PPARγ protein, leading to proteasome-dependent degradation. Stable overexpression or knockdown of CHIP inhibited or promoted adipogenesis, respectively, in 3T3-L1 cells. On the other hand, a CHIP mutant defective in E3 ligase could neither regulate PPARγ protein levels nor suppress adipogenesis, indicating the importance of CHIP-mediated ubiquitylation of PPARγ in adipocyte differentiation. Lastly, a CHIP null embryo fibroblast exhibited augmented adipocyte differentiation with increases in PPARγ and its target protein levels. In conclusion, CHIP acts as an E3 ligase of PPARγ, suppressing PPARγ-mediated adipogenesis. PMID:28059128

  20. Rapid Degradation of Auxin/Indoleacetic Acid Proteins Requires Conserved Amino Acids of Domain II and Is Proteasome Dependent

    PubMed Central

    Ramos, Jason A.; Zenser, Nathan; Leyser, Ottoline; Callis, Judy

    2001-01-01

    Auxin rapidly induces auxin/indoleacetic acid (Aux/IAA) transcription. The proteins encoded are short-lived nucleus-localized transcriptional regulators that share four conserved domains. In a transient assay measuring protein accumulation, an Aux/IAA 13–amino acid domain II consensus sequence was sufficient to target firefly luciferase (LUC) for low protein accumulation equivalent to that observed previously for full-length PSIAA6. Single amino acid substitutions in these 13 amino acids, corresponding to known auxin response mutants, resulted in a sixfold to 20-fold increase in protein accumulation. Naturally occurring variant amino acids had no effect. Residues identified as essential by single alanine substitutions were not sufficient when all flanking amino acids were alanine, indicating the importance of flanking regions. Using direct protein degradation measurements in transgenic Arabidopsis seedlings, full-length IAA1, PSIAA6, and the N-terminal 73 PSIAA6 amino acids targeted LUC for rapid degradation with 8-min half-lives. The C-terminal 109 amino acids did not affect LUC half-life. Smaller regions containing domain II also targeted LUC for rapid degradation, but the rates were not equivalent to those of the full-length protein. A single domain II substitution in the context of full-length PSIAA6 increased half-life 30-fold. Proteasome inhibitors affected Aux/IAA::LUC fusion protein accumulation, demonstrating the involvement of the proteasome. PMID:11595806

  1. Proteasome-mediated degradation of integral inner nuclear membrane protein emerin in fibroblasts lacking A-type lamins

    SciTech Connect

    Muchir, Antoine; Massart, Catherine; Engelen, Baziel G. van; Lammens, Martin; Bonne, Gisele; Worman, Howard J. . E-mail: hjw14@columbia.edu

    2006-12-29

    We previously identified and characterized a homozygous LMNA nonsense mutation leading to the absence of A-type lamins in a premature neonate who died at birth. We show here that the absence of A-type lamins is due to degradation of the aberrant mRNA transcript with a premature termination codon. In cultured fibroblasts from the subject with the homozygous LMNA nonsense mutation, there was a decreased steady-state expression of the integral inner nuclear membrane proteins emerin and nesprin-1{alpha} associated with their mislocalization to the bulk endoplasmic reticulum and a hyperphosphorylation of emerin. To determine if decreased emerin expression occurred post-translationally, we treated cells with a selective proteasome inhibitor and observed an increase in expression. Our results show that mislocalization of integral inner nuclear membrane proteins to the endoplasmic reticulum in human cells lacking A-type lamins leads to their degradation and provides the first evidence that their degradation is mediated by the proteasome.

  2. Regulated endoplasmic reticulum-associated degradation of a polytopic protein: p97 recruits proteasomes to Insig-1 before extraction from membranes.

    PubMed

    Ikeda, Yukio; Demartino, George N; Brown, Michael S; Lee, Joon No; Goldstein, Joseph L; Ye, Jin

    2009-12-11

    Polytopic membrane proteins subjected to endoplasmic reticulum (ER)-associated degradation are extracted from membranes and targeted to proteasomes for destruction. The extraction mechanism is poorly understood. One polytopic ER protein subjected to ER-associated degradation is Insig-1, a negative regulator of cholesterol synthesis. Insig-1 is rapidly degraded by proteasomes when cells are depleted of cholesterol, and its degradation is inhibited when sterols accumulate in cells. Insig-2, a functional homologue of Insig-1, is degraded slowly, and its degradation is not regulated by sterols. Here, we report that a single amino acid substitution in Insig-2, Insig-2(L210A), causes Insig-2 to be degraded in an accelerated and sterol-regulated manner similar to Insig-1. In seeking an explanation for the accelerated degradation, we found that proteasomes bind to wild type Insig-1 and mutant Insig-2(L210A) but not to wild type Insig-2, whereas the proteins are still embedded in cell membranes. This binding depends on at least two factors, ubiquitination of Insig and association with the ATPase p97/VCP complex. These data suggest that p97 recruits proteasomes to polytopic ER proteins even before they are extracted from membranes.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  4. Ubiquitin binding modulates IAP antagonist-stimulated proteasomal degradation of c-IAP1 and c-IAP2(1).

    PubMed

    Blankenship, John W; Varfolomeev, Eugene; Goncharov, Tatiana; Fedorova, Anna V; Kirkpatrick, Donald S; Izrael-Tomasevic, Anita; Phu, Lilian; Arnott, David; Aghajan, Mariam; Zobel, Kerry; Bazan, J Fernando; Fairbrother, Wayne J; Deshayes, Kurt; Vucic, Domagoj

    2009-01-01

    A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.

  5. The Proteasome Inhibitor, MG132, Attenuates Diabetic Nephropathy by Inhibiting SnoN Degradation In Vivo and In Vitro

    PubMed Central

    Huang, Wei; Yang, Chen; Nan, Qinling; Gao, Chenlin; Feng, Hong; Gou, Fang; Chen, Guo; Zhang, Zhihong; Yan, Pijun; Peng, Juan

    2014-01-01

    Transforming growth factor-β (TGF-β) has been shown to be involved in diabetic nephropathy (DN). The SnoN protein can regulate TGF-β signaling through interaction with Smad proteins. Recent studies have shown that SnoN is mainly degraded by the ubiquitin-proteasome pathway. However, the role of SnoN in the regulation of TGF-β/Smad signaling in DN is still unclear. In this study, diabetic rats were randomly divided into a diabetic control group (DC group) and a proteasome inhibitor (MG132) diabetes therapy group (DT group). Kidney damage parameters and the expression of SnoN, Smurf2, and TGF-β were observed. Simultaneously, we cultured rat glomerular mesangial cells (GMCs) stimulated with high glucose, and SnoN and Arkadia expression were measured. Results demonstrated that 24-hour urine protein, ACR, BUN, and the expression of Smurf2 and TGF-β were significantly increased (P < 0.05), whereas SnoN was significantly decreased in the DC group (P < 0.05). However, these changes diminished after treatment with MG132. SnoN expression in GMCs decreased significantly (P < 0.05), but Arkadia expression gradually increased due to high glucose stimulation (P < 0.05), which could be almost completely reversed by MG132 (P < 0.05). The present results support the hypothesis that MG132 may alleviate kidney damage by inhibiting SnoN degradation and TGF-β activation, suggesting that the ubiquitin-proteasome pathway may become a new therapeutic target for DN. PMID:25003128

  6. The proteasome inhibitor, MG132, attenuates diabetic nephropathy by inhibiting SnoN degradation in vivo and in vitro.

    PubMed

    Huang, Wei; Yang, Chen; Nan, Qinling; Gao, Chenlin; Feng, Hong; Gou, Fang; Chen, Guo; Zhang, Zhihong; Yan, Pijun; Peng, Juan; Xu, Yong

    2014-01-01

    Transforming growth factor-β (TGF-β) has been shown to be involved in diabetic nephropathy (DN). The SnoN protein can regulate TGF-β signaling through interaction with Smad proteins. Recent studies have shown that SnoN is mainly degraded by the ubiquitin-proteasome pathway. However, the role of SnoN in the regulation of TGF- β/Smad signaling in DN is still unclear. In this study, diabetic rats were randomly divided into a diabetic control group (DC group) and a proteasome inhibitor (MG132) diabetes therapy group (DT group). Kidney damage parameters and the expression of SnoN, Smurf2, and TGF-β were observed. Simultaneously, we cultured rat glomerular mesangial cells (GMCs) stimulated with high glucose, and SnoN and Arkadia expression were measured. Results demonstrated that 24-hour urine protein, ACR, BUN, and the expression of Smurf2 and TGF- β were significantly increased (P < 0.05), whereas SnoN was significantly decreased in the DC group (P < 0.05). However, these changes diminished after treatment with MG132. SnoN expression in GMCs decreased significantly (P < 0.05), but Arkadia expression gradually increased due to high glucose stimulation (P < 0.05), which could be almost completely reversed by MG132 (P < 0.05). The present results support the hypothesis that MG132 may alleviate kidney damage by inhibiting SnoN degradation and TGF-β activation, suggesting that the ubiquitin-proteasome pathway may become a new therapeutic target for DN.

  7. Role of Promyelocytic Leukemia (Pml) Sumolation in Nuclear Body Formation, 11s Proteasome Recruitment, and as2O3-Induced Pml or Pml/Retinoic Acid Receptor α Degradation

    PubMed Central

    Lallemand-Breitenbach, Valérie; Zhu, Jun; Puvion, Francine; Koken, Marcel; Honoré, Nicole; Doubeikovsky, Alexandre; Duprez, Estelle; Pandolfi, Pier Paolo; Puvion, Edmond; Freemont, Paul; de Thé, Hugues

    2001-01-01

    Promyelocytic leukemia (PML) is the organizer of nuclear matrix domains, PML nuclear bodies (NBs), with a proposed role in apoptosis control. In acute promyelocytic leukemia, PML/retinoic acid receptor (RAR) α expression disrupts NBs, but therapies such as retinoic acid or arsenic trioxide (As2O3) restore them. PML is conjugated by the ubiquitin-related peptide SUMO-1, a process enhanced by As2O3 and proposed to target PML to the nuclear matrix. We demonstrate that As2O3 triggers the proteasome-dependent degradation of PML and PML/RARα and that this process requires a specific sumolation site in PML, K160. PML sumolation is dispensable for its As2O3-induced matrix targeting and formation of primary nuclear aggregates, but is required for the formation of secondary shell-like NBs. Interestingly, only these mature NBs harbor 11S proteasome components, which are further recruited upon As2O3 exposure. Proteasome recruitment by sumolated PML only likely accounts for the failure of PML-K160R to be degraded. Therefore, studying the basis of As2O3-induced PML/RARα degradation we show that PML sumolation directly or indirectly promotes its catabolism, suggesting that mature NBs could be sites of intranuclear proteolysis and opening new insights into NB alterations found in viral infections or transformation. PMID:11413191

  8. Catabolism of endogenous and overexpressed APH1a and PEN2: evidence for artifactual involvement of the proteasome in the degradation of overexpressed proteins

    PubMed Central

    Dunys, Julie; Kawarai, Toshitaka; Wilk, Sherwin; St. George-Hyslop, Peter; Alves Da Costa, Cristine; Checler, Frédéric

    2005-01-01

    PS (presenilin)-dependent γ-secretase occurs as a high-molecular-mass complex composed of either PS1 or PS2 associated with Nct (nicastrin), PEN2 (presenilin enhancer 2 homologue) and APH1 (anterior pharynx defective 1 homologue). Numerous reports have documented the very complicated physical and functional cross-talk between these proteins that ultimately governs the biological activity of the γ-secretase, but very few studies examined the fate of the components of the complex. We show that, in both HEK-293 cells and the TSM1 neuronal cell line, the immunoreactivities of overexpressed myc-tagged-APH1a and -PEN2 were enhanced by the proteasome inhibitors ZIE and lactacystin, whereas a broad range of protease inhibitors had no effect. By contrast, proteasome inhibitors were totally unable to affect the cellular expression of endogenous APH1aL and PEN2 in HEK-293 cells, TSM1 and primary cultured cortical neurons. To explain this apparent discrepancy, we examined the degradation of myc-tagged-APH1a and -PEN2, in vitro, by cell extracts containing endogenous proteasome and by purified 20S proteasome. Strikingly, myc-tagged-APH1a and -PEN2 resist proteolysis by endogenous proteasome and purified 20S proteasome. We also show that endogenous PEN2 expression was drastically higher in wild-type than in PS- and Nct-deficient fibroblasts and was enhanced by proteasome inhibitors only in the two deficient cell systems. However, here again, purified 20S proteasome appeared unable to cleave endogenous PEN2 present in PS-deficient fibroblasts. The levels of endogenous APH1aL-like immunoreactivity were not modified by proteasome inhibitors and were unaffected by PS deficiency. Altogether, our results indicate that endogenous PEN2 and APH1aL do not undergo proteasomal degradation under physiological conditions in HEK-293 cells, TSM1 cells and fibroblasts and that the clearance of PEN2 in PS- and Nct-deficient fibroblasts is not mediated by 20S proteasome. Whether the 26S

  9. 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

  10. The Chaperone-assisted E3 Ligase C Terminus of Hsc70-interacting Protein (CHIP) Targets PTEN for Proteasomal Degradation*

    PubMed Central

    Ahmed, Syed Feroj; Deb, Satamita; Paul, Indranil; Chatterjee, Anirban; Mandal, Tapashi; Chatterjee, Uttara; Ghosh, Mrinal K.

    2012-01-01

    The tumor suppressor, PTEN is key to the regulation of diverse cellular processes, making it a prime candidate to be tightly regulated. The PTEN level is controlled in a major way by E3 ligase-mediated degradation through the Ubiquitin-Proteasome System (UPS). Nedd 4-1, XIAP, and WWP2 have been shown to maintain PTEN turnover. Here, we report that CHIP, the chaperone-associated E3 ligase, induces ubiquitination and regulates the proteasomal turnover of PTEN. It was apparent from our findings that PTEN transiently associates with the molecular chaperones and thereby gets diverted to the degradation pathway through its interaction with CHIP. The TPR domain of CHIP and parts of the N-terminal domain of PTEN are required for their interaction. Overexpression of CHIP leads to elevated ubiquitination and a shortened half-life of endogenous PTEN. On the other hand, depletion of endogenous CHIP stabilizes PTEN. CHIP is also shown to regulate PTEN-dependent transcription presumably through its down-regulation. PTEN shared an inverse correlation with CHIP in human prostate cancer patient samples, thereby triggering the prospects of a more complex mode of PTEN regulation in cancer. PMID:22427670

  11. Accelerated proteasomal activity induced by Pb2+, Ga3+, or Cu2+ exposure does not induce degradation of alpha-synuclein.

    PubMed

    Grunberg-Etkovitz, Nurit; Lev, Nirit; Ickowicz, Debby; Avital, Almog; Offen, Daniel; Malik, Zvi

    2009-01-01

    The involvement of environmental heavy metals in Parkinson's disease (PD) has been suggested by epidemiologic studies; however, the mechanism of this effect is unknown. PD is characterized by the aggregation of alpha-synuclein in Lewy bodies. We previously showed that Pb2+ accelerates proteasomal activity. Therefore, we examined the effect of Pb2+, Ga3+, and Cu2+ on alpha-synuclein in human SH-SY5Y cells. The heavy metals induced an increase in heme-oxygenase-1 levels without significant cell death or ROS generation. The metals inhibited ALA-dehydratase, which is the inhibitory subunit of the proteasome, thereby accelerating proteasomal activity and decreasing protein levels of CDK-1 and PBGD. However, alpha-synuclein protein levels increased after exposure to metals, similar to the effect obtained with the proteasome inhibitor, hemin, suggesting that alpha-synuclein is inaccessible to proteasomal degradation. Indeed, electron microscopy revealed the formation of aggresomes in Pb2+- or hemin-treated cells. Thus, although heavy metals enhance proteasomal activity, alpha-synuclein is protected from degradation, and its protein levels and aggregation are increased.

  12. NAD(P)H quinone oxidoreductase 1 inhibits the proteasomal degradation of homocysteine-induced endoplasmic reticulum protein

    SciTech Connect

    Maeda, Tomoji; Tanabe-Fujimura, Chiaki; Fujita, Yu; Abe, Chihiro; Nanakida, Yoshino; Zou, Kun; Liu, Junjun; Liu, Shuyu; Nakajima, Toshihiro; Komano, Hiroto

    2016-05-13

    Homocysteine-induced endoplasmic reticulum (ER) protein (Herp) is an ER stress-inducible key regulatory component of ER-associated degradation (ERAD) that has been implicated in insulin hypersecretion in diabetic mouse models. Herp expression is tightly regulated. Additionally, Herp is a highly labile protein and interacts with various proteins, which are characteristic features of ubiquitinated protein. Previously, we reported that ubiquitination is not required for Herp degradation. In addition, we found that the lysine residues of Herp (which are ubiquitinated by E3 ubiquitin ligase) are not sufficient for regulation of Herp degradation. In this study, we found that NAD(P)H quinone oxidoreductase 1 (NQO1)-mediated targeting of Herp to the proteasome was involved in Herp degradation. In addition, we found that Herp protein levels were markedly elevated in synoviolin-null cells. The E3 ubiquitin ligase synoviolin is a central component of ERAD and is involved in the degradation of nuclear factor E2-related factor-2 (Nrf2), which regulates cellular reactive oxygen species. Additionally, NQO1 is a target of Nrf2. Thus, our findings indicated that NQO1 could stabilize Herp protein expression via indirect regulation of synoviolin. -- Highlights: •Herp interacts with NQO1. •NQO1 regulates Herp degradation.

  13. CHIP Regulates Osteoclast Formation through Promoting TRAF6 Protein Degradation

    PubMed Central

    Li, Shan; Shu, Bing; Zhang, Yanquan; Li, Jia; Guo, Junwei; Wang, Yinyin; Ren, Fangli; Xiao, Guozhi; Chang, Zhijie; Chen, Di

    2014-01-01

    Objective Carboxyl terminus of Hsp70-interacting protein (CHIP or STUB1) is an E3 ligase and regulates the stability of several proteins which are involved in tumor growth and metastasis. However, the role of CHIP in bone growth and bone remodeling in vivo has not been reported. The objective of this study is to investigate the role and mechanism of CHIP in regulation of bone mass and bone remodeling. Methods The bone phenotype of Chip−/− mice was examined by histology, histomorphometry and micro-CT analyses. The regulatory mechanism of CHIP on the degradation of TRAF6 and the inhibition of NF-κB signaling was examined by immunoprecipitation (IP), western blotting and luciferase reporter assays. Results In this study, we found that deletion of the Chip gene leads to osteopenic phenotype and increased osteoclast formation. We further found that TRAF6, as a novel substrate of CHIP, is up-regulated in Chip−/− osteoclasts. TRAF6 is critical for RANKL-induced osteoclastogenesis. TRAF6 is an adaptor protein which functions as an E3 ligase to regulate the activation of TAK1 and the I-κB kinase (IKK) and is a key regulator of NF-κB signaling. CHIP interacts with TRAF6 to promote TRAF6 ubiquitination and proteasome degradation. CHIP inhibits p65 nuclear translocation, leading to the repression of the TRAF6-mediated NF-κB transcription. Conclusion CHIP inhibits NF-κB signaling via promoting TRAF6 degradation and plays an important role in osteoclastogenesis and bone remodeling, suggesting that it may be a novel therapeutic target for the treatment of bone loss associated diseases. PMID:24578159

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

    PubMed Central

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

    2011-01-01

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

  15. Prefoldin subunits are protected from ubiquitin-proteasome system-mediated degradation by forming complex with other constituent subunits.

    PubMed

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

    2011-06-03

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

  16. Protein Kinase Cδ Stimulates Proteasome-Dependent Degradation of C/EBPα during Apoptosis Induction of Leukemic Cells

    PubMed Central

    Zhao, Meng; Duan, Xu-Fang; Zhao, Xu-Yun; Zhang, Bo; Lu, Ying; Liu, Wei; Cheng, Jin-Ke; Chen, Guo-Qiang

    2009-01-01

    Background The precise regulation and maintenance of balance between cell proliferation, differentiation and death in metazoan are critical for tissue homeostasis. CCAAT/enhancer-binding protein alpha (C/EBPα) has been implicated as a key regulator of differentiation and proliferation in various cell types. Here we investigated the potential dynamic change and role of C/EBPα protein during apoptosis induction. Methodology/Principal Findings Upon onset of apoptosis induced by various kinds of inducers such as NSC606985, etoposide and others, C/EBPα expression presented a profound down-regulation in leukemic cell lines and primary cells via induction of protein degradation and inhibition of transcription, as assessed respectively by cycloheximide inhibition test, real-time quantitative RT-PCR and luciferase reporter assay. Applying chemical inhibition, forced expression of dominant negative mutant and catalytic fragment (CF) of protein kinase Cdelta (PKCδ), which was proteolytically activated during apoptosis induction tested, we showed that the active PKCδ protein contributed to the increased degradation of C/EBPα protein. Three specific proteasome inhibitors antagonized C/EBPα degradation during apoptosis induction. More importantly, ectopic expression of PKCδ-CF stimulated the ubiquitination of C/EBPα protein, while the chemical inhibition of PKCδ action significantly inhibited the enhanced ubiquitination of C/EBPα protein under NSC606985 treatment. Additionally, silencing of C/EBPα expression by small interfering RNAs enhanced, while inducible expression of C/EBPα inhibited NSC606985/etoposide-induced apoptosis in leukemic cells. Conclusions/Significance These observations indicate that the activation of PKCδ upon apoptosis results in the increased proteasome-dependent degradation of C/EBPα, which partially contributes to PKCδ-mediated apoptosis. PMID:19662097

  17. The fission yeast nucleoporin Alm1 is required for proteasomal degradation of kinetochore components.

    PubMed

    Salas-Pino, Silvia; Gallardo, Paola; Barrales, Ramón R; Braun, Sigurd; Daga, Rafael R

    2017-10-03

    Kinetochores (KTs) are large multiprotein complexes that constitute the interface between centromeric chromatin and the mitotic spindle during chromosome segregation. In spite of their essential role, little is known about how centromeres and KTs are assembled and how their precise stoichiometry is regulated. In this study, we show that the nuclear pore basket component Alm1 is required to maintain both the proteasome and its anchor, Cut8, at the nuclear envelope, which in turn regulates proteostasis of certain inner KT components. Consistently, alm1-deleted cells show increased levels of KT proteins, including CENP-C(Cnp3), spindle assembly checkpoint activation, and chromosome segregation defects. Our data demonstrate a novel function of the nucleoporin Alm1 in proteasome localization required for KT homeostasis. © 2017 Salas-Pino et al.

  18. The Adenovirus E4-ORF3 Protein Stimulates SUMOylation of General Transcription Factor TFII-I to Direct Proteasomal Degradation

    PubMed Central

    Bridges, Rebecca G.; Sohn, Sook-Young; Wright, Jordan

    2016-01-01

    ABSTRACT Modulation of host cell transcription, translation, and posttranslational modification processes is critical for the ability of many viruses to replicate efficiently within host cells. The human adenovirus (Ad) early region 4 open reading frame 3 (E4-ORF3) protein forms unique inclusions throughout the nuclei of infected cells and inhibits the antiviral Mre11-Rad50-Nbs1 DNA repair complex through relocalization. E4-ORF3 also induces SUMOylation of Mre11 and Nbs1. We recently identified additional cellular targets of E4-ORF3 and found that E4-ORF3 stimulates ubiquitin-like modification of 41 cellular proteins involved in a wide variety of processes. Among the proteins most abundantly modified in an E4-ORF3-dependent manner was the general transcription factor II–I (TFII-I). Analysis of Ad-infected cells revealed that E4-ORF3 induces TFII-I relocalization and SUMOylation early during infection. In the present study, we explored the relationship between E4-ORF3 and TFII-I. We found that Ad infection or ectopic E4-ORF3 expression leads to SUMOylation of TFII-I that precedes a rapid decline in TFII-I protein levels. We also show that E4-ORF3 is required for ubiquitination of TFII-I and subsequent proteasomal degradation. This is the first evidence that E4-ORF3 regulates ubiquitination. Interestingly, we found that E4-ORF3 modulation of TFII-I occurs in diverse cell types but only E4-ORF3 of Ad species C regulates TFII-I, providing critical insight into the mechanism by which E4-ORF3 targets TFII-I. Finally, we show that E4-ORF3 stimulates the activity of a TFII-I-repressed viral promoter during infection. Our results characterize a novel mechanism of TFII-I regulation by Ad and highlight how a viral protein can modulate a critical cellular transcription factor during infection. PMID:26814176

  19. Host Protein Ku70 Binds and Protects HIV-1 Integrase from Proteasomal Degradation and Is Required for HIV Replication*

    PubMed Central

    Zheng, Yingfeng; Ao, Zhujun; Wang, Binchen; Jayappa, Kallesh Danappa; Yao, Xiaojian

    2011-01-01

    HIV-1 integrase (IN) is a key viral enzymatic protein acting in several viral replication steps, including integration. IN has been shown to be an unstable protein degraded by the N-end rule pathway through the host ubiquitin-proteasome machinery. However, it is still not fully understood how this viral protein is protected from the host ubiquitin-proteasome system within cells during HIV replication. In the present study, we provide evidence that the host protein Ku70 interacts with HIV-1 IN and protects it from the Lys48-linked polyubiquitination proteasomal pathway. Moreover, Ku70 is able to down-regulate the overall protein polyubiquitination level within the host cells and to specifically deubiquitinate IN through their interaction. Mutagenic studies revealed that the C terminus of IN (residues 230–288) is required for IN binding to the N-terminal part of Ku70 (Ku70(1–430)), and their interaction is independent of Ku70/80 heterodimerization. Finally, knockdown of Ku70 expression in both virus-producing and target CD4+ T cells significantly disrupted HIV-1 replication and rendered two-long terminal repeat circles and integration undetectable, indicating that Ku70 is required for both the early and the late stages of the HIV-1 life cycle. Interestingly, Ku70 was incorporated into the progeny virus in an IN-dependent way. We proposed that Ku70 may interact with IN during viral assembly and accompany HIV-1 IN upon entry into the new target cells, acting to 1) protect IN from the host defense system and 2) assist IN integration activity. Overall, this report provides another example of how HIV-1 hijacks host cellular machinery to protect the virus itself and to facilitate its replication. PMID:21454661

  20. Autophagy promotes ferroptosis by degradation of ferritin.

    PubMed

    Hou, Wen; Xie, Yangchun; Song, Xinxin; Sun, Xiaofang; Lotze, Michael T; Zeh, Herbert J; Kang, Rui; Tang, Daolin

    2016-08-02

    Macroautophagy/autophagy is an evolutionarily conserved degradation pathway that maintains homeostasis. Ferroptosis, a novel form of regulated cell death, is characterized by a production of reactive oxygen species from accumulated iron and lipid peroxidation. However, the relationship between autophagy and ferroptosis at the genetic level remains unclear. Here, we demonstrated that autophagy contributes to ferroptosis by degradation of ferritin in fibroblasts and cancer cells. Knockout or knockdown of Atg5 (autophagy-related 5) and Atg7 limited erastin-induced ferroptosis with decreased intracellular ferrous iron levels, and lipid peroxidation. Remarkably, NCOA4 (nuclear receptor coactivator 4) was a selective cargo receptor for the selective autophagic turnover of ferritin (namely ferritinophagy) in ferroptosis. Consistently, genetic inhibition of NCOA4 inhibited ferritin degradation and suppressed ferroptosis. In contrast, overexpression of NCOA4 increased ferritin degradation and promoted ferroptosis. These findings provide novel insight into the interplay between autophagy and regulated cell death.

  1. cAMP signaling increases histone deacetylase 8 expression by inhibiting JNK-dependent degradation via autophagy and the proteasome system in H1299 lung cancer cells.

    PubMed

    Park, Ji-Yeon; Juhnn, Yong-Sung

    2016-02-05

    This study aimed to investigate the roles of autophagy and the ubiquitin-proteasome system in the degradation of histone deacetylase 8 (HDAC8) and to clarify the mechanism by which cAMP signaling regulates this degradation. cAMP signaling was activated by treating H1299 non-small cell lung cancer cells with isoproterenol or forskolin/3-isobutyl-1-methylxanthine, and HDAC8 expression was assessed by western blot analysis. The inhibition of autophagy and ubiquitin-proteasome-dependent degradation increased HDAC8 expression. cAMP signaling inhibited JNK activation, which decreased the phosphorylation of Bcl-2, thereby reducing autophagy, and the phosphorylation of Itch, thereby reducing ubiquitination. These results suggest that the HDAC8 protein is degraded via autophagy and the ubiquitin-proteasome system and that cAMP signaling increases HDAC8 protein levels by reducing JNK-mediated autophagy and ubiquitin-proteasome-dependent degradation of the HDAC8 protein in H1299 lung cancer cells.

  2. Protein kinase Cβ activates fat mass and obesity-associated protein by influencing its ubiquitin/proteasome degradation.

    PubMed

    Tai, Haoran; Wang, Xiaobo; Zhou, Jiao; Han, Xiaojuan; Fang, Tingting; Gong, Hui; Huang, Ning; Chen, Honghan; Qin, Jianqiong; Yang, Ming; Wei, Xiawei; Yang, Li; Xiao, Hengyi

    2017-10-01

    Protein kinase Cβ (PKCβ) is a serine-threonine kinase associated with obesity and diabetic complications; its activation contributes to weight gain, and deletion of its gene results in resistance to genetic- and diet-induced obesity. Fat mass and obesity-associated (FTO) protein is a recently identified RNA demethylase, and its overexpression in mice leads to increased body weight as well as fat mass. Although sharing some features in anabolism regulation, PKCβ and FTO have not been investigated together; therefore, their relationship has not been established. We report that PKCβ positively regulates FTO on the posttranslation level, evidenced by the facts that PKCβ activation contributes to high-glucose-induced FTO up-regulation, and overexpression of PKCβ suppresses ubiquitin-proteasome degradation of FTO, whereas PKCβ inactivation acts in the opposite manner. It was also found that PKCβ can phosphorylate FTO on threonine, and this phosphorylation requires both catalytic and regulatory domains of PKCβ. Moreover, PKCβ inhibition can suppress 3T3-L1 cell differentiation in normal and FTO-overexpressing cells but not in FTO-silenced or -inhibited cells. We propose that PKCβ acts to suppress the degradation of FTO protein and reveals the associated role of PKCβ and FTO in adipogenesis, suggesting a new pathway that affects the development of obesity and metabolic diseases.-Tai, H., Wang, X., Zhou, J., Han, X., Fang, T., Gong, H., Huang, N., Chen, H., Qin, J., Yang, M., Wei, X., Yang, L., Xiao, H. Protein kinase Cβ activates fat mass and obesity-associated protein by influencing its ubiquitin/proteasome degradation. © FASEB.

  3. 4-Hydroxynonenal differentially regulates adiponectin gene expression and secretion via activating PPARγ and accelerating ubiquitin–proteasome degradation

    PubMed Central

    Wanga, Zhigang; Dou, Xiaobing; Gu, Dongfang; Shen, Chen; Yao, Tong; Nguyen, Van; Braunschweig, Carol; Song, Zhenyuan

    2011-01-01

    Although well-established, the underlying mechanisms involved in obesity-related plasma adiponectin decline remain elusive. Oxidative stress is associated with obesity and insulin resistance and considered to contribute to the progression toward obesity-related metabolic disorders. In this study, we investigated the effects of 4-hydroxynonenal (4-HNE), the most abundant lipid peroxidation end product, on adiponectin production and its potential implication in obesity-related adiponectin decrease. Long-term high-fat diet feeding led to obesity in mouse, accompanied by decreased plasma adiponectin and increased adipose tissue 4-HNE content. Exposure of adipocytes to exogenous 4-HNE resulted in decreased adiponectin secretion in a dose-dependent manner, which was consistent with significantly decreased intracellular adiponectin protein abundance. In contrast, adiponectin gene expression was significantly elevated by 4-HNE treatment, which was concomitant with increased peroxisome proliferator-activated receptor gamma (PPAR-γ) gene expression and transactivity. The effect was abolished by T0070907, a PPAR-γ antagonist, suggesting that PPAR-γ activation plays a critical role in this process. To gain insight into mechanisms involved in adiponectin protein decrease, we examined the effects of 4-HNE on adiponectin protein degradation. Cycloheximide (CHX)-chase assay revealed that 4-HNE exposure accelerated adiponectin protein degradation, which was prevented by MG132, a potent proteasome inhibitor. Immunoprecipitation assay showed that 4-HNE exposure increased ubiquitinated adiponectin protein levels. These data altogether indicated that 4-HNE enhanced adiponectin protein degradation via ubiquitin–proteasome system. Finally, we demonstrated that supplementation of HF diet with betaine, an antioxidant and methyl donor, alleviated high-fat-induced adipose tissue 4-HNE increase and attenuated plasma adiponectin decline. Taken together, our findings suggest that the lipid

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

    PubMed

    Hsu, Hsien-Yeh; Lin, Tung-Yi; Wu, Yu-Chung; Tsao, Shu-Ming; Hwang, Pai-An; Shih, Yu-Wei; Hsu, Jason

    2014-09-15

    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 β (TGFβ) 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 TGFβ 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 TGFβ receptors.

  5. Rapid Proteasomal Degradation of Mutant Proteins Is the Primary Mechanism Leading to Tumorigenesis in Patients With Missense AIP Mutations

    PubMed Central

    Hernández-Ramírez, Laura C.; Martucci, Federico; Morgan, Rhodri M. L.; Trivellin, Giampaolo; Tilley, Daniel; Ramos-Guajardo, Nancy; Iacovazzo, Donato; D'Acquisto, Fulvio; Prodromou, Chrisostomos

    2016-01-01

    Context: The pathogenic effect of mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene (AIPmuts) in pituitary adenomas is incompletely understood. We have identified the primary mechanism of loss of function for missense AIPmuts. Objective: This study sought to analyze the mechanism/speed of protein turnover of wild-type and missense AIP variants, correlating protein half-life with clinical parameters. Design and Setting: Half-life and protein–protein interaction experiments and cross-sectional analysis of AIPmut positive patients' data were performed in a clinical academic research institution. Patients: Data were obtained from our cohort of pituitary adenoma patients and literature-reported cases. Interventions: Protein turnover of endogenous AIP in two cell lines and fifteen AIP variants overexpressed in HEK293 cells was analyzed via cycloheximide chase and proteasome inhibition. Glutathione-S-transferase pull-down and quantitative mass spectrometry identified proteins involved in AIP degradation; results were confirmed by coimmunoprecipitation and gene knockdown. Relevant clinical data was collected. Main Outcome Measures: Half-life of wild-type and mutant AIP proteins and its correlation with clinical parameters. Results: Endogenous AIP half-life was similar in HEK293 and lymphoblastoid cells (43.5 and 32.7 h). AIP variants were divided into stable proteins (median, 77.7 h; interquartile range [IQR], 60.7–92.9 h), and those with short (median, 27 h; IQR, 21.6–28.7 h) or very short (median, 7.7 h; IQR, 5.6–10.5 h) half-life; proteasomal inhibition rescued the rapid degradation of mutant proteins. The experimental half-life significantly correlated with age at diagnosis of acromegaly/gigantism (r = 0.411; P = .002). The FBXO3-containing SKP1–CUL1–F-box protein complex was identified as the E3 ubiquitin-ligase recognizing AIP. Conclusions: AIP is a stable protein, driven to ubiquitination by the SKP1–CUL1–F-box protein complex

  6. Ubiquitin recognition by the proteasome.

    PubMed

    Saeki, Yasushi

    2017-02-01

    The 26S proteasome is a 2.5-MDa complex responsible for the selective, ATP-dependent degradation of ubiquitylated proteins in eukaryotic cells. Substrates in hundreds cellular pathways are timely ubiquitylated and converged to the proteasome by direct recognition or by multiple shuttle factors. Engagement of substrate protein triggers conformational changes of the proteasome, which drive substrate unfolding, deubiquitylation and translocation of substrates to proteolytic sites. Recent studies have challenged the previous paradigm that Lys48-linked tetraubiquitin is a minimal degradation signal: in addition, monoubiquitylation or multiple short ubiquitylations can serve as the targeting signal for proteasomal degradation. In this review, I highlight recent advances in our understanding of the proteasome structure, the ubiquitin topology in proteasome targeting, and the cellular factors that regulate proteasomal degradation. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  7. BCL11B is frequently downregulated in HTLV-1-infected T-cells through Tax-mediated proteasomal degradation.

    PubMed

    Permatasari, Happy Kurnia; Nakahata, Shingo; Ichikawa, Tomonaga; Morishita, Kazuhiro

    2017-08-26

    Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia-lymphoma (ATLL). The HTLV-1-encoded protein Tax plays important roles in the proliferation of HTLV-1-infected T-cells by affecting cellular proteins. In this study, we showed that Tax transcriptionally and post-transcriptionally downregulates the expression of the tumor suppressor gene B-cell leukemia/lymphoma 11B (BCL11B), which encodes a lymphoid-related transcription factor. BCL11B expression was downregulated in HTLV-1-infected T-cell lines at the mRNA and protein levels, and forced expression of BCL11B suppressed the proliferation of these cells. The proteasomal inhibitor MG132 increased BCL11B expression in HTLV-1-infected cell lines, and colocalization of Tax with BCL11B was detected in the cytoplasm of HTLV-1-infected T-cells following MG132 treatment. shRNA knock-down of Tax expression also increased the expression of BCL11B in HTLV-1-infected cells. Moreover, we found that Tax physically binds to BCL11B protein and induces the polyubiquitination of BCL11B and proteasome-dependent degradation of BCL11B. Thus, inactivation of BCL11B by Tax protein may play an important role in the Tax-mediated leukemogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. 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. © 2013 Wiley Periodicals, Inc.

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

    PubMed Central

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

    2014-01-01

    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

  10. Induced Degradation of Tat by Nucleocapsid (NC) via the Proteasome Pathway and Its Effect on HIV Transcription

    PubMed Central

    Hong, Hye-Won; Lee, Seong-Wook; Myung, Heejoon

    2013-01-01

    Human Immunodeficiency Virus type 1 (HIV-1) is a retrovirus that causes acquired immunodeficiency syndrome (AIDS). HIV-1 Tat protein upregulates transcriptional transactivation. The nucleocapsid protein NC of HIV-1 is a component of virion and plays a key role in genome packaging. Herein, we have demonstrated the interaction between NC and Tat by means of a yeast two-hybrid assay, GST pull-down analysis, co-immunoprecipitation and subcellular colocalization analysis. We observed that the level of Tat was significantly reduced in the presence of NC. But NC did not affect mRNA expression level of Tat. The level of Tat in the presence of NC was increased by treating cells with a proteasome inhibitor, MG132. The ubiquitination state of Tat was not seen to increase in the presence of NC, suggesting the proteasomal degradation was independent of ubiquitination. Lowered level of Tat in the presence of NC led to a decrease in Tat-mediated transcriptional transactivation. PMID:23611845

  11. Akt Phosphorylates Wnt Coactivator and Chromatin Effector Pygo2 at Serine 48 to Antagonize Its Ubiquitin/Proteasome-mediated Degradation*

    PubMed Central

    Li, Qiuling; Li, Yuewei; Gu, Bingnan; Fang, Lei; Zhou, Pengbo; Bao, Shilai; Huang, Lan; Dai, Xing

    2015-01-01

    Pygopus 2 (Pygo2/PYGO2) is an evolutionarily conserved coactivator and chromatin effector in the Wnt/β-catenin signaling pathway that regulates cell growth and differentiation in various normal and malignant tissues. Although PYGO2 is highly overexpressed in a number of human cancers, the molecular mechanism underlying its deregulation is largely unknown. Here we report that Pygo2 protein is degraded through the ubiquitin/proteasome pathway and is posttranslationally stabilized through phosphorylation by activated phosphatidylinositol 3-kinase/Akt signaling. Specifically, Pygo2 is stabilized upon inhibition of the proteasome, and its intracellular level is regulated by Cullin 4 (Cul4) and DNA damage-binding protein 1 (DDB1), components of the Cul4-DDB1 E3 ubiquitin ligase complex. Furthermore, Pygo2 is phosphorylated at multiple residues, and Akt-mediated phosphorylation at serine 48 leads to its decreased ubiquitylation and increased stability. Finally, we provide evidence that Akt and its upstream growth factors act in parallel with Wnt to stabilize Pygo2. Taken together, our findings highlight chromatin regulator Pygo2 as a common node downstream of oncogenic Wnt and Akt signaling pathways and underscore posttranslational modification, particularly phosphorylation and ubiquitylation, as a significant mode of regulation of Pygo2 protein expression. PMID:26170450

  12. Propofol Prevents Hippocampal Neuronal Loss and Memory Impairment in Cerebral Ischemia Injury Through Promoting PTEN Degradation.

    PubMed

    Chen, Xin; Du, Ye-Mu; Xu, Feng; Liu, Dai; Wang, Yuan-Lin

    2016-09-01

    Neuroprotective effect of propofol against cerebral ischemia injury was widely investigated. However, its mechanisms remain unclear. Phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway is supposed as a cell survival pathway, and phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a negative regulator of AKT phosphorylation. Whether PTEN was involved in the protective effect of propofol against cerebral ischemia injury was not elucidated. In this study, the function of PTEN in the acute phase of cerebral ischemia injury was investigated. Our data showed that propofol promoted the PTEN degradation in the acute phase of cerebral ischemia injury and concurrently activated AKT phosphorylation. The increase of ubiquitinated PTEN caused by cerebral ischemia injury were degraded in propofol-pretreated rats. Moreover, we evidenced that proteasome activity was stimulated in propofol-treated rats. These data pointed that PTEN degradation was facilitated in the acute phase after propofol treatment possibly through activating ubiquitin-proteasome system. Therefore, we applied PTEN inhibitor-bpV before cerebral ischemia injury. Like propofol, bpV pretreatment also mitigated cerebral ischemia injury-induced cell loss in CA1 region and memory impairment. Taken together, our data suggest that PTEN degradation is neuroprotective against cerebral ischemia injury and propofol facilitates PTEN degradation to prevent hippocampal neuronal loss and memory deficit in cerebral ischemia injury.

  13. STAT3 Potentiates SIAH-1 Mediated Proteasomal Degradation of β-Catenin in Human Embryonic Kidney Cells

    PubMed Central

    Shin, Minkyung; Yi, Eun Hee; Kim, Byung-Hak; Shin, Jae-Cheon; Park, Jung Youl; Cho, Chung-Hyun; Park, Jong-Wan; Choi, Kang-Yell; Ye, Sang-Kyu

    2016-01-01

    The β-catenin functions as an adhesion molecule and a component of the Wnt signaling pathway. In the absence of the Wnt ligand, β-catenin is constantly phosphorylated, which designates it for degradation by the APC complex. This process is one of the key regulatory mechanisms of β-catenin. The level of β-catenin is also controlled by the E3 ubiquitin protein ligase SIAH-1 via a phosphorylation-independent degradation pathway. Similar to β-catenin, STAT3 is responsible for various cellular processes, such as survival, proliferation, and differentiation. However, little is known about how these molecules work together to regulate diverse cellular processes. In this study, we investigated the regulatory relationship between STAT3 and β-catenin in HEK293T cells. To our knowledge, this is the first study to report that β-catenin-TCF-4 transcriptional activity was suppressed by phosphorylated STAT3; furthermore, STAT3 inactivation abolished this effect and elevated activated β-catenin levels. STAT3 also showed a strong interaction with SIAH-1, a regulator of active β-catenin via degradation, which stabilized SIAH-1 and increased its interaction with β-catenin. These results suggest that activated STAT3 regulates active β-catenin protein levels via stabilization of SIAH-1 and the subsequent ubiquitin-dependent proteasomal degradation of β-catenin in HEK293T cells. PMID:27871173

  14. A Role for Protein Phosphorylation in Cytochrome P450 3A4 Ubiquitin-dependent Proteasomal Degradation*S⃞

    PubMed Central

    Wang, YongQiang; Liao, Mingxiang; Hoe, Nicholas; Acharya, Poulomi; Deng, Changhui; Krutchinsky, Andrew N.; Correia, Maria Almira

    2009-01-01

    Cytochromes P450 (P450s) incur phosphorylation. Although the precise role of this post-translational modification is unclear, marking P450s for degradation is plausible. Indeed, we have found that after structural inactivation, CYP3A4, the major human liver P450, and its rat orthologs are phosphorylated during their ubiquitin-dependent proteasomal degradation. Peptide mapping coupled with mass spectrometric analyses of CYP3A4 phosphorylated in vitro by protein kinase C (PKC) previously identified two target sites, Thr264 and Ser420. We now document that liver cytosolic kinases additionally target Ser478 as a major site. To determine whether such phosphorylation is relevant to in vivo CYP3A4 degradation, wild type and CYP3A4 with single, double, or triple Ala mutations of these residues were heterologously expressed in Saccharomyces cerevisiae pep4Δ strains. We found that relative to CYP3A4wt, its S478A mutant was significantly stabilized in these yeast, and this was greatly to markedly enhanced for its S478A/T264A, S478A/S420A, and S478A/T264A/S420A double and triple mutants. Similar relative S478A/T264A/S420A mutant stabilization was also observed in HEK293T cells. To determine whether phosphorylation enhances CYP3A4 degradation by enhancing its ubiquitination, CYP3A4 ubiquitination was examined in an in vitro UBC7/gp78-reconstituted system with and without cAMP-dependent protein kinase A and PKC, two liver cytosolic kinases involved in CYP3A4 phosphorylation. cAMP-dependent protein kinase A/PKC-mediated phosphorylation of CYP3A4wt but not its S478A/T264A/S420A mutant enhanced its ubiquitination in this system. Together, these findings indicate that phosphorylation of CYP3A4 Ser478, Thr264, and Ser420 residues by cytosolic kinases is important both for its ubiquitination and proteasomal degradation and suggest a direct link between P450 phosphorylation, ubiquitination, and degradation. PMID:19095658

  15. Cap-dependent mRNA translation and the ubiquitin-proteasome system cooperate to promote ERBB2-dependent esophageal cancer phenotype.

    PubMed

    Issaenko, O A; Bitterman, P B; Polunovsky, V A; Dahlberg, P S

    2012-09-01

    Pathological post-transcriptional control of the proteome composition is a central feature of malignancy. Two steps in this pathway, eIF4F-driven cap-dependent mRNA translation and the ubiquitin-proteasome system (UPS), are deregulated in most if not all cancers. We tested a hypothesis that eIF4F is aberrantly activated in human esophageal adenocarcinoma (EAC) and requires elevated rates of protein turnover and proteolysis and thereby activated UPS for its pro-neoplastic function. Here, we show that 80% of tumors and cell lines featuring amplified ERBB2 display an aberrantly activated eIF4F. Direct genetic targeting of the eIF4F in ERBB2-amplified EAC cells with a constitutively active form of the eIF4F repressor 4E-BP1 decreased colony formation and proliferation and triggered apoptosis. In contrast, suppression of m-TOR-kinase activity towards 4E-BP1with rapamycin only modestly inhibited eIF4F-driven cap-dependent translation and EAC malignant phenotype; and promoted feedback activation of other cancer pathways. Our data show that co-treatment with 2 FDA-approved agents, the m-TOR inhibitor rapamycin and the proteasome inhibitor bortezomib, leads to strong synergistic growth-inhibitory effects. Moreover, direct targeting of eIF4F with constitutively active 4E-BP1 is significantly more potent in collaboration with bortezomib than rapamycin. These data support the hypothesis that a finely tuned balance between eIF4F-driven protein synthesis and proteasome-mediated protein degradation is required for the maintenance of ERBB2-mediated EAC malignant phenotype. Altogether, our study supports the development of pharmaceuticals to directly target eIF4F as most efficient strategy; and provides a clear rationale for the clinical evaluation of combination therapy with m-TOR inhibitors and bortezomib for EAC treatment.

  16. Microtubule-disrupting chemotherapeutics result in enhanced proteasome-mediated degradation and disappearance of tubulin in neural cells.

    PubMed

    Huff, Lyn M; Sackett, Dan L; Poruchynsky, Marianne S; Fojo, Tito

    2010-07-15

    We sought to examine the effects of microtubule-targeting agents (MTA) on neural cells to better understand the problem of neurotoxicity, their principal side effect, and to possibly develop a model of clinical toxicity. Studies showed that microtubule-depolymerizing agents (MDA) not only disassembled microtubules in neural HCN2 cells but also led to rapid disappearance of tubulin, and that this was specific for MDAs. Tubulin levels decreased to 20% as early as 8 hours after adding vincristine, and to 1% to 30% (mean, 9.8 +/- 7.6%; median of 7%) after 100 nmol/L vincristine for 24 hours. This disappearance was reversible. An increase in both glu-terminated and acetylated tubulin, markers of stable tubulin, preceded reaccumulation of soluble tubulin, suggesting a priority for stabilizing tubulin first as microtubules before replenishing the soluble pool. Similar results were shown with other MDAs. Furthermore, microtubule reassembly did not arise from a central focus but instead appeared to involve dispersed nucleation, as evidenced by the appearance of small, stable microtubule stubs throughout the cytoplasm. In contrast, experiments with four nonneural "normal" cell lines and four cancer cell lines resulted in microtubule destabilization but only modest tubulin degradation. Evidence for proteasome-mediated degradation was obtained by demonstrating that adding a proteasome inhibitor before vincristine prevented tubulin disappearance. In summary, MDAs lead to rapid disappearance of tubulin in neural but not in other normal or cancer cells. These results underscore the fine control that occurs in neural cells and may further our understanding of neurotoxicity following MDAs.

  17. Silymarin induces cyclin D1 proteasomal degradation via its phosphorylation of threonine-286 in human colorectal cancer cells.

    PubMed

    Eo, Hyun Ji; Park, Gwang Hun; Song, Hun Min; Lee, Jin Wook; Kim, Mi Kyoung; Lee, Man Hyo; Lee, Jeong Rak; Koo, Jin Suk; Jeong, Jin Boo

    2015-01-01

    Silymarin from milk thistle (Silybum marianum) plant has been reported to show anti-cancer, anti-inflammatory, antioxidant and hepatoprotective effects. For anti-cancer activity, silymarin is known to regulate cell cycle progression through cyclin D1 downregulation. However, the mechanism of silymarin-mediated cyclin D1 downregulation still remains unanswered. The current study was performed to elucidate the molecular mechanism of cyclin D1 downregulation by silymarin in human colorectal cancer cells. The treatment of silymarin suppressed the cell proliferation in HCT116 and SW480 cells and decreased cellular accumulation of exogenously-induced cyclin D1 protein. However, silymarin did not change the level of cyclin D1 mRNA. Inhibition of proteasomal degradation by MG132 attenuated silymarin-mediated cyclin D1 downregulation and the half-life of cyclin D1 was decreased in the cells treated with silymarin. In addition, silymarin increased phosphorylation of cyclin D1 at threonine-286 and a point mutation of threonine-286 to alanine attenuated silymarin-mediated cyclin D1 downregulation. Inhibition of NF-κB by a selective inhibitor, BAY 11-7082 suppressed cyclin D1 phosphorylation and downregulation by silymarin. From these results, we suggest that silymarin-mediated cyclin D1 downregulation may result from proteasomal degradation through its threonine-286 phosphorylation via NF-κB activation. The current study provides new mechanistic link between silymarin, cyclin D1 downregulation and cell growth in human colorectal cancer cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Zinc Mesoporphyrin Induces Rapid Proteasomal Degradation of Hepatitis C Nonstructural 5A Protein in Human Hepatoma Cells

    PubMed Central

    Hou, Weihong; Tian, Qing; Zheng, Jianyu; Bonkovsky, Herbert L.

    2009-01-01

    Background & Aims The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV), plays a critical role in HCV replication and is an attractive target for the therapy of HCV infection. So far, little is known about the post-translational regulation of NS5A protein and its precise role in HCV RNA replication. Our objectives were to elucidate the down-regulation of NS5A protein and HCV RNA replication by zinc mesoporphyrin (ZnMP), and the mechanism by which this process occurs. Methods Human hepatoma cells expressing HCV proteins were used to investigate the post-translational regulation of ZnMP on NS5A protein by Western blots (WB) and immunoprecipitation (IP). Quantitative RT-PCR (qRT-PCR) was used to determine the effects of ZnMP on HCV RNA replication. Results ZnMP selectively and markedly down-regulated NS5A protein levels by increasing degradation of NS5A protein [half life fell from 18.7 h to 2.7 h]. The proteasome inhibitors, epoxomicin and MG132, significantly abrogated degradation of NS5A protein by ZnMP without affecting levels of NS5A in the absence of ZnMP. Analysis of immunoprecipitates with an anti-ubiquitin antibody revealed polyubiquitination of NS5A, suggesting that ZnMP induces ubiquitination of NS5A protein. In addition, 10 μM of ZnMP reduced HCV replication by ~63% in the Con1 replicon cells, ~70% in J6/JFH1 HCV transfected cells, and ~90% in J6/JFH1 HCV infected cells without affecting cell viability. Conclusions ZnMP produces a rapid and profound down-regulation of the NS5A protein by enhancing its polyubiquitination and proteasome-dependent catabolism. Zinc mesoporphyrin may hold promise as a novel agent to treat HCV infection. PMID:19909748

  19. 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

  20. Foxp3 enhances HIF-1α target gene expression in human bladder cancer through decreasing its ubiquitin-proteasomal degradation

    PubMed Central

    Lin, Chang-Te; Tung, Chun-Liang; Shen, Cheng-Huang; Tsai, Hsin-Tzu; Yang, Wen-Horng; Chang, Hung-I; Chen, Syue-Yi; Tzai, Tzong-Shin

    2016-01-01

    Hypoxia-inducible factor-1α (HIF-1α) can control a transcriptional factor forkhead box P3 (Foxp3) protein expression in T lymphocyte differentiation through proteasome-mediated degradation. In this study, we unveil a reverse regulatory mechanism contributing to bladder cancer progression; Foxp3 expression attenuates HIF-1α degradation. We first demonstrated that Foxp3 expression positively correlates with the metastatic potential in T24 cells and can increase the expression of HIF-1α-target genes, such as vascular endothelial growth factor (VEGF) and glucose transporter (GLUT). Foxp3 protein can bind with HIF-1α, particularly under hypoxia. In vivo ubiquination assay demonstrated that Foxp3 can decrease HIF-1α degradation in a dose-dependent manner. Knocking-down of Foxp3 expression blocks in vivo tumor growth in mice and prolongs mice's survival, which is associated with von Willebrand factor expression. Thirty-three of 145 (22.8 %) bladder tumors exhibit Foxp3 expression. Foxp3 expression is an independent predictor for disease progression in superficial bladder cancer patients (p = 0.032), associated with less number of intratumoral CD8+ lymphocyte. The metaanalysis from 2 published datasets showed Foxp3 expression is positively associated with GLUT−4, −9, and VEGF-A, B-, D expression. This reverse post-translational regulation of HIF-1α protein by Foxp3 provides a new potential target for developing new therapeutic strategy for bladder cancer. PMID:27557492

  1. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

    SciTech Connect

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; Bai, Lin; Hu, Kuan; Merkx, Remco; Huang, Jessica; Chatterjee, Champak; Ovaa, Huib; Gygi, Steven P.; Li, Huilin; Darwin, K. Heran

    2015-03-23

    Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world’s most devastating pathogens.

  2. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

    DOE PAGES

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; ...

    2015-03-23

    Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and proteinmore » degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world’s most devastating pathogens.« less

  3. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis

    PubMed Central

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; Bai, Lin; Hu, Kuan; Merkx, Remco; Huang, Jessica; Chatterjee, Champak; Ovaa, Huib; Gygi, Steven P.; Li, Huilin; Darwin, K. Heran

    2015-01-01

    Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world's most devastating pathogens. PMID:25831519

  4. An adenosine triphosphate-independent proteasome activator contributes to the virulence of Mycobacterium tuberculosis.

    PubMed

    Jastrab, Jordan B; Wang, Tong; Murphy, J Patrick; Bai, Lin; Hu, Kuan; Merkx, Remco; Huang, Jessica; Chatterjee, Champak; Ovaa, Huib; Gygi, Steven P; Li, Huilin; Darwin, K Heran

    2015-04-07

    Mycobacterium tuberculosis encodes a proteasome that is highly similar to eukaryotic proteasomes and is required to cause lethal infections in animals. The only pathway known to target proteins for proteasomal degradation in bacteria is pupylation, which is functionally analogous to eukaryotic ubiquitylation. However, evidence suggests that the M. tuberculosis proteasome contributes to pupylation-independent pathways as well. To identify new proteasome cofactors that might contribute to such pathways, we isolated proteins that bound to proteasomes overproduced in M. tuberculosis and found a previously uncharacterized protein, Rv3780, which formed rings and capped M. tuberculosis proteasome core particles. Rv3780 enhanced peptide and protein degradation by proteasomes in an adenosine triphosphate (ATP)-independent manner. We identified putative Rv3780-dependent proteasome substrates and found that Rv3780 promoted robust degradation of the heat shock protein repressor, HspR. Importantly, an M. tuberculosis Rv3780 mutant had a general growth defect, was sensitive to heat stress, and was attenuated for growth in mice. Collectively, these data demonstrate that ATP-independent proteasome activators are not confined to eukaryotes and can contribute to the virulence of one the world's most devastating pathogens.

  5. Aurora Kinase A Promotes AR Degradation via the E3 Ligase CHIP.

    PubMed

    Sarkar, Sukumar; Brautigan, David L; Larner, James M

    2017-08-01

    Reducing the levels of the androgen receptor (AR) is one of the most viable approaches to combat castration-resistant prostate cancer. Previously, we observed that proteasomal-dependent degradation of AR in response to 2-methoxyestradiol (2-ME) depends primarily on the E3 ligase C-terminus of HSP70-interacting protein (STUB1/CHIP). Here, 2-ME stimulation activates CHIP by phosphorylation via Aurora kinase A (AURKA). Aurora A kinase inhibitors and RNAi knockdown of Aurora A transcript selectively blocked CHIP phosphorylation and AR degradation. Aurora A kinase is activated by 2-ME in the S-phase as well as during mitosis, and phosphorylates CHIP at S273. Prostate cancer cells expressing an S273A mutant of CHIP have attenuated AR degradation upon 2-ME treatment compared with cells expressing wild-type CHIP, supporting the idea that CHIP phosphorylation by Aurora A activates its E3 ligase activity for the AR. These results reveal a novel 2-ME→Aurora A→CHIP→AR pathway that promotes AR degradation via the proteasome that may offer novel therapeutic opportunities for prostate cancer. Mol Cancer Res; 15(8); 1063-72. ©2017 AACR. ©2017 American Association for Cancer Research.

  6. Chidamide Inhibits Aerobic Metabolism to Induce Pancreatic Cancer Cell Growth Arrest by Promoting Mcl-1 Degradation

    PubMed Central

    Wang, Yanbing; Kuai, Qiyuan; Li, Changlan; Wang, Yu; Jiang, Xingwei; Wang, Xuanlin; Li, Weijing; He, Min; Ren, Suping; Yu, Qun

    2016-01-01

    Pancreatic cancer is a fatal malignancy worldwide and urgently requires valid therapies. Previous research showed that the HDAC inhibitor chidamide is a promising anti-cancer agent in pancreatic cancer cell lines. In this study, we elucidate a probable underlying anti-cancer mechanism of chidamide involving the degradation of Mcl-1. Mcl-1 is frequently upregulated in human cancers, which has been demonstrated to participate in oxidative phosphorylation, in addition to its anti-apoptotic actions as a Bcl-2 family member. The pancreatic cancer cell lines BxPC-3 and PANC-1 were treated with chidamide, resulting in Mcl-1 degradation accompanied by induction of Mcl-1 ubiquitination. Treatment with MG132, a proteasome inhibitor reduced Mcl-1 degradation stimulated by chidamide. Chidamide decreased O2 consumption and ATP production to inhibit aerobic metabolism in both pancreatic cancer cell lines and primary cells, similar to knockdown of Mcl-1, while overexpression of Mcl-1 in pancreatic cancer cells could restore the aerobic metabolism inhibited by chidamide. Furthermore, chidamide treatment or Mcl-1 knockdown significantly induced cell growth arrest in pancreatic cancer cell lines and primary cells, and Mcl-1 overexpression could reduce this cell growth inhibition. In conclusion, our results suggest that chidamide promotes Mcl-1 degradation through the ubiquitin-proteasome pathway, suppressing the maintenance of mitochondrial aerobic respiration by Mcl-1, and resulting in inhibition of pancreatic cancer cell proliferation. Our work supports the claim that chidamide has therapeutic potential for pancreatic cancer treatment. PMID:27875574

  7. ATP Binding by Proteasomal ATPases Regulates Cellular Assembly and Substrate-induced Functions of the 26 S Proteasome*

    PubMed Central

    Kim, Young-Chan; Li, Xiaohua; Thompson, David; DeMartino, George N.

    2013-01-01

    We examined the role of ATP binding by six different ATPase subunits (Rpt1–6) in the cellular assembly and molecular functions of mammalian 26 S proteasome. Four Rpt subunits (Rpt1–4) with ATP binding mutations were incompetent for cellular assembly into 26 S proteasome. In contrast, analogous mutants of Rpt5 and Rpt6 were incorporated normally into 26 S proteasomes in both intact cells and an in vitro assembly assay. Surprisingly, purified 26 S proteasomes containing either mutant Rpt5 or Rpt6 had normal basal ATPase activity and substrate gate opening for hydrolysis of short peptides. However, these mutant 26 S proteasomes were severely defective for ATP-dependent in vitro degradation of ubiquitylated and non-ubiquitylated proteins and did not display substrate-stimulated ATPase and peptidase activities characteristic of normal proteasomes. These results reveal differential roles of ATP binding by various Rpt subunits in proteasome assembly and function. They also indicate that substrate-stimulated ATPase activity and gating depend on the concerted action of a full complement of Rpt subunits competent for ATP binding and that this regulation is essential for normal proteolysis. Thus, protein substrates appear to promote their own degradation by stimulating proteasome functions involved in proteolysis. PMID:23212908

  8. 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 (K48Ub(2)) 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 K48Ub(2)-oleosin aggregates. These results indicate that K48Ub(2)-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. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  9. Genetics of Proteasome Diseases

    PubMed Central

    Gomes, Aldrin V.

    2013-01-01

    The proteasome is a large, multiple subunit complex that is capable of degrading most intracellular proteins. Polymorphisms in proteasome subunits are associated with cardiovascular diseases, diabetes, neurological diseases, and cancer. One polymorphism in the proteasome gene PSMA6 (−8C/G) is associated with three different diseases: type 2 diabetes, myocardial infarction, and coronary artery disease. One type of proteasome, the immunoproteasome, which contains inducible catalytic subunits, is adapted to generate peptides for antigen presentation. It has recently been shown that mutations and polymorphisms in the immunoproteasome catalytic subunit PSMB8 are associated with several inflammatory and autoinflammatory diseases including Nakajo-Nishimura syndrome, CANDLE syndrome, and intestinal M. tuberculosis infection. This comprehensive review describes the disease-related polymorphisms in proteasome genes associated with human diseases and the physiological modulation of proteasome function by these polymorphisms. Given the large number of subunits and the central importance of the proteasome in human physiology as well as the fast pace of detection of proteasome polymorphisms associated with human diseases, it is likely that other polymorphisms in proteasome genes associated with diseases will be detected in the near future. While disease-associated polymorphisms are now readily discovered, the challenge will be to use this genetic information for clinical benefit. PMID:24490108

  10. Disruption of glycosylation enhances ubiquitin-mediated proteasomal degradation of Shadoo in Scrapie-infected rodents and cultured cells.

    PubMed

    Zhang, Jin; Guo, Yan; Xie, Wu-Ling; Xu, Yin; Ren, Ke; Shi, Qi; Zhang, Bao-Yun; Chen, Cao; Tian, Chan; Gao, Chen; Dong, Xiao-Ping

    2014-06-01

    Shadoo (Sho) is an N-glycosylated glycophosphatidylinositol-anchored protein that is expressed in the brain and exhibits neuroprotective properties. Recently, research has shown that a reduction of Sho levels may reflect the presence of PrPSc in the brain. However, the possible mechanism by which prion infection triggers down-regulation of Sho remains unclear. In the present study, Western blot and immunohistochemical assays revealed that Sho, especially glycosylated Sho, declined markedly in the brains of five scrapie agent-infected hamsters and mice at the terminal stages. Analyses of the down-regulation of Sho levels with the emergence of PrPSc C2 proteolytic fragments did not identify close association in all tested scrapie-infected models. To further investigate the mechanism of depletion of Sho in prion disease, a Sho-expressing plasmid with HA tag was introduced into a scrapie-infected cell line, SMB-S15, and its normal cell line, SMB-PS. Western blot assay revealed dramatically decreased Sho in SMB-S15 cells, especially its glycosylated form. Proteasome inhibitor MG132 reversed the decrease of nonglycosylated Sho, but had little effect on glycosylated Sho. N-acetylglucosamine transferase inhibitor tunicamycin efficiently reduced the glycosylations of Sho and PrPC in SMB-PS cells, while two other endoplasmic reticulum stress inducers showed clear inhibition of diglycosylated PrPC, but did not change the expression level and profile of Sho. Furthermore, immunoprecipitation of HA-Sho illustrated ubiquitination of Sho in SMB-S15 cells, but not in SMB-PS cells. We propose that the depletions of Sho in scrapie-infected cell lines due to inhibition of glycosylation mediate protein destabilization and subsequently proteasome degradation after modification by ubiquitination.

  11. Urban renewal in the nucleus: is protein turnover by proteasomes absolutely required for nuclear receptor-regulated transcription?

    PubMed

    Nawaz, Zafar; O'Malley, Bert W

    2004-03-01

    The importance of the ubiquitin proteasome pathway in higher eukaryotes has been well established in cell cycle regulation, signal transduction, and cell differentiation, but has only recently been linked to nuclear hormone receptor-regulated gene transcription. Characterization of a number of ubiquitin proteasome pathway enzymes as coactivators and observations that several nuclear receptors are ubiquitinated and degraded in the course of their nuclear activities provide evidence that ubiquitin proteasome-mediated protein degradation plays an integral role in eukaryotic transcription. In addition to receptors, studies have revealed that coactivators are ubiquitinated and degraded via the proteasome. The notion that the ubiquitin proteasome pathway is involved in gene transcription is further strengthened by the fact that ubiquitin proteasome pathway enzymes are recruited to the promoters of target genes and that proteasome-dependent degradation of nuclear receptors is required for efficient transcriptional activity. These findings suggest that protein degradation is coupled with nuclear receptor coactivation activity. It is possible that the ubiquitin proteasome pathway modulates transcription by promoting remodeling and turnover of the nuclear receptor-transcription complex. In this review, we discus the possible role of the ubiquitin proteasome pathway in nuclear hormone receptor-regulated gene transcription.

  12. A Novel Retinoblastoma Protein (RB) E3 Ubiquitin Ligase (NRBE3) Promotes RB Degradation and Is Transcriptionally Regulated by E2F1 Transcription Factor.

    PubMed

    Wang, Yingshuang; Zheng, Zongfang; Zhang, Jingyi; Wang, You; Kong, Ruirui; Liu, Jiangying; Zhang, Ying; Deng, Hongkui; Du, Xiaojuan; Ke, Yang

    2015-11-20

    Retinoblastoma protein (RB) plays critical roles in tumor suppression and is degraded through the proteasomal pathway. However, E3 ubiquitin ligases responsible for proteasome-mediated degradation of RB are largely unknown. Here we characterize a novel RB E3 ubiquitin ligase (NRBE3) that binds RB and promotes RB degradation. NRBE3 contains an LXCXE motif and bound RB in vitro. NRBE3 interacted with RB in cells when proteasome activity was inhibited. NRBE3 promoted RB ubiquitination and degradation via the ubiquitin-proteasome pathway. Importantly, purified NRBE3 ubiquitinated recombinant RB in vitro, and a U-box was identified as essential for its E3 activity. Surprisingly, NRBE3 was transcriptionally activated by E2F1/DP1. Consequently, NRBE3 affected the cell cycle by promoting G1/S transition. Moreover, NRBE3 was up-regulated in breast cancer tissues. Taken together, we identified NRBE3 as a novel ubiquitin E3 ligase for RB that might play a role as a potential oncoprotein in human cancers. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Proteasomal degradation of ubiquitinated Insig proteins is determined by serine residues flanking ubiquitinated lysines

    PubMed Central

    Lee, Joon No; Gong, Yi; Zhang, Xiangyu; Ye, Jin

    2006-01-01

    Insig-1 and Insig-2 are closely related proteins of the endoplasmic reticulum that play crucial roles in cholesterol homeostasis by inhibiting excessive cholesterol synthesis and uptake. In sterol-depleted cells Insig-1 is degraded at least 15 times more rapidly than Insig-2, owing to ubiquitination of Lys-156 and Lys-158 in Insig-1. In this study, we use domain-swapping methods to localize amino acid residues responsible for this differential degradation. In the case of Insig-2, Glu-214 stabilizes the protein by preventing ubiquitination. When Glu-214 is changed to alanine, Insig-2 becomes ubiquitinated, but it is still not degraded as rapidly as ubiquitinated Insig-1. The difference in the degradation rates is traced to two amino acids: Ser-149 in Insig-1 and Ser-106 in Insig-2. Ser-149, which lies NH2-terminal to the ubiquitination sites, accelerates the degradation of ubiquitinated Insig-1. Ser-106, which is COOH-terminal to the ubiquitination sites, retards the degradation of ubiquitinated Insig-2. The current studies indicate that the degradation of ubiquitinated Insigs is controlled by serine residues flanking the sites of ubiquitination. PMID:16549805

  14. Down-Regulation of Desmosomes in Cultured Cells: The Roles of PKC, Microtubules and Lysosomal/Proteasomal Degradation

    PubMed Central

    McHarg, Selina; Hopkins, Gemma; Lim, Lusiana; Garrod, David

    2014-01-01

    Desmosomes are intercellular adhesive junctions of major importance for tissue integrity. To allow cell motility and migration they are down-regulated in epidermal wound healing. Electron microscopy indicates that whole desmosomes are internalised by cells in tissues, but the mechanism of down-regulation is unclear. In this paper we provide an overview of the internalisation of half-desmosomes by cultured cells induced by calcium chelation. Our results show that: (i) half desmosome internalisation is dependent on conventional PKC isoforms; (ii) microtubules transport internalised half desmosomes to the region of the centrosome by a kinesin-dependent mechanism; (iii) desmosomal proteins remain colocalised after internalisation and are not recycled to the cell surface; (iv) internalised desmosomes are degraded by the combined action of lysosomes and proteasomes. We also confirm that half desmosome internalisation is dependent upon the actin cytoskeleton. These results suggest that half desmosomes are not disassembled and recycled during or after internalisation but instead are transported to the centrosomal region where they are degraded. These findings may have significance for the down-regulation of desmosomes in wounds. PMID:25291180

  15. Proteasomal degradation of preemptive quality control (pQC) substrates is mediated by an AIRAPL–p97 complex

    PubMed Central

    Braunstein, Ilana; Zach, Lolita; Allan, Susanne; Kalies, Kai-Uwe; Stanhill, Ariel

    2015-01-01

    The initial folding of secreted proteins occurs in the ER lumen, which contains specific chaperones and where posttranslational modifications may occur. Therefore lack of translocation, regardless of entry route or protein identity, is a highly toxic event, as the newly synthesized polypeptide is misfolded and can promiscuously interact with cytosolic factors. Mislocalized proteins bearing a signal sequence that did not successfully translocate through the translocon complex are subjected to a preemptive quality control (pQC) pathway and are degraded by the ubiquitin-proteasome system (UPS). In contrast to UPS-mediated, ER-associated degradation, few components involved in pQC have been identified. Here we demonstrate that on specific translocation inhibition, a p97–AIRAPL complex directly binds and regulates the efficient processing of polyubiquitinated pQC substrates by the UPS. We also demonstrate p97’s role in pQC processing of preproinsulin in cases of naturally occurring mutations within the signal sequence of insulin. PMID:26337389

  16. Newcastle disease virus degrades HIF-1α through proteasomal pathways independent of VHL and p53

    PubMed Central

    Abd-Aziz, Noraini; Stanbridge, Eric J.

    2016-01-01

    Newcastle disease virus (NDV) is a candidate agent for oncolytic virotherapy. Despite its potential, the exact mechanism of its oncolysis is still not known. Recently, we reported that NDV exhibited an increased oncolytic activity in hypoxic cancer cells. These types of cells negatively affect therapeutic outcome by overexpressing pro-survival genes under the control of the hypoxia-inducible factor (HIF). HIF-1 is a heterodimeric transcriptional factor consisting of a regulated α (HIF-1α) and a constitutive β subunit (HIF-1β). To investigate the effects of NDV infection on HIF-1α in cancer cells, the osteosarcoma (Saos-2), breast carcinoma (MCF-7), colon carcinoma (HCT116) and fibrosarcoma (HT1080) cell lines were used in the present study. Data obtained showed that a velogenic NDV infection diminished hypoxia-induced HIF-1α accumulation, leading to a decreased activation of its downstream target gene, carbonic anhydrase 9. This NDV-induced downregulation of HIF-1α occurred post-translationally and was partially abrogated by proteasomal inhibition. The process appeared to be independent of the tumour suppressor protein p53. These data revealed a correlation between NDV infection and HIF-1α downregulation, which highlights NDV as a promising agent to eliminate hypoxic cancer cells. PMID:27902314

  17. Cardiac fibrosis in mouse expressing DsRed tetramers involves chronic autophagy and proteasome degradation insufficiency

    PubMed Central

    Chen, Tsung-Hsien; Chen, Mei-Ru; Chen, Tzu-Yin; Wu, Tzu-Chin; Liu, Shan-Wen; Hsu, Ching-Han; Liou, Gan-Guang; Kao, Yu-Ying; Dong, Guo-Chung; Chu, Pao-Hsien; Liao, Jiunn-Wang; Lin, Kurt Ming-Chao

    2016-01-01

    Proteinopathy in the heart which often manifests excessive misfolded/aggregated proteins in cardiac myocytes can result in severe fibrosis and heart failure. Here we developed a mouse model, which transgenically express tetrameric DsRed, a red fluorescent protein (RFP), in an attempt to mimic the pathological mechanisms ofcardiac fibrosis. Whilst DsRed is expressed and forms aggregation in most mouse organs, certain pathological defects are specifically recapitulated in cardiac muscle cells including mitochondria damages, aggresome-like residual bodies, excessive ubiquitinated proteins, and the induction of autophagy. The proteinopathy and cellular injuries caused by DsRed aggregates may be due to impaired or overburdened ubiquitin-proteasome system and autophagy-lysosome systems. We further identified that DsRed can be ubiquitinated and associated with MuRF1, a muscle-specific E3 ligase. Concomitantly, an activation of NF-κB signaling and a strong TIMP1 induction were noted, suggesting that RFP-induced fibrosis was augmented by a skewed balance between TIMP1 and MMPs. Taken together, our study highlights the molecular consequences of uncontrolled protein aggregation leading to congestive heart failure, and provides novel insights into fibrosis formation that can be exploited for improved therapy. PMID:27494843

  18. Newcastle disease virus degrades HIF-1α through proteasomal pathways independent of VHL and p53.

    PubMed

    Abd-Aziz, Noraini; Stanbridge, Eric J; Shafee, Norazizah

    2016-12-01

    Newcastle disease virus (NDV) is a candidate agent for oncolytic virotherapy. Despite its potential, the exact mechanism of its oncolysis is still not known. Recently, we reported that NDV exhibited an increased oncolytic activity in hypoxic cancer cells. These types of cells negatively affect therapeutic outcome by overexpressing pro-survival genes under the control of the hypoxia-inducible factor (HIF). HIF-1 is a heterodimeric transcriptional factor consisting of a regulated α (HIF-1α) and a constitutive β subunit (HIF-1β). To investigate the effects of NDV infection on HIF-1α in cancer cells, the osteosarcoma (Saos-2), breast carcinoma (MCF-7), colon carcinoma (HCT116) and fibrosarcoma (HT1080) cell lines were used in the present study. Data obtained showed that a velogenic NDV infection diminished hypoxia-induced HIF-1α accumulation, leading to a decreased activation of its downstream target gene, carbonic anhydrase 9. This NDV-induced downregulation of HIF-1α occurred post-translationally and was partially abrogated by proteasomal inhibition. The process appeared to be independent of the tumour suppressor protein p53. These data revealed a correlation between NDV infection and HIF-1α downregulation, which highlights NDV as a promising agent to eliminate hypoxic cancer cells.

  19. Binding-induced folding of prokaryotic ubiquitin-like protein on the Mycobacterium proteasomal ATPase targets substrates for degradation

    PubMed Central

    Wang, Tao; Darwin, K. Heran; Li, Huilin

    2010-01-01

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analogue of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein bearing little sequence or structural resemblance to the highly structured ubiquitin. Thus it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled-coils that recognize Pup. Mpa binds unstructured Pup via hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an α-helix in Pup. Our work revealed a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This critical difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment of tuberculosis. PMID:20953180

  20. Binding-induced Folding of Prokaryotic Ubiquitin-like Protein on the Mycobacterium Proteasomal ATPase Targets Substrates for Degradation

    SciTech Connect

    T Wang; K Heran Darwin; H Li

    2011-12-31

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

  1. Binding-induced folding of prokaryotic ubiquitin-like protein on the mycobacterium proteasomal ATPase targets substrates for degradation

    SciTech Connect

    Wang, T.; Li, H.; Darwin, K. H.

    2010-11-01

    Mycobacterium tuberculosis uses a proteasome system that is analogous to the eukaryotic ubiquitin-proteasome pathway and is required for pathogenesis. However, the bacterial analog of ubiquitin, prokaryotic ubiquitin-like protein (Pup), is an intrinsically disordered protein that bears little sequence or structural resemblance to the highly structured ubiquitin. Thus, it was unknown how pupylated proteins were recruited to the proteasome. Here, we show that the Mycobacterium proteasomal ATPase (Mpa) has three pairs of tentacle-like coiled coils that recognize Pup. Mpa bound unstructured Pup through hydrophobic interactions and a network of hydrogen bonds, leading to the formation of an {alpha}-helix in Pup. Our work describes a binding-induced folding recognition mechanism in the Pup-proteasome system that differs mechanistically from substrate recognition in the ubiquitin-proteasome system. This key difference between the prokaryotic and eukaryotic systems could be exploited for the development of a small molecule-based treatment for tuberculosis.

  2. 20S proteasome activity is modified via S-glutathionylation based on intracellular redox status of the yeast Saccharomyces cerevisiae: implications for the degradation of oxidized proteins.

    PubMed

    Demasi, Marilene; Hand, Adrian; Ohara, Erina; Oliveira, Cristiano L P; Bicev, Renata N; Bertoncini, Clelia A; Netto, Luis E S

    2014-09-01

    Protein S-glutathionylation is a post-translational modification that controls many cellular pathways. Recently, we demonstrated that the α5-subunit of the 20S proteasome is S-glutathionylated in yeast cells grown to the stationary phase in rich medium containing glucose, stimulating 20S core gate opening and increasing the degradation of oxidized proteins. In the present study, we evaluated the correlation between proteasomal S-glutathionylation and the intracellular redox status. The redox status was controlled by growing yeast cells in distinct carbon sources which induced respiratory (glycerol/ethanol) or fermentative (glucose) metabolism. Cells grown under glycerol/ethanol displayed higher reductive power when compared to cells grown under glucose. When purified from cells grown in glucose, 20S proteasome α5-subunit exhibited an intense anti-glutathione labeling. A higher frequency of the open catalytic chamber gate was observed in the S-glutathionylated preparations as demonstrated by transmission electron microscopy. Therefore, cells that had been grown in glucose displayed an increased ability to degrade oxidized proteins. The results of the present study suggest that 20S proteasomal S-glutathionylation is a relevant adaptive response to oxidative stress that is capable to sense the intracellular redox environment, leading to the removal of oxidized proteins via a process that is not dependent upon ubiquitylation and ATP consumption. Copyright © 2014 Elsevier Inc. All rights reserved.

  3. Light-Regulated Hypocotyl Elongation Involves Proteasome-Dependent Degradation of the Microtubule Regulatory Protein WDL3 in Arabidopsis[C][W][OA

    PubMed Central

    Liu, Xiaomin; Qin, Tao; Ma, Qianqian; Sun, Jingbo; Liu, Ziqiang; Yuan, Ming; Mao, Tonglin

    2013-01-01

    Light significantly inhibits hypocotyl cell elongation, and dark-grown seedlings exhibit elongated, etiolated hypocotyls. Microtubule regulatory proteins function as positive or negative regulators that mediate hypocotyl cell elongation by altering microtubule organization. However, it remains unclear how plants coordinate these regulators to promote hypocotyl growth in darkness and inhibit growth in the light. Here, we demonstrate that WAVE-DAMPENED 2–LIKE3 (WDL3), a microtubule regulatory protein of the WVD2/WDL family from Arabidopsis thaliana, functions in hypocotyl cell elongation and is regulated by a ubiquitin-26S proteasome–dependent pathway in response to light. WDL3 RNA interference Arabidopsis seedlings grown in the light had much longer hypocotyls than controls. Moreover, WDL3 overexpression resulted in overall shortening of hypocotyl cells and stabilization of cortical microtubules in the light. Cortical microtubule reorganization occurred slowly in cells from WDL3 RNA interference transgenic lines but was accelerated in cells from WDL3-overexpressing seedlings subjected to light treatment. More importantly, WDL3 protein was abundant in the light but was degraded through the 26S proteasome pathway in the dark. Overexpression of WDL3 inhibited etiolated hypocotyl growth in regulatory particle non-ATPase subunit-1a mutant (rpn1a-4) plants but not in wild-type seedlings. Therefore, a ubiquitin-26S proteasome–dependent mechanism regulates the levels of WDL3 in response to light to modulate hypocotyl cell elongation. PMID:23653471

  4. Stress-responsive mitogen-activated protein kinases interact with the EAR motif of a poplar zinc finger protein and mediate its degradation through the 26S proteasome.

    PubMed

    Hamel, Louis-Philippe; Benchabane, Meriem; Nicole, Marie-Claude; Major, Ian T; Morency, Marie-Josée; Pelletier, Gervais; Beaudoin, Nathalie; Sheen, Jen; Séguin, Armand

    2011-11-01

    Mitogen-activated protein kinases (MAPKs) contribute to the establishment of plant disease resistance by regulating downstream signaling components, including transcription factors. In this study, we identified MAPK-interacting proteins, and among the newly discovered candidates was a Cys-2/His-2-type zinc finger protein named PtiZFP1. This putative transcription factor belongs to a family of transcriptional repressors that rely on an ERF-associated amphiphilic repression (EAR) motif for their repression activity. Amino acids located within this repression motif were also found to be essential for MAPK binding. Close examination of the primary protein sequence revealed a functional bipartite MAPK docking site that partially overlaps with the EAR motif. Transient expression assays in Arabidopsis (Arabidopsis thaliana) protoplasts suggest that MAPKs promote PtiZFP1 degradation through the 26S proteasome. Since features of the MAPK docking site are conserved among other EAR repressors, our study suggests a novel mode of defense mechanism regulation involving stress-responsive MAPKs and EAR repressors.

  5. Human cytomegalovirus inhibits apoptosis by proteasome-mediated degradation of Bax at endoplasmic reticulum-mitochondrion contacts.

    PubMed

    Zhang, Aiping; Hildreth, Richard L; Colberg-Poley, Anamaris M

    2013-05-01

    Human cytomegalovirus (HCMV) encodes the UL37 exon 1 protein (pUL37x1), which is the potent viral mitochondrion-localized inhibitor of apoptosis (vMIA), to increase survival of infected cells. HCMV vMIA traffics from the endoplasmic reticulum (ER) to ER subdomains, which are physically linked to mitochondria known as mitochondrion-associated membranes (MAM), and to mitochondria. The antiapoptotic function of vMIA is thought to primarily result from its ability to inhibit Bax-mediated permeabilization of the outer mitochondrial membrane (OMM). Here, we establish that vMIA retargets Bax to the MAM as well as to the OMM from immediate early through late times of infection. However, MAM localization of Bax results in its increased ubiquitination and proteasome-mediated degradation. Surprisingly, HCMV infection does not increase OMM-associated degradation (OMMAD) of Bax, even though the ER and mitochondria are physically connected at the MAM. It was recently found that lipid rafts at the plasma membrane can connect extrinsic and intrinsic apoptotic pathways and can serve as sites of apoptosome assembly. In transfected permissive human fibroblasts, vMIA mediates, through its cholesterol affinity, association of Bax and apoptosome components with MAM lipid rafts. While Bax association with MAM lipid rafts was detected in HCMV-infected cells, association of apoptosome components was not. These results establish that Bax recruitment to the MAM and its MAM-associated degradation (MAMAD) are a newly described antiapoptotic mechanism used by HCMV infection to increase cell survival for its growth.

  6. T cell activation induces proteasomal degradation of Argonaute and rapid remodeling of the microRNA repertoire

    PubMed Central

    Bronevetsky, Yelena; Villarino, Alejandro V.; Eisley, Christopher J.; Barbeau, Rebecca; Barczak, Andrea J.; Heinz, Gitta A.; Kremmer, Elisabeth; Heissmeyer, Vigo; McManus, Michael T.; Erle, David J.; Rao, Anjana

    2013-01-01

    Activation induces extensive changes in the gene expression program of naive CD4+ T cells, promoting their differentiation into helper T cells that coordinate immune responses. MicroRNAs (miRNAs) play a critical role in this process, and miRNA expression also changes dramatically during T cell differentiation. Quantitative analyses revealed that T cell activation induces global posttranscriptional miRNA down-regulation in vitro and in vivo. Argonaute (Ago) proteins, the core effector proteins of the miRNA-induced silencing complex (miRISC), were also posttranscriptionally down-regulated during T cell activation. Ago2 was inducibly ubiquitinated in activated T cells and its down-regulation was inhibited by the proteasome inhibitor MG132. Therefore, activation-induced miRNA down-regulation likely occurs at the level of miRISC turnover. Measurements of miRNA-processing intermediates uncovered an additional layer of activation-induced, miRNA-specific transcriptional regulation. Thus, transcriptional and posttranscriptional mechanisms cooperate to rapidly reprogram the miRNA repertoire in differentiating T cells. Altering Ago2 expression in T cells revealed that Ago proteins are limiting factors that determine miRNA abundance. Naive T cells with reduced Ago2 and miRNA expression differentiated more readily into cytokine-producing helper T cells, suggesting that activation-induced miRNA down-regulation promotes acquisition of helper T cell effector functions by relaxing the repression of genes that direct T cell differentiation. PMID:23382546

  7. The SPRY domain–containing SOCS box protein SPSB2 targets iNOS for proteasomal degradation

    PubMed Central

    Kuang, Zhihe; Lewis, Rowena S.; Curtis, Joan M.; Zhan, Yifan; Saunders, Bernadette M.; Babon, Jeffrey J.; Kolesnik, Tatiana B.; Low, Andrew; Masters, Seth L.; Willson, Tracy A.; Kedzierski, Lukasz; Yao, Shenggen; Handman, Emanuela

    2010-01-01

    Inducible nitric oxide (NO) synthase (iNOS; NOS2) produces NO and related reactive nitrogen species, which are critical effectors of the innate host response and are required for the intracellular killing of pathogens such as Mycobacterium tuberculosis and Leishmania major. We have identified SPRY domain–containing SOCS (suppressor of cytokine signaling) box protein 2 (SPSB2) as a novel negative regulator that recruits an E3 ubiquitin ligase complex to polyubiquitinate iNOS, resulting in its proteasomal degradation. SPSB2 interacts with the N-terminal region of iNOS via a binding interface on SPSB2 that has been mapped by nuclear magnetic resonance spectroscopy and mutational analyses. SPSB2-deficient macrophages showed prolonged iNOS expression, resulting in a corresponding increase in NO production and enhanced killing of L. major parasites. These results lay the foundation for the development of small molecule inhibitors that could disrupt the SPSB–iNOS interaction and thus prolong the intracellular lifetime of iNOS, which may be beneficial in chronic and persistent infections. PMID:20603330

  8. Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation.

    PubMed

    Yabe, Ryotaro; Miura, Akane; Usui, Tatsuya; Mudrak, Ingrid; Ogris, Egon; Ohama, Takashi; Sato, Koichi

    2015-01-01

    Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.

  9. SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage

    PubMed Central

    Hong, Xuehui; Liu, Wenyu; Song, Ruipeng; Shah, Jamie J.; Feng, Xing; Tsang, Chi Kwan; Morgan, Katherine M.; Bunting, Samuel F.; Inuzuka, Hiroyuki; Zheng, X. F. Steven; Shen, Zhiyuan; Sabaawy, Hatem E.; Liu, LianXin; Pine, Sharon R.

    2016-01-01

    SOX9 encodes a transcription factor that governs cell fate specification throughout development and tissue homeostasis. Elevated SOX9 is implicated in the genesis and progression of human tumors by increasing cell proliferation and epithelial-mesenchymal transition. We found that in response to UV irradiation or genotoxic chemotherapeutics, SOX9 is actively degraded in various cancer types and in normal epithelial cells, through a pathway independent of p53, ATM, ATR and DNA-PK. SOX9 is phosphorylated by GSK3β, facilitating the binding of SOX9 to the F-box protein FBW7α, an E3 ligase that functions in the DNA damage response pathway. The binding of FBW7α to the SOX9 K2 domain at T236-T240 targets SOX9 for subsequent ubiquitination and proteasomal destruction. Exogenous overexpression of SOX9 after genotoxic stress increases cell survival. Our findings reveal a novel regulatory mechanism for SOX9 stability and uncover a unique function of SOX9 in the cellular response to DNA damage. This new mechanism underlying a FBW7-SOX9 axis in cancer could have implications in therapy resistance. PMID:27566146

  10. SOX9 is targeted for proteasomal degradation by the E3 ligase FBW7 in response to DNA damage.

    PubMed

    Hong, Xuehui; Liu, Wenyu; Song, Ruipeng; Shah, Jamie J; Feng, Xing; Tsang, Chi Kwan; Morgan, Katherine M; Bunting, Samuel F; Inuzuka, Hiroyuki; Zheng, X F Steven; Shen, Zhiyuan; Sabaawy, Hatem E; Liu, LianXin; Pine, Sharon R

    2016-10-14

    SOX9 encodes a transcription factor that governs cell fate specification throughout development and tissue homeostasis. Elevated SOX9 is implicated in the genesis and progression of human tumors by increasing cell proliferation and epithelial-mesenchymal transition. We found that in response to UV irradiation or genotoxic chemotherapeutics, SOX9 is actively degraded in various cancer types and in normal epithelial cells, through a pathway independent of p53, ATM, ATR and DNA-PK. SOX9 is phosphorylated by GSK3β, facilitating the binding of SOX9 to the F-box protein FBW7α, an E3 ligase that functions in the DNA damage response pathway. The binding of FBW7α to the SOX9 K2 domain at T236-T240 targets SOX9 for subsequent ubiquitination and proteasomal destruction. Exogenous overexpression of SOX9 after genotoxic stress increases cell survival. Our findings reveal a novel regulatory mechanism for SOX9 stability and uncover a unique function of SOX9 in the cellular response to DNA damage. This new mechanism underlying a FBW7-SOX9 axis in cancer could have implications in therapy resistance.

  11. Inhibition of ERK1/2 Pathway Suppresses Adiponectin Secretion via Accelerating Protein Degradation by Ubiquitin-Proteasome System: Relevance to Obesity-related Adiponectin Decline

    PubMed Central

    Gu, Dongfang; Wang, Zhigang; Dou, Xiaobing; Zhang, Ximei; Li, Songtao; Vu, Lyndsey; Yao, Tong; Song, Zhenyuan

    2013-01-01

    Objective Predominantly secreted by adipose tissue, adiponectin possesses insulin-sensitizing, anti-atherogenic, anti-inflammatory, and anti-angiogenic properties. Paradoxically, obesity is associated with declined plasma adiponectin levels; however, the underlying mechanisms remain elusive. In this study, we investigated the mechanistic involvement of MEK/ERK1/2 pathway in obesity-related adiponectin decrease. Materials/Methods C57 BL/6 mice exposed to a high-fat diet (HFD) were employed as animal obesity model. Both fully-differentiated 3T3-L1 and mouse primary adipocytes were used in the in vitro experiments. Results Obesity and plasma adiponectin decline induced by prolonged HFD exposure was associated with suppressed ERK1/2 activation in adipose tissue. In adipocytes, specific inhibition of MEK/ERK1/2 pathway decreased intracellular and secretory adiponectin levels, whereas adiponectin gene expression was increased, suggesting that MEK/ERK1/2 inhibition may promote adiponectin protein degradation. Cycloheximide (CHX)-chase assay revealed that MEK/ERK1/2 inhibition accelerated adiponectin protein degradation, which was prevented by MG132, a potent proteasome inhibitor. Immunoprecipitation assay showed that intracellular MEK/ERK1/2 activity was negatively associated with ubiqutinated adiponectin protein levels. Consistently, long-term HFD feeing in mice increased ubiquitinated adiponectin levels in the epididymal fat pads. Conclusions Adipose tissue MEK/ERK1/2 activity can differentially regulates adiponectin gene expression and protein abundance and its suppression in obesity may play a mechanistic role in obesity-related plasma adiponectin decline. PMID:23490586

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

    PubMed

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

    2016-10-15

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

  13. Antiepileptic drugs reduce the efficacy of methotrexate chemotherapy through accelerated degradation of the reduced folate carrier by the ubiquitin-proteasome pathway.

    PubMed

    Halwachs, Sandra; Schaefer, Ingo; Seibel, Peter; Honscha, Walther

    2011-01-01

    Concurrent treatment with methotrexate (MTX) and enzyme-inducing antiepileptic drugs including phenobarbital (PB) reduces the efficacy of MTX chemotherapy in cancer patients. We have shown that Reduced folate carrier (Rfc1)-mediated uptake of MTX, an essential determinant of MTX chemotherapy, is significantly reduced by PB via protein kinase C (PKC). However, whether PB treatment affects Rfc1 activity through regulation of carrier protein stability and the mechanisms involved remain unclear. Protein turnover assays using hepatocytoma cells demonstrated that Rfc1 is a long-lived protein that is mainly degraded by the ubiquitin-proteasome proteolytic pathway under basal conditions. Pretreatment with PB significantly reduced Rfc1-mediated MTX uptake and shortened the carrier protein half-life. This effect was abolished by the specific PKC inhibitor Gö6976. Inhibition of proteasomes with MG-132 significantly elevated Rfc1 protein levels and induced colocalization of Rfc1 and ubiquitin particularly in submembranous cellular compartments. Finally, we demonstrated that PB treatment resulted in enhanced levels of Rfc1 polyubiquitin conjugates. Our results demonstrate that PB treatment causes downregulation of Rfc1 activity through PKC-dependent accelerated degradation of the Rfc1 protein by the ubiqutin-proteasome pathway. This regulatory mechanism may therefore involve clinically relevant drug resistance in patients concurrently receiving MTX and enzyme-inducing antiepileptic drugs. Copyright © 2011 S. Karger AG, Basel.

  14. 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

  15. RSK1 protects P-glycoprotein/ABCB1 against ubiquitin–proteasomal degradation by downregulating the ubiquitin-conjugating enzyme E2 R1

    PubMed Central

    Katayama, Kazuhiro; Fujiwara, Chiaki; Noguchi, Kohji; Sugimoto, Yoshikazu

    2016-01-01

    P-glycoprotein (P-gp) is a critical determinant of multidrug resistance in cancer. We previously reported that MAPK inhibition downregulates P-gp expression and that P-gp undergoes ubiquitin–proteasomal degradation regulated by UBE2R1 and SCFFbx15. Here, we investigated the crosstalk between MAPK inhibition and the ubiquitin–proteasomal degradation of P-gp. Proteasome inhibitors or knockdown of FBXO15 and/or UBE2R1 cancelled MEK inhibitor-induced P-gp downregulation. RSK1 phosphorylated Thr162 on UBE2R1 but did not phosphorylate FBXO15. MEK and RSK inhibitors increased UBE2R1-WT but not UBE2R1-T162D and -T162A expression. UBE2R1-T162D showed higher self-ubiquitination and destabilisation than UBE2R1-WT and -T162A. Unlike UBE2R1-WT and -T162A, UBE2R1-T162D did not induce P-gp ubiquitination. UBE2R1-WT or -T162A downregulated P-gp expression and upregulated rhodamine 123 level and sensitivity to vincristine and doxorubicin. However, UBE2R1-T162D did not confer any change in P-gp expression, rhodamine 123 accumulation and sensitivity to the drugs. These results suggest that RSK1 protects P-gp against ubiquitination by reducing UBE2R1 stability. PMID:27786305

  16. S-nitrosylation-dependent proteasomal degradation restrains Cdk5 activity to regulate hippocampal synaptic strength

    PubMed Central

    Zhang, Peng; Fu, Wing-Yu; Fu, Amy K. Y.; Ip, Nancy Y.

    2015-01-01

    Precise regulation of synaptic strength requires coordinated activity and functions of synaptic proteins, which is controlled by a variety of post-translational modification. Here we report that S-nitrosylation of p35, the activator of cyclin-dependent kinase 5 (Cdk5), by nitric oxide (NO) is important for the regulation of excitatory synaptic strength. While blockade of NO signalling results in structural and functional synaptic deficits as indicated by reduced mature dendritic spine density and surface expression of glutamate receptor subunits, phosphorylation of numerous synaptic substrates of Cdk5 and its activity are aberrantly upregulated following reduced NO production. The results show that the NO-induced reduction in Cdk5 activity is mediated by S-nitrosylation of p35, resulting in its ubiquitination and degradation by the E3 ligase PJA2. Silencing p35 protein in hippocampal neurons partially rescues the NO blockade-induced synaptic deficits. These findings collectively demonstrate that p35 S-nitrosylation by NO signalling is critical for regulating hippocampal synaptic strength. PMID:26503494

  17. Proteasome dynamics.

    PubMed

    Enenkel, Cordula

    2014-01-01

    Proteasomes are highly conserved multisubunit protease complexes and occur in the cyto- and nucleoplasm of eukaryotic cells. In dividing cells proteasomes exist as holoenzymes and primarily localize in the nucleus. During quiescence they dissociate into proteolytic core and regulatory complexes and are sequestered into motile cytosolic clusters. Proteasome clusters rapidly clear upon the exit from quiescence, where proteasome core and regulatory complexes reassemble and localize to the nucleus again. The mechanisms underlying proteasome transport and assembly are not yet understood. Here, I summarize our present knowledge about nuclear transport and assembly of proteasomes in yeast and project our studies in this eukaryotic model organism to the mammalian cell system. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

  18. Chromium–Insulin Reduces Insulin Clearance and Enhances Insulin Signaling by Suppressing Hepatic Insulin-Degrading Enzyme and Proteasome Protein Expression in KKAy Mice

    PubMed Central

    Wang, Zhong Q.; Yu, Yongmei; Zhang, Xian H.; Komorowski, James

    2014-01-01

    JDS–chromium–insulin (CRI)-003 is a novel form of insulin that has been directly conjugated with chromium (Cr) instead of zinc. Our hypothesis was that CRI enhances insulin’s effects by altering insulin-degrading enzyme (IDE) and proteasome enzymes. To test this hypothesis, we measured hepatic IDE content and proteasome parameters in a diabetic animal model. Male KKAy mice were randomly divided into three groups (n = 8/group); Sham (saline), human regular insulin (Reg-In), and chromium conjugated human insulin (CRI), respectively. Interventions were initiated at doses of 2 U insulin/kg body weight daily for 8-weeks. Plasma glucose and insulin were measured. Hepatic IDE, proteasome, and insulin signaling proteins were determined by western blotting. Insulin tolerance tests at week 7 showed that both insulin treatments significantly reduced glucose concentrations and increased insulin levels compared with the Sham group, CRI significantly reduced glucose at 4 and 6 h relative to Reg-In (P < 0.05), suggesting the effects of CRI on reducing glucose last longer than Reg-In. CRI treatment significantly increased hepatic IRS-1 and Akt1 and reduced IDE, 20S as well as 19S protein abundance (P < 0.01, P < 0.05, and P < 0.001, respectively), but Reg-In only significantly increased Akt1 (P < 0.05). Similar results were also observed in Reg-In- and CRI-treated HepG2 cells. This study, for the first time, demonstrates that CRI reduces plasma insulin clearance by inhibition of hepatic IDE protein expression and enhances insulin signaling as well as prevents degradation of IRS-1 and IRS-2 by suppressing ubiquitin-proteasome pathway in diabetic mice. PMID:25071716

  19. Human Sex Determination at the Edge of Ambiguity: INHERITED XY SEX REVERSAL DUE TO ENHANCED UBIQUITINATION AND PROTEASOMAL DEGRADATION OF A MASTER TRANSCRIPTION FACTOR.

    PubMed

    Racca, Joseph D; Chen, Yen-Shan; Yang, Yanwu; Phillips, Nelson B; Weiss, Michael A

    2016-10-14

    A general problem is posed by analysis of transcriptional thresholds governing cell fate decisions in metazoan development. A model is provided by testis determination in therian mammals. Its key step, Sertoli cell differentiation in the embryonic gonadal ridge, is initiated by SRY, a Y-encoded architectural transcription factor. Mutations in human SRY cause gonadal dysgenesis leading to XY female development (Swyer syndrome). Here, we have characterized an inherited mutation compatible with either male or female somatic phenotypes as observed in an XY father and XY daughter, respectively. The mutation (a crevice-forming substitution at a conserved back surface of the SRY high mobility group box) markedly destabilizes the domain but preserves specific DNA affinity and induced DNA bend angle. On transient transfection of diverse human and rodent cell lines, the variant SRY exhibited accelerated proteasomal degradation (relative to wild type) associated with increased ubiquitination; in vitro susceptibility to ubiquitin-independent ("default") cleavage by the 20S core proteasome was unchanged. The variant's gene regulatory activity (as assessed in a cellular model of the rat embryonic XY gonadal ridge) was reduced by 2-fold relative to wild-type SRY at similar levels of mRNA expression. Chemical proteasome inhibition restored native-like SRY expression and transcriptional activity in association with restored occupancy of a sex-specific enhancer element in principal downstream gene Sox9, demonstrating that the variant SRY exhibits essentially native activity on a per molecule basis. Our findings define a novel mechanism of impaired organogenesis, accelerated ubiquitin-directed proteasomal degradation of a master transcription factor leading to a developmental decision poised at the edge of ambiguity.

  20. scyllo-Inositol promotes robust mutant Huntingtin protein degradation.

    PubMed

    Lai, Aaron Y; Lan, Cynthia P; Hasan, Salwa; Brown, Mary E; McLaurin, Joanne

    2014-02-07

    Huntington disease is characterized by neuronal aggregates and inclusions containing polyglutamine-expanded huntingtin protein and peptide fragments (polyQ-Htt). We have used an established cell-based assay employing a PC12 cell line overexpressing truncated exon 1 of Htt with a 103-residue polyQ expansion that yields polyQ-Htt aggregates to investigate the fate of polyQ-Htt-drug complexes. scyllo-Inositol is an endogenous inositol stereoisomer known to inhibit accumulation and toxicity of the amyloid-β peptide and α-synuclein. In light of these properties, we investigated the effect of scyllo-inositol on polyQ-Htt accumulation. We show that scyllo-inositol lowered the number of visible polyQ-Htt aggregates and robustly decreased polyQ-Htt protein abundance without concomitant cellular toxicity. We found that scyllo-inositol-induced polyQ-Htt reduction was by rescue of degradation pathways mediated by the lysosome and by the proteasome but not autophagosomes. The rescue of degradation pathways was not a direct result of scyllo-inositol on the lysosome or proteasome but due to scyllo-inositol-induced reduction in mutant polyQ-Htt protein levels.

  1. PLK1 and HOTAIR accelerate proteasomal degradation of SUZ12 and ZNF198 during hepatitis B virus-induced liver carcinogenesis

    PubMed Central

    Zhang, Hao; Diab, Ahmed; Fan, Huitao; Mani, Saravana Kumar Kailasam; Hullinger, Ronald; Merle, Philippe; Andrisani, Ourania

    2015-01-01

    Elucidating mechanisms of hepatitis B virus (HBV)-mediated hepatocarcinogenesis is needed to gain insights into the etiology and treatment of liver cancer. Cells where HBV is replicating exhibit increased expression of Plk1 kinase and reduced levels of two transcription repression factors, SUZ12 and ZNF198. SUZ12 is an essential subunit of the transcription repressive complex PRC2. ZNF198 stabilizes the transcription repressive complex composed of LSD1, Co-REST and HDAC1. These two transcription repressive complexes are held together by binding the long noncoding RNA HOTAIR. In this study we linked these regulatory events mechanistically, by showing that Plk1 induces proteasomal degradation of SUZ12 and ZNF198 by site-specific phosphorylation. Plk1-dependent ubiquitination of SUZ12 and ZNF198 was enhanced by expression of HOTAIR, significantly reducing SUZ12 and ZNF198 stability. In cells expressing the HBV X protein (HBx) downregulation of SUZ12 and ZNF198 mediated global changes in histone modifications. In turn, HBx-expressing cells propagated an altered chromatin landscape after cell division, as exemplified by changes in histone modifications of the EpCAM promoter, a target of PRC2 and LSD1/Co-REST/HDAC1 complexes. Notably, liver tumors from X/cmyc bitransgenic mice exhibited downregulation of SUZ12 and ZNF198 along with elevated expression of Plk1, HOTAIR, and EpCAM. Clinically, similar effects were documented in a set of HBV-related liver tumors consistent with the likelihood that downregulation of SUZ12 and ZNF198 leads to epigenetic reprogramming of infected hepatocytes. Since both Plk1 and HOTAIR are elevated in many human cancers, we propose that their combined effects are involved in epigenetic reprogramming associated broadly with oncogenic transformation. PMID:25855382

  2. PLK1 and HOTAIR Accelerate Proteasomal Degradation of SUZ12 and ZNF198 during Hepatitis B Virus-Induced Liver Carcinogenesis.

    PubMed

    Zhang, Hao; Diab, Ahmed; Fan, Huitao; Mani, Saravana Kumar Kailasam; Hullinger, Ronald; Merle, Philippe; Andrisani, Ourania

    2015-06-01

    Elucidating mechanisms of hepatitis B virus (HBV)-mediated hepatocarcinogenesis is needed to gain insights into the etiology and treatment of liver cancer. Cells where HBV is replicating exhibit increased expression of Plk1 kinase and reduced levels of two transcription repression factors, SUZ12 and ZNF198. SUZ12 is an essential subunit of the transcription repressive complex PRC2. ZNF198 stabilizes the transcription repressive complex composed of LSD1, Co-REST, and HDAC1. These two transcription repressive complexes are held together by binding the long noncoding RNA HOTAIR. In this study, we linked these regulatory events mechanistically by showing that Plk1 induces proteasomal degradation of SUZ12 and ZNF198 by site-specific phosphorylation. Plk1-dependent ubiquitination of SUZ12 and ZNF198 was enhanced by expression of HOTAIR, significantly reducing SUZ12 and ZNF198 stability. In cells expressing the HBV X protein (HBx), downregulation of SUZ12 and ZNF198 mediated global changes in histone modifications. In turn, HBx-expressing cells propagated an altered chromatin landscape after cell division, as exemplified by changes in histone modifications of the EpCAM promoter, a target of PRC2 and LSD1/Co-REST/HDAC1 complexes. Notably, liver tumors from X/c-myc bitransgenic mice exhibited downregulation of SUZ12 and ZNF198 along with elevated expression of Plk1, HOTAIR, and EpCAM. Clinically, similar effects were documented in a set of HBV-related liver tumors consistent with the likelihood that downregulation of SUZ12 and ZNF198 leads to epigenetic reprogramming of infected hepatocytes. Because both Plk1 and HOTAIR are elevated in many human cancers, we propose that their combined effects are involved in epigenetic reprogramming associated broadly with oncogenic transformation.

  3. Honokiol induces proteasomal degradation of AML1-ETO oncoprotein via increasing ubiquitin conjugase UbcH8 expression in leukemia.

    PubMed

    Zhou, Bin; Li, Haiying; Xing, Chongyun; Ye, Haige; Feng, Jianhua; Wu, Jianbo; Lu, Zhongqiu; Fang, Jing; Gao, Shenmeng

    2017-03-15

    AML1-ETO is the most common oncoprotein leading to acute myeloid leukemia (AML), in which 5-year survival rate is only about 30%. However, currently there are no specific therapies for AML patients with AML1-ETO. Here, we report that AML1-ETO protein is rapidly degraded by Honokiol (HNK), a natural phenolic compound isolated from the plant Magnolia officinalis. HNK induced the degradation of AML1-ETO in a concentration- and time-dependent manner in leukemic cell lines and primary AML blasts with t(8;21) translocation. Mechanistically, HNK obviously increased the expression of UbcH8, an E2-conjugase for the degradation of AML1-ETO, through triggering accumulation of acetylated histones in the promoter region of UbcH8. Knockdown of UbcH8 by small hairpin RNAs (shRNAs) prevented HNK-induced degradation of AML-ETO, suggesting that UbcH8 plays a critical role in the degradation of AML1-ETO. HNK inhibited cell proliferation and induced apoptotic death without activation of caspase-3, which was reported to cleave and degrade AML1-ETO protein. Thus, HNK-induced degradation of AML1-ETO is independent of activation of caspase-3. Finally, HNK reduced the angiogenesis and migration in Kasumi-1-injected zebrafish, decreased xenograft tumor size in a xenograft leukemia mouse model, and prolonged the survival time in mouse C1498 AML model. Collectively, HNK might be a potential treatment for t(8;21) leukemia by targeting AML1-ETO oncoprotein. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Pseudomonas aeruginosa Cif Protein Enhances the Ubiquitination and Proteasomal Degradation of the Transporter Associated with Antigen Processing (TAP) and Reduces Major Histocompatibility Complex (MHC) Class I Antigen Presentation*

    PubMed Central

    Bomberger, Jennifer M.; Ely, Kenneth H.; Bangia, Naveen; Ye, Siying; Green, Kathy A.; Green, William R.; Enelow, Richard I.; Stanton, Bruce A.

    2014-01-01

    Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8+ T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation. PMID:24247241

  5. Pseudomonas aeruginosa Cif protein enhances the ubiquitination and proteasomal degradation of the transporter associated with antigen processing (TAP) and reduces major histocompatibility complex (MHC) class I antigen presentation.

    PubMed

    Bomberger, Jennifer M; Ely, Kenneth H; Bangia, Naveen; Ye, Siying; Green, Kathy A; Green, William R; Enelow, Richard I; Stanton, Bruce A

    2014-01-03

    Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8(+) T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.

  6. Intermediate filament transcription in astrocytes is repressed by proteasome inhibition

    PubMed Central

    Middeldorp, Jinte; Kamphuis, Willem; Sluijs, Jacqueline A.; Achoui, Dalila; Leenaars, Cathalijn H. C.; Feenstra, Matthijs G. P.; van Tijn, Paula; Fischer, David F.; Berkers, Celia; Ovaa, Huib; Quinlan, Roy A.; Hol, Elly M.

    2009-01-01

    Increased expression of the astrocytic intermediate filament protein glial fibrillary acidic protein (GFAP) is a characteristic of astrogliosis. This process occurs in the brain during aging and neurodegeneration and coincides with impairment of the ubiquitin proteasome system. Inhibition of the proteasome impairs protein degradation; therefore, we hypothesized that the increase in GFAP may be the result of impaired proteasomal activity in astrocytes. We investigated the effect of proteasome inhibitors on GFAP expression and other intermediate filament proteins in human astrocytoma cells and in a rat brain model for astrogliosis. Extensive quantitative RT-PCR, immunocytochemistry, and Western blot analysis resulted unexpectedly in a strong decrease of GFAP mRNA to <4% of control levels [Control (DMSO) 100±19.2%; proteasome inhibitor (epoxomicin) 3.5±1.3%, n=8; P≤0.001] and a loss of GFAP protein in astrocytes in vitro. We show that the proteasome alters GFAP promoter activity, possibly mediated by transcription factors as demonstrated by a GFAP promoter-luciferase assay and RT2 Profiler PCR array for human transcription factors. Most important, we demonstrate that proteasome inhibitors also reduce GFAP and vimentin expression in a rat model for induced astrogliosis in vivo. Therefore, proteasome inhibitors could serve as a potential therapy to modulate astrogliosis associated with CNS injuries and disease.—Middeldorp, J., Kamphuis, W., Sluijs, J. A., Achoui, D., Leenaars, C. H. C., Feenstra, M. G. P., van Tijn, P., Fischer, D. F., Berkers, C., Ovaa, H., Quinlan, R. A., Hol, E. M. Intermediate filament transcription in astrocytes is repressed by proteasome inhibition. PMID:19332645

  7. Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production

    PubMed Central

    Brehm, Anja; Liu, Yin; Sheikh, Afzal; Marrero, Bernadette; Omoyinmi, Ebun; Zhou, Qing; Montealegre, Gina; Biancotto, Angelique; Reinhardt, Adam; Almeida de Jesus, Adriana; Pelletier, Martin; Tsai, Wanxia L.; Remmers, Elaine F.; Kardava, Lela; Hill, Suvimol; Kim, Hanna; Lachmann, Helen J.; Megarbane, Andre; Chae, Jae Jin; Brady, Jilian; Castillo, Rhina D.; Brown, Diane; Casano, Angel Vera; Gao, Ling; Chapelle, Dawn; Huang, Yan; Stone, Deborah; Chen, Yongqing; Sotzny, Franziska; Lee, Chyi-Chia Richard; Kastner, Daniel L.; Torrelo, Antonio; Zlotogorski, Abraham; Moir, Susan; Gadina, Massimo; McCoy, Phil; Wesley, Robert; Rother, Kristina; Hildebrand, Peter W.; Brogan, Paul; Krüger, Elke; Aksentijevich, Ivona; Goldbach-Mansky, Raphaela

    2015-01-01

    Autosomal recessive mutations in proteasome subunit β 8 (PSMB8), which encodes the inducible proteasome subunit β5i, cause the immune-dysregulatory disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), which is classified as a proteasome-associated autoinflammatory syndrome (PRAAS). Here, we identified 8 mutations in 4 proteasome genes, PSMA3 (encodes α7), PSMB4 (encodes β7), PSMB9 (encodes β1i), and proteasome maturation protein (POMP), that have not been previously associated with disease and 1 mutation in PSMB8 that has not been previously reported. One patient was compound heterozygous for PSMB4 mutations, 6 patients from 4 families were heterozygous for a missense mutation in 1 inducible proteasome subunit and a mutation in a constitutive proteasome subunit, and 1 patient was heterozygous for a POMP mutation, thus establishing a digenic and autosomal dominant inheritance pattern of PRAAS. Function evaluation revealed that these mutations variably affect transcription, protein expression, protein folding, proteasome assembly, and, ultimately, proteasome activity. Moreover, defects in proteasome formation and function were recapitulated by siRNA-mediated knockdown of the respective subunits in primary fibroblasts from healthy individuals. Patient-isolated hematopoietic and nonhematopoietic cells exhibited a strong IFN gene-expression signature, irrespective of genotype. Additionally, chemical proteasome inhibition or progressive depletion of proteasome subunit gene transcription with siRNA induced transcription of type I IFN genes in healthy control cells. Our results provide further insight into CANDLE genetics and link global proteasome dysfunction to increased type I IFN production. PMID:26524591

  8. SIAH-1 Interacts with the Kaposi's Sarcoma-Associated Herpesvirus-Encoded ORF45 Protein and Promotes Its Ubiquitylation and Proteasomal Degradation▿

    PubMed Central

    Abada, Rinat; Dreyfuss-Grossman, Tsofia; Herman-Bachinsky, Yifat; Geva, Haim; Masa, Shiri-Rivka; Sarid, Ronit

    2008-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus 8, is a potentially tumorigenic virus implicated in the etiology of Kaposi's sarcoma, primary effusion lymphoma, and some forms of multicentric Castleman's disease. The open reading frame 45 (ORF45) protein, encoded by the KSHV genome, is capable of inhibiting virus-dependent interferon induction and appears to be essential for both early and late stages of infection. In the present study, we show, both in yeast two-hybrid assays and in mammalian cells, that the ORF45 protein interacts with the cellular ubiquitin E3 ligase family designated seven in absentia homologue (SIAH). We provide evidence that SIAH-1 promotes the degradation of KSHV ORF45 through a RING domain-dependent mechanism and via the ubiquitin-proteasome system. Furthermore, our data indicate the involvement of SIAH-1 in the regulation of the expression of ORF45 in KSHV-infected cells. Since the availability of KSHV ORF45 is expected to influence the course of KSHV infection, our findings identify a novel biological role for SIAH proteins as modulators of virus infection. PMID:18077711

  9. Reversible phosphorylation of the 26S proteasome.

    PubMed

    Guo, Xing; Huang, Xiuliang; Chen, Mark J

    2017-04-01

    The 26S proteasome at the center of the ubiquitin-proteasome system (UPS) is essential for virtually all cellular processes of eukaryotes. A common misconception about the proteasome is that, once made, it remains as a static and uniform complex with spontaneous and constitutive activity for protein degradation. Recent discoveries have provided compelling evidence to support the exact opposite insomuch as the 26S proteasome undergoes dynamic and reversible phosphorylation under a variety of physiopathological conditions. In this review, we summarize the history and current understanding of proteasome phosphorylation, and advocate the idea of targeting proteasome kinases/phosphatases as a new strategy for clinical interventions of several human diseases.

  10. The degradation of mixed lineage kinase domain-like protein promotes neuroprotection after ischemic brain injury

    PubMed Central

    Zhou, Yanlong; Zhou, Beiqun; Tu, Hui; Tang, Yan; Xu, Chen; Chen, Yanbo; Zhao, Zhong; Miao, Zhigang

    2017-01-01

    Mixed lineage kinase domain-like (MLKL) protein was recently found to play a critical role in necrotic cell death. To explore its role in neurological diseases, we measured MLKL protein expression after ischemia injury in a mouse model. We found that MLKL expression significantly increased 12 h after ischemia/reperfusion (I/R) injury with peak levels at 48 h. Inhibition of MLKL by intraperitoneal administration of NSA significantly reduced infarct volume and improved neurological deficits after 75 min of ischemia and 24 h of reperfusion. Further, we found NSA reduced MLKL levels via the ubiquitination proteasome pathway, but not by inhibiting RNA transcription. Interestingly, NSA administration increased cleaved PARP-1 levels, indicating the protective effects of MLKL inhibition is not related to apoptosis. These findings suggest MLKL is a new therapeutic target for neurological pathologies like stroke. Therefore, promoting degradation of MLKL may be a novel avenue to reduce necrotic cell death after ischemic brain injury. PMID:28978125

  11. The degradation of mixed lineage kinase domain-like protein promotes neuroprotection after ischemic brain injury.

    PubMed

    Zhou, Yanlong; Zhou, Beiqun; Tu, Hui; Tang, Yan; Xu, Chen; Chen, Yanbo; Zhao, Zhong; Miao, Zhigang

    2017-09-15

    Mixed lineage kinase domain-like (MLKL) protein was recently found to play a critical role in necrotic cell death. To explore its role in neurological diseases, we measured MLKL protein expression after ischemia injury in a mouse model. We found that MLKL expression significantly increased 12 h after ischemia/reperfusion (I/R) injury with peak levels at 48 h. Inhibition of MLKL by intraperitoneal administration of NSA significantly reduced infarct volume and improved neurological deficits after 75 min of ischemia and 24 h of reperfusion. Further, we found NSA reduced MLKL levels via the ubiquitination proteasome pathway, but not by inhibiting RNA transcription. Interestingly, NSA administration increased cleaved PARP-1 levels, indicating the protective effects of MLKL inhibition is not related to apoptosis. These findings suggest MLKL is a new therapeutic target for neurological pathologies like stroke. Therefore, promoting degradation of MLKL may be a novel avenue to reduce necrotic cell death after ischemic brain injury.

  12. 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

  13. Insulin/insulin-like growth factor (IGF) stimulation abrogates an association between a deubiquitinating enzyme USP7 and insulin receptor substrates (IRSs) followed by proteasomal degradation of IRSs.

    PubMed

    Yoshihara, Hidehito; Fukushima, Toshiaki; Hakuno, Fumihiko; Saeki, Yasushi; Tanaka, Keiji; Ito, Akihiro; Yoshida, Minoru; Iemura, Shun-ichiro; Natsume, Tohru; Asano, Tomoichiro; Chida, Kazuhiro; Girnita, Leonard; Takahashi, Shin-Ichiro

    2012-06-22

    Insulin receptor substrates (IRSs) play central roles in insulin/insulin-like growth factor (IGF) signaling and mediate a variety of their bioactivities. IRSs are tyrosine-phosphorylated by activated insulin receptor/IGF-I receptor tyrosine kinase in response to insulin/IGF, and are recognized by signaling molecules possessing the SH2 domain such as phosphatidylinositol 3-kinase (PI3K), leading to the activation of downstream pathways. Recent studies have suggested that degradation of IRSs by the ubiquitin-proteasome pathway leads to impaired insulin/IGF signaling, but the precise mechanism underlying the process is still unclear. In this study, we identified deubiquitinating enzyme ubiquitin specific protease 7 (USP7) as an IRS-2-interacting protein and demonstrated that deubiquitinase activity of USP7 plays important roles in IRS-2 stabilization through the ubiquitin-proteasome pathway. In addition, insulin treatment dissociated USP7 from IRS-2, leading to degradation of IRS-2. This dissociation was prevented by treatment with LY294002, a PI3K inhibitor, indicating that insulin activation of the PI3K pathway leads to dissociation of IRS-2 from USP7 and IRS-2 degradation. We obtained similar results for IRS-1 in cells treated with insulin and for IRS-2 in cells treated with IGF-I. Taken together, this is the first report demonstrating that USP7 is an IRS-1/2 deubiquitinating enzyme forming a negative feedback loop in insulin/IGF signaling. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Increased S-Nitrosylation and Proteasomal Degradation of Caspase-3 during Infection Contribute to the Persistence of Adherent Invasive Escherichia coli (AIEC) in Immune Cells

    PubMed Central

    Dunne, Karl A.; Allam, Amr; McIntosh, Anne; Houston, Stephanie A.; Cerovic, Vuk; Goodyear, Carl S.; Roe, Andrew J.; Beatson, Scott A.; Milling, Simon W.; Walker, Daniel; Wall, Daniel M.

    2013-01-01

    Adherent invasive Escherichia coli (AIEC) have been implicated as a causative agent of Crohn’s disease (CD) due to their isolation from the intestines of CD sufferers and their ability to persist in macrophages inducing granulomas. The rapid intracellular multiplication of AIEC sets it apart from other enteric pathogens such as Salmonella Typhimurium which after limited replication induce programmed cell death (PCD). Understanding the response of infected cells to the increased AIEC bacterial load and associated metabolic stress may offer insights into AIEC pathogenesis and its association with CD. Here we show that AIEC persistence within macrophages and dendritic cells is facilitated by increased proteasomal degradation of caspase-3. In addition S-nitrosylation of pro- and active forms of caspase-3, which can inhibit the enzymes activity, is increased in AIEC infected macrophages. This S-nitrosylated caspase-3 was seen to accumulate upon inhibition of the proteasome indicating an additional role for S-nitrosylation in inducing caspase-3 degradation in a manner independent of ubiquitination. In addition to the autophagic genetic defects that are linked to CD, this delay in apoptosis mediated in AIEC infected cells through increased degradation of caspase-3, may be an essential factor in its prolonged persistence in CD patients. PMID:23861899

  15. ER stress suppresses DNA double-strand break repair and sensitizes tumor cells to ionizing radiation by stimulating proteasomal degradation of Rad51.

    PubMed

    Yamamori, Tohru; Meike, Shunsuke; Nagane, Masaki; Yasui, Hironobu; Inanami, Osamu

    2013-10-11

    In this study, we provide evidence that endoplasmic reticulum (ER) stress suppresses DNA double-strand break (DSB) repair and increases radiosensitivity of tumor cells by altering Rad51 levels. We show that the ER stress inducer tunicamycin stimulates selective degradation of Rad51 via the 26S proteasome, impairing DSB repair and enhancing radiosensitivity in human lung cancer A549 cells. We also found that glucose deprivation, which is a physiological inducer of ER stress, triggered similar events. These findings suggest that ER stress caused by the intratumoral environment influences tumor radiosensitivity, and that it has potential as a novel target to improve cancer radiotherapy.

  16. Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN

    SciTech Connect

    Stroschein, Shannon L.; Bonni, Shirin; Wrana, Jeffrey L.; Luo, Kunxin

    2001-09-11

    Smad proteins mediate transforming growth factor-b signaling to regulate cell growth and differentiation. SnoN is an important negative regulator of TGFb signaling that functions to maintain the repressed state of TGFb target genes in the absence of ligand. Upon TGFb stimulation, Smad3 and Smad2 translocate into the nucleus and induce a rapid degradation of SnoN, allowing activation of TGFb target genes. Here we show that Smad2- or Smad3-induced degradation of SnoN requires the ubiquitin-dependent proteasome and can be mediated by the anaphase promoting complex (APC) and the UbcH5 family of ubiquitin conjugating enzymes. Smad3 and to a lesser extent, Smad2, interact with both the APC and SnoN, resulting in the recruitment of the APC to SnoN and subsequent ubiquitination of SnoN in a destruction box-dependent manner. In addition to the destruction box, efficient degradation of SnoN also requires the Smad3 binding site in SnoN as well as key lysine residues necessary for ubiquitin attachment. Mutation of either the Smad3 binding site or lysine residues results in stabilization of SnoN and in enhanced antagonism of TGFb signaling. Our studies elucidate an important pathway for the degradation of SnoN and reveal a novel role of the APC in regulation of TGFb signaling.

  17. The ubiquitin-proteasome system: opportunities for therapeutic intervention in solid tumors

    PubMed Central

    Johnson, Daniel E.

    2014-01-01

    The destruction of proteins via the ubiquitin-proteasome system is a multi-step, complex process involving polyubiquitination of substrate proteins, followed by proteolytic degradation by the macromolecular 26S proteasome complex. Inhibitors of the proteasome promote the accumulation of proteins that are deleterious to cell survival, and represent promising anti-cancer agents. In multiple myeloma and mantle cell lymphoma, treatment with the first generation proteasome inhibitor bortezomib, or the second generation inhibitor carfilzomib, has demonstrated significant therapeutic benefit in humans. This has prompted US FDA approval of these agents and development of additional second generation compounds with improved properties. There is considerable interest in extending the benefits of proteasome inhibitors to the treatment of solid tumor malignancies. Herein we review progress that has been made in the preclinical development and clinical evaluation of different proteasome inhibitors in solid tumors. In addition, we describe several novel approaches that are currently being pursued for the treatment of solid tumors, including drug combinatorial strategies incorporating proteasome inhibitors, and the targeting of components of the ubiquitin-proteasome system that are distinct from the 26S proteasome complex. PMID:24659480

  18. Proteasomes play an essential role in thymocyte apoptosis.

    PubMed Central

    Grimm, L M; Goldberg, A L; Poirier, G G; Schwartz, L M; Osborne, B A

    1996-01-01

    Cell death in many different organisms requires the activation of proteolytic cascades involving cytosolic proteases. Here we describe a novel requirement in thymocyte cell death for the 20S proteasome, a highly conserved multicatalytic protease found in all eukaryotes. Specific inhibitors of proteasome function blocked cell death induced by ionizing radiation, glucocorticoids or phorbol ester. In addition to inhibiting apoptosis, these signals prevented the cleavage of poly(ADP-ribose) polymerase that accompanies many cell deaths. Since overall rates of protein degradation were not altered significantly during cell death in thymocytes, these results suggest that the proteasome may either degrade regulatory protein(s) that normally inhibit the apoptotic pathway or may proteolytically activate protein(s) than promote cell death. Images PMID:8670888

  19. Receptor for Activated C Kinase 1 (RACK1) Promotes Dishevelled Protein Degradation via Autophagy and Antagonizes Wnt Signaling.

    PubMed

    Cheng, Minzhang; Xue, Hua; Cao, Weipeng; Li, Wenxia; Chen, Hua; Liu, Bofeng; Ma, Benyu; Yan, Xiaohua; Chen, Ye-Guang

    2016-06-10

    Wnt signaling plays a critical role in embryonic development, tissue homeostasis, and cancer development. Dishevelled (Dvl) is an essential and central component in Wnt signaling, and its stability and activity is tightly regulated. It has been shown that Dvl can be degraded via both the proteasome and autophagy-lysosome pathways. Here we report that receptor for activated C kinase 1 (RACK1) negatively regulates Dishevelled stability and Wnt signaling. RACK1 interacts with Dvl proteins and promotes their lysosomal degradation, and this effect is enhanced by autophagy induction. RACK1 also interacts with LC3 and enhances the association of LC3 with Dvl2, thereby leading to degradation of Dvl proteins through autophagy. These findings reveal a novel regulatory function of RACK1 in Wnt signaling by modulating Dvl stability. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Inhibition of PCSK9 transcription by berberine involves down-regulation of hepatic HNF1α protein expression through the ubiquitin-proteasome degradation pathway.

    PubMed

    Dong, Bin; Li, Hai; Singh, Amar Bahadur; Cao, Aiqin; Liu, Jingwen

    2015-02-13

    Our previous in vitro studies have identified hepatocyte nuclear factor 1α (HNF1α) as an obligated trans-activator for PCSK9 gene expression and demonstrated its functional involvement in the suppression of PCSK9 expression by berberine (BBR), a natural cholesterol-lowering compound. In this study, we investigated the mechanism underlying the inhibitory effect of BBR on HNF1α-mediated PCSK9 transcription. Administration of BBR to hyperlipidemic mice and hamsters lowered circulating PCSK9 concentrations and hepatic PCSK9 mRNA levels without affecting the gene expression of HNF1α. However, hepatic HNF1α protein levels were markedly reduced in BBR-treated animals as compared with the control. Using HepG2 cells as a model system, we obtained evidence that BBR treatment let to accelerated degradation of HNF1α protein. By applying inhibitors to selectively block the ubiquitin proteasome system (UPS) and autophagy-lysosomal pathway, we show that HNF1α protein content in HepG2 cells was not affected by bafilomycin A1 treatment, but it was dose-dependently increased by UPS inhibitors bortezomib and MG132. Bortezomib treatment elevated HNF1α and PCSK9 cellular levels with concomitant reductions of LDL receptor protein. Moreover, HNF1α protein displayed a multiubiquitination ladder pattern in cells treated with BBR or overexpressing ubiquitin. By expressing GFP-HNF1α fusion protein in cells, we observed that blocking UPS resulted in accumulation of GFP-HNF1α in cytoplasm. Importantly, we show that the BBR reducing effects on HNF1α protein and PCSK9 gene transcription can be eradicated by proteasome inhibitors. Altogether, our studies using BBR as a probe uncovered a new aspect of PCSK9 regulation by ubiquitin-induced proteasomal degradation of HNF1α.

  1. Autoregulation of the 26S proteasome by in situ ubiquitination

    PubMed Central

    Jacobson, Andrew D.; MacFadden, Andrea; Wu, Zhiping; Peng, Junmin; Liu, Chang-Wei

    2014-01-01

    The 26S proteasome degrades ubiquitinated proteins, and proteasomal degradation controls various cellular events. Here we report that the human 26S proteasome is ubiquitinated, by which the ubiquitin receptors Adrm1 and S5a, the ATPase subunit Rpt5, and the deubiquitinating enzyme Uch37 are ubiquitinated in situ by proteasome-associating ubiquitination enzymes. Ubiquitination of these subunits significantly impairs the 26S proteasome's ability to bind, deubiquitinate, and degrade ubiquitinated proteins. Moreover, ubiquitination of the 26S proteasome can be antagonized by proteasome-residing deubiquitinating enzymes, by the binding of polyubiquitin chains, and by certain cellular stress, indicating that proteasome ubiquitination is dynamic and regulated in cells. We propose that in situ ubiquitination of the 26S proteasome regulates its activity, which could function to adjust proteasomal activity in response to the alteration of cellular ubiquitination levels. PMID:24743594

  2. Blue light induces degradation of the negative regulator phytochrome interacting factor 1 to promote photomorphogenic development of Arabidopsis seedlings.

    PubMed

    Castillon, Alicia; Shen, Hui; Huq, Enamul

    2009-05-01

    Phytochrome interacting factors (PIFs) are nuclear basic helix-loop-helix (bHLH) transcription factors that negatively regulate photomorphogenesis both in the dark and in the light in Arabidopsis. The phytochrome (phy) family of photoreceptors induces the rapid phosphorylation and degradation of PIFs in response to both red and far-red light conditions to promote photomorphogenesis. Although phys have been shown to function under blue light conditions, the roles of PIFs under blue light have not been investigated in detail. Here we show that PIF1 negatively regulates photomorphogenesis at the seedling stage under blue light conditions. pif1 seedlings displayed more open cotyledons and slightly reduced hypocotyl length compared to wild type under diurnal (12 hr light/12 hr dark) blue light conditions. Double-mutant analyses demonstrated that pif1phyA, pif1phyB, pif1cry1, and pif1cry2 have enhanced cotyledon opening compared to the single photoreceptor mutants under diurnal blue light conditions. Blue light induced the rapid phosphorylation, polyubiquitination, and degradation of PIF1 through the ubi/26S proteasomal pathway. PIF1 interacted with phyA and phyB in a blue light-dependent manner, and the interactions with phys are necessary for the blue light-induced degradation of PIF1. phyA played a dominant role under pulses of blue light, while phyA, phyB, and phyD induced the degradation of PIF1 in an additive manner under prolonged continuous blue light conditions. Interestingly, the absence of cry1 and cry2 enhanced the degradation of PIF1 under blue light conditions. Taken together, these data suggest that PIF1 functions as a negative regulator of photomorphogenesis under blue light conditions and that blue light-activated phys induce the degradation of PIF1 through the ubi/26S proteasomal pathway to promote photomorphogenesis.

  3. HIV-2 and SIVmac accessory virulence factor Vpx down-regulates SAMHD1 enzyme catalysis prior to proteasome-dependent degradation.

    PubMed

    DeLucia, Maria; Mehrens, Jennifer; Wu, Ying; Ahn, Jinwoo

    2013-06-28

    SAMHD1, a dGTP-regulated deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase, down-regulates dNTP pools in terminally differentiated and quiescent cells, thereby inhibiting HIV-1 infection at the reverse transcription step. HIV-2 and simian immunodeficiency virus (SIV) counteract this restriction via a virion-associated virulence accessory factor, Vpx (Vpr in some SIVs), which loads SAMHD1 onto CRL4-DCAF1 E3 ubiquitin ligase for polyubiquitination, programming it for proteasome-dependent degradation. However, the detailed molecular mechanisms of SAMHD1 recruitment to the E3 ligase have not been defined. Further, whether divergent, orthologous Vpx proteins, encoded by distinct HIV/SIV strains, bind SAMHD1 in a similar manner, at a molecular level, is not known. We applied surface plasmon resonance analysis to assess the requirements for and kinetics of binding between various primate SAMHD1 proteins and Vpx proteins from SIV or HIV-2 strains. Our data indicate that Vpx proteins, bound to DCAF1, interface with the C terminus of primate SAMHD1 proteins with nanomolar affinity, manifested by rapid association and slow dissociation. Further, we provide evidence that Vpx binding to SAMHD1 inhibits its catalytic activity and induces disassembly of a dGTP-dependent oligomer. Our studies reveal a previously unrecognized biochemical mechanism of Vpx-mediated SAMHD1 inhibition: direct down-modulation of its catalytic activity, mediated by the same binding event that leads to SAMHD1 recruitment to the E3 ubiquitin ligase for proteasome-dependent degradation.

  4. Cigarette smoke induces proteasomal-mediated degradation of DNA methyltransferases and methyl CpG-/CpG domain-binding proteins in embryonic orofacial cells.

    PubMed

    Mukhopadhyay, Partha; Greene, Robert M; Pisano, M Michele

    2015-12-01

    Orofacial clefts, the most prevalent of developmental anomalies, occur with a frequency of 1 in 700 live births. Maternal cigarette smoking during pregnancy represents a risk factor for having a child with a cleft lip and/or cleft palate. Using primary cultures of first branchial arch-derived cells (1-BA cells), which contribute to the formation of the lip and palate, the present study addressed the hypothesis that components of cigarette smoke alter global DNA methylation, and/or expression of DNA methyltransferases (Dnmts) and various methyl CpG-binding proteins. Primary cultures of 1-BA cells, exposed to 80μg/mL cigarette smoke extract (CSE) for 24h, exhibited a >13% decline in global DNA methylation and triggered proteasomal-mediated degradation of Dnmts (DNMT-1 and -3a), methyl CpG binding protein 2 (MeCP2) and methyl-CpG binding domain protein 3 (MBD-3). Pretreatment of 1-BA cells with the proteasomal inhibitor MG-132 completely reversed such degradation. Collectively, these data allow the suggestion of a potential epigenetic mechanism underlying maternal cigarette smoke exposure-induced orofacial clefting.

  5. Ubiquitin–proteasome degradation of serum- and glucocorticoid-regulated kinase-1 (SGK-1) is mediated by the chaperone-dependent E3 ligase CHIP

    PubMed Central

    Belova, Larissa; Sharma, Sanjay; Brickley, Deanna R.; Nicolarsen, Jeremy R.; Patterson, Cam; Conzen, Suzanne D.

    2006-01-01

    SGK-1 (serum- and glucocorticoid-regulated kinase-1) is a stress-induced serine/threonine kinase that is phosphorylated and activated downstream of PI3K (phosphoinositide 3-kinase). SGK-1 plays a critical role in insulin signalling, cation transport and cell survival. SGK-1 mRNA expression is transiently induced following cellular stress, and SGK-1 protein levels are tightly regulated by rapid proteasomal degradation. In the present study we report that SGK-1 forms a complex with the stress-associated E3 ligase CHIP [C-terminus of Hsc (heat-shock cognate protein) 70-interacting protein]; CHIP is required for both the ubiquitin modification and rapid proteasomal degradation of SGK-1. We also show that CHIP co-localizes with SGK-1 at or near the endoplasmic reticulum. CHIP-mediated regulation of SGK-1 steady-state levels alters SGK-1 kinase activity. These data suggest a model that integrates CHIP function with regulation of the PI3K/SGK-1 pathway in the stress response. PMID:16895519

  6. CIGARETTE SMOKE INDUCES PROTEASOMAL-MEDIATED DEGRADATION OF DNA METHYLTRANSFERASES AND METHYL CpG-/CpG DOMAIN-BINDING PROTEINS IN EMBRYONIC OROFACIAL CELLS

    PubMed Central

    Mukhopadhyay, Partha; Greene, Robert M.; Pisano, M. Michele

    2015-01-01

    Orofacial clefts, the most prevalent of developmental anomalies, occur with a frequency of 1 in 700 live births. Maternal cigarette smoking during pregnancy represents a risk factor for having a child with a cleft lip and/or cleft palate. Using primary cultures of first branchial arch-derived cells (1-BA cells), which contribute to the formation of the lip and palate, the present study addressed the hypothesis that components of cigarette smoke alter global DNA methylation, and/or expression of DNA methyltransferases (Dnmts) and various methyl CpG-binding proteins. Primary cultures of 1-BA cells, exposed to 80 μg/ml cigarette smoke extract (CSE) for 24 hrs, exhibited a >13% decline in global DNA methylation and triggered proteasomal-mediated degradation of Dnmts (DNMT-1, and - 3a), methyl CpG binding protein 2 (MeCP2) and methyl-CpG binding domain protein 3 (MBD-3). Pretreatment of 1-BA cells with the proteasomal inhibitor MG-132 completely reversed such degradation. Collectively, these data allow the suggestion of a potential epigenetic mechanism underlying maternal cigarette smoke exposure-induced orofacial clefting. PMID:26482727

  7. The Rho Family Member RhoE Interacts with Skp2 and Is Degraded at the Proteasome during Cell Cycle Progression*

    PubMed Central

    Lonjedo, Marta; Poch, Enric; Mocholí, Enric; Hernández-Sánchez, Marta; Ivorra, Carmen; Franke, Thomas F.; Guasch, Rosa M.; Pérez-Roger, Ignacio

    2013-01-01

    RhoE/Rnd3 is an atypical member of the Rho family of small GTPases. In addition to regulating actin cytoskeleton dynamics, RhoE is involved in the regulation of cell proliferation, survival, and metastasis. We examined RhoE expression levels during cell cycle and investigated mechanisms controlling them. We show that RhoE accumulates during G1, in contact-inhibited cells, and when the Akt pathway is inhibited. Conversely, RhoE levels rapidly decrease at the G1/S transition and remain low for most of the cell cycle. We also show that the half-life of RhoE is shorter than that of other Rho proteins and that its expression levels are regulated by proteasomal degradation. The expression patterns of RhoE overlap with that of the cell cycle inhibitor p27. Consistently with an involvement of RhoE in cell cycle regulation, RhoE and p27 levels decrease after overexpression of the F-box protein Skp2. We have identified a region between amino acids 231 and 240 of RhoE as the Skp2-interacting domain and Lys235 as the substrate for ubiquitylation. Based on our results, we propose a mechanism according to which proteasomal degradation of RhoE by Skp2 regulates its protein levels to control cellular proliferation. PMID:24045951

  8. Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells.

    PubMed

    Li, Hewang; Armando, Ines; Yu, Peiying; Escano, Crisanto; Mueller, Susette C; Asico, Laureano; Pascua, Annabelle; Lu, Quansheng; Wang, Xiaoyan; Villar, Van Anthony M; Jones, John E; Wang, Zheng; Periasamy, Ammasi; Lau, Yuen-Sum; Soares-da-Silva, Patricio; Creswell, Karen; Guillemette, Gaétan; Sibley, David R; Eisner, Gilbert; Gildea, John J; Felder, Robin A; Jose, Pedro A

    2008-06-01

    Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R.

  9. Dopamine 5 receptor mediates Ang II type 1 receptor degradation via a ubiquitin-proteasome pathway in mice and human cells

    PubMed Central

    Li, Hewang; Armando, Ines; Yu, Peiying; Escano, Crisanto; Mueller, Susette C.; Asico, Laureano; Pascua, Annabelle; Lu, Quansheng; Wang, Xiaoyan; Villar, Van Anthony M.; Jones, John E.; Wang, Zheng; Periasamy, Ammasi; Lau, Yuen-Sum; Soares-da-Silva, Patricio; Creswell, Karen; Guillemette, Gaétan; Sibley, David R.; Eisner, Gilbert; Felder, Robin A.; Jose, Pedro A.

    2008-01-01

    Hypertension is a multigenic disorder in which abnormal counterregulation between dopamine and Ang II plays a role. Recent studies suggest that this counterregulation results, at least in part, from regulation of the expression of both the antihypertensive dopamine 5 receptor (D5R) and the prohypertensive Ang II type 1 receptor (AT1R). In this report, we investigated the in vivo and in vitro interaction between these GPCRs. Disruption of the gene encoding D5R in mice increased both blood pressure and AT1R protein expression, and the increase in blood pressure was reversed by AT1R blockade. Activation of D5R increased the degradation of glycosylated AT1R in proteasomes in HEK cells and human renal proximal tubule cells heterologously and endogenously expressing human AT1R and D5R. Confocal microscopy, Förster/fluorescence resonance energy transfer microscopy, and fluorescence lifetime imaging microscopy revealed that activation of D5R initiated ubiquitination of the glycosylated AT1R at the plasma membrane. The regulated degradation of AT1R via a ubiquitin/proteasome pathway by activation of D5R provides what we believe to be a novel mechanism whereby blood pressure can be regulated by the interaction of 2 counterregulatory GPCRs. Our results therefore suggest that treatments for hypertension might be optimized by designing compounds that can target the AT1R and the D5R. PMID:18464932

  10. Calcium-dependent protein kinase CPK28 targets the methionine adenosyltransferases for degradation by the 26S proteasome and affects ethylene biosynthesis and lignin deposition in Arabidopsis.

    PubMed

    Jin, Yu; Ye, Nenghui; Zhu, Fuyuan; Li, Haoxuan; Wang, Juan; Jiang, Liwen; Zhang, Jianhua

    2017-04-01

    S-adenosylmethionine (AdoMet) is synthesized by methionine adenosyltransferase (MAT), and plays an essential role in ethylene biosynthesis and other methylation reactions. Despite increasing knowledge of MAT regulation at transcriptional levels, how MAT is post-translationally regulated remains unknown in plant cells. Phosphorylation is an important post-translational modification for regulating the activity of enzymes, protein function and signaling transduction. Using molecular and biochemical approaches, we have identified the phosphorylation of MAT proteins by calcium-dependent protein kinase (CPK28). Phenotypically, both MAT2-overexpressing transgenic plants and cpk28 mutants display short hypocotyls and ectopic lignifications. Their shortened hypocotyl phenotypes are caused by ethylene overproduction and rescued by ethylene biosynthesis inhibitor aminoethoxyvinylglycine treatment. Genetic evidence reveals that MAT2 mutation restores the phenotype of ectopic lignification in CPK28-deficient plants. We find that total MAT proteins and AdoMet are increased in cpk28 mutants, but decreased in CPK28-overexpressing seedlings. We also find that MATs in OE::CPK28 are degraded through the 26S proteasome pathway. Our work suggests that CPK28 targets MATs (MAT1, MAT2 and MAT3) for degradation by the 26S proteasome pathway, and thus affects ethylene biosynthesis and lignin deposition in Arabidopsis. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

  11. C-terminal maturation fragments of presenilin 1 and 2 control secretion of APP alpha and A beta by human cells and are degraded by proteasome.

    PubMed

    da Costa, C A; Ancolio, K; Checler, F

    1999-03-01

    Most early-onset forms of Alzheimer's disease are due to missense mutations located on two homologous proteins named presenilin 1 and 2 (PS1 and PS2). Several lines of evidence indicate that PS1 and PS2 undergo various post-transcriptional events including endoproteolytic cleavages, giving rise to 28-30 kD N-terminal (NTF) and 18-20 kD C-terminal (CTF) fragments that accumulate in vivo. Whether the biological activity of presenilins is borne by the processed fragments or their holoprotein precursor remains in question. We have examined the putative control of beta APP maturation by CTF-PS1/PS2 and the catabolic process of the latter proteins by the multicatalytic complex, proteasome. We transiently and stably transfected HEK293 cells with CTF-PS1 or CTF-PS2 cDNA. We examined these transfectants for their production of A beta 40, A beta 42, and APP alpha by immunoprecipitation using specific polyclonals. The effect of a series of proteases inhibitors on the immunoreactivity of CTF-PS1/PS2 was examined by Western blot. Finally, the influence of proteasome inhibitors on the generation of beta APP fragments by CTF-expressing cells was assessed by combined immunoprecipitation and densitometric analyses. We showed that transient and stable transfection of CTF-PS1 and CTF-PS2 cDNAs in human cells leads to increased secretion of APP alpha and A beta, the maturation products of beta APP. Furthermore, we demonstrated that two proteasome inhibitors, lactacystin and Z-IE(Ot-Bu)A-Leucinal, prevent the degradation of both CTFs. Accordingly, we established that proteasome inhibitors drastically potentiate the phenotypic increased production of APP alpha and A beta elicited by CTF-PS1/PS2. Our data establish that the C-terminal products of PS1 and PS2 maturation exhibit biological activity and in particular control beta APP maturation upstream to alpha-and beta/gamma-secretase cleavages. This function is directly controlled by the proteasome that modulates the intracellular

  12. Differential regulation of proteasome functionality in reproductive vs. somatic tissues of Drosophila during aging or oxidative stress

    PubMed Central

    Tsakiri, Eleni N.; Sykiotis, Gerasimos P.; Papassideri, Issidora S.; Gorgoulis, Vassilis G.; Bohmann, Dirk; Trougakos, Ioannis P.

    2013-01-01

    Proteasome is central to proteostasis maintenance, as it degrades both normal and damaged proteins. Herein, we undertook a detailed analysis of proteasome regulation in the in vivo setting of Drosophila melanogaster. We report that a major hallmark of somatic tissues of aging flies is the gradual accumulation of ubiquitinated and carbonylated proteins; these effects correlated with a ∼50% reduction of proteasome expression and catalytic activities. In contrast, gonads of aging flies were relatively free of proteome oxidative damage and maintained substantial proteasome expression levels and highly active proteasomes. Moreover, gonads of young flies were found to possess more abundant and more active proteasomes than somatic tissues. Exposure of flies to oxidants induced higher proteasome activities specifically in the gonads, which were, independently of age, more resistant than soma to oxidative challenge and, as analyses in reporter transgenic flies showed, retained functional antioxidant responses. Finally, inducible Nrf2 activation in transgenic flies promoted youthful proteasome expression levels in the aged soma, suggesting that age-dependent Nrf2 dysfunction is causative of decreasing somatic proteasome expression during aging. The higher investment in proteostasis maintenance in the gonads plausibly facilitates proteome stability across generations; it also provides evidence in support of the trade-off theories of aging.—Tsakiri, E. N., Sykiotis, G. P., Papassideri, I. S., Gorgoulis, V. G., Bohmann, D., Trougakos, I. P. Differential regulation of proteasome functionality in reproductive vs. somatic tissues of Drosophila during aging or oxidative stress. PMID:23457214

  13. Disulfiram promotes the conversion of carcinogenic cadmium to a proteasome inhibitor with pro-apoptotic activity in human cancer cells

    SciTech Connect

    Li Lihua; Yang Huanjie; Chen Di; Cui, Cindy; Ping Dou, Q.

    2008-06-01

    The ubiquitin-proteasome system is involved in various cellular processes, including transcription, apoptosis, and cell cycle. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as anticancer drugs. Cadmium (Cd) is a widespread environmental pollutant that has been classified as a human carcinogen. Recent study in our laboratory suggested that the clinically used anti-alcoholism drug disulfiram (DSF) could form a complex with tumor cellular copper, resulting in inhibition of the proteasomal chymotrypsin-like activity and induction of cancer cell apoptosis. In the current study, we report, for the first time, that DSF is able to convert the carcinogen Cd to a proteasome-inhibitor and cancer cell apoptosis inducer. Although the DSF-Cd complex inhibited the chymotrypsin-like activity of a purified 20S proteasome with an IC{sub 50} value of 32 {mu}mol/L, this complex was much more potent in inhibiting the chymotrypsin-like activity of prostate cancer cellular 26S proteasome. Inhibition of cellular proteasome activity by the DSF-Cd complex resulted in the accumulation of ubiquitinated proteins and the natural proteasome substrate p27, which was followed by activation of calpain and induction of apoptosis. Importantly, human breast cancer MCF10DCIS cells were much more sensitive to the DSF-Cd treatment than immortalized but non-tumorigenic human breast MCF-10A cells, demonstrating that the DSF-Cd complex could selectively induce proteasome inhibition and apoptosis in human tumor cells. Our work suggests the potential use of DSF for treatment of cells with accumulated levels of carcinogen Cd.

  14. Assembly of the 20S Proteasome

    PubMed Central

    Kunjappu, Mary J.; Hochstrasser, Mark

    2013-01-01

    The proteasome is a cellular protease responsible for the selective degradation of the majority of the intracellular proteome. It recognizes, unfolds, and cleaves proteins that are destined for removal, usually by prior attachment to polymers of ubiquitin. This macromolecular machine is composed of two subcomplexes, the 19S regulatory particle (RP) and the 20S core particle (CP), which together contain at least 33 different and precisely positioned subunits. How these subunits assemble into functional complexes is an area of active exploration. Here we describe the current status of studies on the assembly of the 20S proteasome (CP). The 28-subunit CP is found in all three domains of life and its cylindrical stack of four heptameric rings is well conserved. Though several CP subunits possess self-assembly properties, a consistent theme in recent years has been the need for dedicated assembly chaperones that promote on-pathway assembly. To date, a minimum of three accessory factors have been implicated in aiding the construction of the 20S proteasome. These chaperones interact with different assembling proteasomal precursors and usher subunits into specific slots in the growing structure. This review will focus largely on chaperone-dependent CP assembly and its regulation. PMID:23507199

  15. The Ubiquitin-Proteasome Pathway and Proteasome Inhibitors

    PubMed Central

    Myung, Jayhyuk; Kim, Kyung Bo

    2008-01-01

    The ubiquitin-proteasome pathway has emerged as a central player in the regulation of several diverse cellular processes. Here, we describe the important components of this complex biochemical machinery as well as several important cellular substrates targeted by this pathway and examples of human diseases resulting from defects in various components of the ubiquitin-proteasome pathway. In addition, this review covers the chemistry of synthetic and natural proteasome inhibitors, emphasizing their mode of actions toward the 20S proteasome. Given the importance of proteasome-mediated protein degradation in various intracellular processes, inhibitors of this pathway will continue to serve as both molecular probes of major cellular networks as well as potential therapeutic agents for various human diseases. PMID:11410931

  16. HSF-1 activates the ubiquitin proteasome system to promote non-apoptotic developmental cell death in C. elegans.

    PubMed

    Kinet, Maxime J; Malin, Jennifer A; Abraham, Mary C; Blum, Elyse S; Silverman, Melanie R; Lu, Yun; Shaham, Shai

    2016-03-08

    Apoptosis is a prominent metazoan cell death form. Yet, mutations in apoptosis regulators cause only minor defects in vertebrate development, suggesting that another developmental cell death mechanism exists. While some non-apoptotic programs have been molecularly characterized, none appear to control developmental cell culling. Linker-cell-type death (LCD) is a morphologically conserved non-apoptotic cell death process operating in Caenorhabditis elegans and vertebrate development, and is therefore a compelling candidate process complementing apoptosis. However, the details of LCD execution are not known. Here we delineate a molecular-genetic pathway governing LCD in C. elegans. Redundant activities of antagonistic Wnt signals, a temporal control pathway, and mitogen-activated protein kinase kinase signaling control heat shock factor 1 (HSF-1), a conserved stress-activated transcription factor. Rather than protecting cells, HSF-1 promotes their demise by activating components of the ubiquitin proteasome system, including the E2 ligase LET-70/UBE2D2 functioning with E3 components CUL-3, RBX-1, BTBD-2, and SIAH-1. Our studies uncover design similarities between LCD and developmental apoptosis, and provide testable predictions for analyzing LCD in vertebrates.

  17. HSF-1 activates the ubiquitin proteasome system to promote non-apoptotic developmental cell death in C. elegans

    PubMed Central

    Kinet, Maxime J; Malin, Jennifer A; Abraham, Mary C; Blum, Elyse S; Silverman, Melanie R; Lu, Yun; Shaham, Shai

    2016-01-01

    Apoptosis is a prominent metazoan cell death form. Yet, mutations in apoptosis regulators cause only minor defects in vertebrate development, suggesting that another developmental cell death mechanism exists. While some non-apoptotic programs have been molecularly characterized, none appear to control developmental cell culling. Linker-cell-type death (LCD) is a morphologically conserved non-apoptotic cell death process operating in Caenorhabditis elegans and vertebrate development, and is therefore a compelling candidate process complementing apoptosis. However, the details of LCD execution are not known. Here we delineate a molecular-genetic pathway governing LCD in C. elegans. Redundant activities of antagonistic Wnt signals, a temporal control pathway, and mitogen-activated protein kinase kinase signaling control heat shock factor 1 (HSF-1), a conserved stress-activated transcription factor. Rather than protecting cells, HSF-1 promotes their demise by activating components of the ubiquitin proteasome system, including the E2 ligase LET-70/UBE2D2 functioning with E3 components CUL-3, RBX-1, BTBD-2, and SIAH-1. Our studies uncover design similarities between LCD and developmental apoptosis, and provide testable predictions for analyzing LCD in vertebrates. DOI: http://dx.doi.org/10.7554/eLife.12821.001 PMID:26952214

  18. Fulvestrant-induced cell death and proteasomal degradation of estrogen receptor α protein in MCF-7 cells require the CSK c-Src tyrosine kinase.

    PubMed

    Yeh, Wei-Lan; Shioda, Keiko; Coser, Kathryn R; Rivizzigno, Danielle; McSweeney, Kristen R; Shioda, Toshi

    2013-01-01

    Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.

  19. Nuclear Export Signal of Androgen Receptor (NESAR) Regulation of Androgen Receptor Level in Human Prostate Cell Lines via Ubiquitination and Proteasome-Dependent Degradation

    PubMed Central

    Gong, Yanqing; Wang, Dan; Dar, Javid A.; Singh, Prabhpreet; Graham, Lara; Liu, Weijun; Ai, Junkui; Xin, Zhongcheng

    2012-01-01

    Androgen receptor (AR) plays a key role in prostate development and carcinogenesis. Increased expression and/or stability of AR is associated with sensitization of prostate cancer cells to low levels of androgens, leading to castration resistance. Hence, understanding the mechanisms regulating AR protein stability is clinically relevant and may lead to new approaches to prevent and/or treat prostate cancer. Using fluorescence microscopy, Western blot, and pulse chase assay, we showed that nuclear export signal (NES)AR, a nuclear export signal in the ligand binding domain (LBD) of AR, can significantly enhance the degradation of fusion protein constructs in PC3 prostate cancer cells. The half-life of GFP-NESAR was less than 3 h, which was 10 times shorter than that of green fluorescent protein (GFP) control. Further analysis showed that NESAR can signal for polyubiquitination and that degradation of NESAR-containing fusion proteins can be blocked by proteasome inhibitor MG132. Ubiquitination of GFP-AR or GFP-LBD was suppressed in the presence of dihydrotestosterone, which is known to suppress NESAR while inducing nuclear localization signal 2 in AR or LBD, suggesting that the export activity of NESAR is required for NESAR-mediated polyubiquitination. Treatment with MG132 also induced aggresome formation of NESAR-containing fusion proteins in perinuclear regions of the transfected PC3 cells, indicating a role for NESAR in inducing unfolded protein responses. The above observations suggest that NESAR plays a key role in AR ubiquitination and proteasome-dependent degradation in prostate cancer cells. PMID:23041672

  20. Disulfiram promotes the conversion of carcinogenic cadmium to a proteasome inhibitor with pro-apoptotic activity in human cancer cells

    PubMed Central

    Li, Lihua; Yang, Huanjie; Chen, Di; Cui, Cindy; Dou, Q. Ping

    2013-01-01

    The ubiquitinproteasome system is involved in various cellular processes, including transcription, apoptosis, and cell cycle. In vitro, in vivo, and clinical studies suggest the potential use of proteasome inhibitors as anticancer drugs. Cadmium (Cd) is a widespread environmental pollutant that has been classified as a human carcinogen. Recent study in our laboratory suggested that the clinically used anti-alcoholism drug disulfiram (DSF) could form a complex with tumor cellular copper, resulting in inhibition of the proteasomal chymotrypsin-like activity and induction of cancer cell apoptosis. In the current study, we report, for the first time, that DSF is able to convert the carcinogen Cd to a proteasome-inhibitor and cancer cell apoptosis inducer. Although the DSF–Cd complex inhibited the chymotrypsin-like activity of a purified 20S proteasome with an IC50 value of 32 μmol/L, this complex was much more potent in inhibiting the chymotrypsin-like activity of prostate cancer cellular 26S proteasome. Inhibition of cellular proteasome activity by the DSF–Cd complex resulted in the accumulation of ubiquitinated proteins and the natural proteasome substrate p27, which was followed by activation of calpain and induction of apoptosis. Importantly, human breast cancer MCF10DCIS cells were much more sensitive to the DSF–Cd treatment than immortalized but non-tumorigenic human breast MCF-10A cells, demonstrating that the DSF–Cd complex could selectively induce proteasome inhibition and apoptosis in human tumor cells. Our work suggests the potential use of DSF for treatment of cells with accumulated levels of carcinogen Cd. PMID:18304598

  1. Degradation of the encephalomyocarditis virus and hepatitis A virus 3C proteases by the ubiquitin/26S proteasome system in vivo

    SciTech Connect

    Schlax, Peter E.; Zhang Jin; Lewis, Elizabeth; Planchart, Antonio; Lawson, T. Glen . E-mail: tlawson@bates.edu

    2007-04-10

    We have isolated stably transfected mouse embryonic fibroblast cell lines that inducibly express either the mature encephalomyocarditis virus (EMCV) or hepatitis A virus (HAV) 3C protease and have used these cells to demonstrate that both proteins are subject to degradation in vivo by the ubiquitin/26S proteasome system. The detection of 3C protease expression in these cells requires inducing conditions and the presence of one of several proteasome inhibitors. Both 3C proteases are incorporated into conjugates with ubiquitin in vivo. HAV 3C protease expression has deleterious effects on cell viability, as determined by observation and counting of cells cultured in the absence or presence of inducing conditions. The EMCV 3C protease was found to be preferentially localized to the nucleus of induced cells, while the HAV 3C protease remains in the cytoplasm. The absence of polyubiquitinated EMCV 3C protease conjugates in nuclear fraction preparations suggests that localization to the nucleus can protect this protein from ubiquitination.

  2. Cyclin G2 Is Degraded through the Ubiquitin-Proteasome Pathway and Mediates the Antiproliferative Effect of Activin Receptor-like Kinase 7

    PubMed Central

    Xu, Guoxiong; Bernaudo, Stefanie; Fu, Guodong; Lee, Daniel Y.; Yang, Burton B.

    2008-01-01

    We have previously reported that Nodal, a member of the TGF-β superfamily, acts through activin receptor-like kinase 7 (ALK7) to inhibit ovarian cancer cell proliferation. To determine the mechanism underlying their effects, a cell cycle gene array was performed and cyclin G2 mRNA was found to be strongly up-regulated by Nodal and ALK7. To study the function and regulation of cyclin G2 in ovarian cancer cells, expression constructs were generated. We found that cyclin G2 protein level decreased rapidly after transfection, and this decrease was prevented by 26S proteasome inhibitors. Immunoprecipitation and pull-down studies showed that ubiquitin, Skp1, and Skp2 formed complexes with cyclin G2. Knockdown of Skp2 by siRNA increased, whereas overexpression of Skp2 decreased cyclin G2 levels. Nodal and ALK7 decreased the expression of Skp1 and Skp2 and increased cyclin G2 levels. Overexpression of cyclin G2 inhibited cell proliferation whereas cyclin G2-siRNA reduced the antiproliferative effect of Nodal and ALK7. Taken together, these findings provide strong evidence that cyclin G2 is degraded by the ubiquitin–proteasome pathway and that Skp2 plays a role in regulating cyclin G2 levels. Furthermore, our results also demonstrate that the antiproliferative effect of Nodal/ALK7 on ovarian cancer cells is in part mediated by cyclin G2. PMID:18784254

  3. Palmitate induces insulin resistance in human HepG2 hepatocytes by enhancing ubiquitination and proteasomal degradation of key insulin signaling molecules.

    PubMed

    Ishii, Megumi; Maeda, Ayumi; Tani, Shuji; Akagawa, Mitsugu

    2015-01-15

    Obesity-associated insulin resistance is a major pathogenesis of type 2 diabetes mellitus and is characterized by defects in insulin signaling. High concentrations of plasma free fatty acids (FFAs) are involved in the etiology of obesity-associated insulin resistance. However, the detailed mechanism by which FFAs contribute to the development of insulin resistance is not yet fully understood. We investigated the molecular basis of insulin resistance elicited by FFAs using the human hepatocyte cell line HepG2. Among major human FFAs, palmitate markedly inhibited insulin-stimulated phosphorylation of key insulin signaling molecules such as insulin receptor, insulin receptor substrate-1, and Akt, indicating that palmitate is the principal inducer of insulin resistance. We revealed that palmitate facilitates ubiquitination of the key insulin signaling molecules, and subsequently elicits their proteasomal degradation. Furthermore, we demonstrated that inhibition of ubiquitination by the ubiquitin-activating enzyme E1 inhibitor PYR41 significantly prevents palmitate-inducible insulin resistance but not by the proteasome inhibitor MG132, implying that ubiquitinated signaling molecules may be dysfunctional. In conclusion, inhibition of ubiquitination of the key insulin signaling molecules may be a potential strategy for preventing and treating obesity-associated insulin resistance.

  4. Peroxisome proliferator-activated receptor gamma agonists induce proteasome-dependent degradation of cyclin D1 and estrogen receptor alpha in MCF-7 breast cancer cells.

    PubMed

    Qin, Chunhua; Burghardt, Robert; Smith, Roger; Wormke, Mark; Stewart, Jessica; Safe, Stephen

    2003-03-01

    Treatment of MCF-7 cells with the peroxisome proliferator-activated receptor (PPAR) gamma agonists ciglitazone or 15-deoxy-Delta 12,14-prostaglandin J2 resulted in a concentration- and time-dependent decrease of cyclin D1 and estrogen receptor (ER) alpha proteins, and this was accompanied by decreased cell proliferation and G(1)-G(0)-->S-phase progression. Down-regulation of cyclin D1 and ER alpha by PPARgamma agonists was inhibited in cells cotreated with the proteasome inhibitors MG132 and PSII, but not in cells cotreated with the protease inhibitors calpain II and calpeptin. Moreover, after treatment of MCF-7 cells with 15-deoxy-Delta 12,14-prostaglandin J2 and immunoprecipitation with cyclin D1 or ER alpha antibodies, there was enhanced formation of ubiquitinated cyclin D1 and ER alpha bands. Thus, PPARgamma-induced inhibition of breast cancer cell growth is due, in part, to proteasome-dependent degradation of cyclin D1 (and ER alpha), and this pathway may be important for other cancer cell lines.

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

    PubMed

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

    2015-09-04

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

  6. Phosphorylation of WRINKLED1 by KIN10 Results in Its Proteasomal Degradation, Providing a Link between Energy Homeostasis and Lipid Biosynthesis[OPEN

    PubMed Central

    Liu, Hui

    2017-01-01

    WRINKLED1 (WRI1), a member of the APETALA2 (AP2) class of transcription factors, positively regulates glycolysis and lipid biosynthesis in Arabidopsis thaliana. Here, we identify mechanistic links between KIN10, the major SUCROSE NON-FERMENTATION1-RELATED KINASE1 involved in sugar/energy homeostasis, and the posttranslational regulation of WRI1. Transient expression of WRI1 with OLEOSIN1 in Nicotiana benthamiana stimulates triacylglycerol accumulation, but their coexpression with KIN10 abrogates this effect by inducing proteasomal degradation of WRI1. While WRI1 lacks canonical KIN10 target sequences, we demonstrated direct KIN10-dependent phosphorylation of WRI1 using purified Escherichia coli-expressed components. The resulting phosphorylated WRI1 was more rapidly degraded than native WRI1 in cell-free degradation assays. WRI1 phosphorylation was localized to two variants of the canonical KIN10 recognition sequence, one in each of its two AP2 DNA binding domains. Conversion of the phosphorylation sites at Thr-70 and Ser-166 to Ala resulted in a loss of KIN10-dependent phosphorylation, and when coexpressed with KIN10 the WRI1 double mutant accumulated to 2- to 3-fold higher levels than native WRI1. KIN10-dependent degradation of WRI1 provides a homeostatic mechanism that favors lipid biosynthesis when intracellular sugar levels are elevated and KIN10 is inhibited; conversely, glycolysis and lipid biosynthesis are curtailed as sugar levels decrease and KIN10 regains activity. PMID:28314829

  7. Characterizing the Dynamics of Proteasome Complexes by Proteomics Approaches

    PubMed Central

    Kaake, Robyn M.; Kao, Athit; Yu, Clinton

    2014-01-01

    Abstract Significance: The proteasome is the degradation machine of the ubiquitin-proteasome system, which is critical in controlling many essential biological processes. Aberrant regulation of proteasome-dependent protein degradation can lead to various human diseases, and general proteasome inhibitors have shown efficacy for cancer treatments. Though clinically effective, current proteasome inhibitors have detrimental side effects and, thus, better therapeutic strategies targeting proteasomes are needed. Therefore, a comprehensive characterization of proteasome complexes will provide the molecular details that are essential for developing new and improved drugs. Recent Advances: New mass spectrometry (MS)-based proteomics approaches have been developed to study protein interaction networks and structural topologies of proteasome complexes. The results have helped define the dynamic proteomes of proteasome complexes, thus providing new insights into the mechanisms underlying proteasome function and regulation. Critical Issues: The proteasome exists as heterogeneous populations in tissues/cells, and its proteome is highly dynamic and complex. In addition, proteasome complexes are regulated by various mechanisms under different physiological conditions. Consequently, complete proteomic profiling of proteasome complexes remains a major challenge for the field. Future Directions: We expect that proteomic methodologies enabling full characterization of proteasome complexes will continue to evolve. Further advances in MS instrumentation and protein separation techniques will be needed to facilitate the detailed proteomic analysis of low-abundance components and subpopulations of proteasome complexes. The results will help us understand proteasome biology as well as provide new therapeutic targets for disease diagnostics and treatment. Antioxid. Redox Signal. 21, 2444–2456. PMID:24423446

  8. Lipid Droplet-Associated Hydrolase Promotes Lipid Droplet Fusion and Enhances ATGL Degradation and Triglyceride Accumulation.

    PubMed

    Goo, Young-Hwa; Son, Se-Hee; Paul, Antoni

    2017-06-02

    Lipid droplet (LD)-associated hydrolase (LDAH) is a newly identified LD protein abundantly expressed in tissues that predominantly store triacylglycerol (TAG). However, how LDAH regulates TAG metabolism remains unknown. We found that upon oleic acid loading LDAH translocalizes from the ER to newly formed LDs, and induces LD coalescence in a tubulin-dependent manner. LDAH overexpression and downregulation in HEK293 cells increase and decrease, respectively, TAG levels. Pulse and chase experiments show that LDAH enhances TAG biogenesis, but also decreases TAG turnover and fatty acid release from cells. Mutations in predicted catalytic and acyltransferase motifs do not influence TAG levels, suggesting that the effect is independent of LDAH's enzymatic activity. However, a LDAH alternative-splicing variant missing 90 amino acids at C-terminus does not promote LD fusion or TAG accumulation, while it still localizes to LDs. Interestingly, LDAH enhances polyubiquitination and proteasomal degradation of adipose triglyceride lipase (ATGL), a rate limiting enzyme of TAG hydrolysis. Co-expression of ATGL reverses the changes in LD phenotype induced by LDAH, and both proteins counterbalance their effects on TAG stores. Together, these studies support that under conditions of TAG storage in LDs LDAH plays a primarily lipogenic role, inducing LD growth and enhancing degradation of ATGL.

  9. Promotion of CHIP-mediated p53 degradation protects the heart from ischemic injury.

    PubMed

    Naito, Atsuhiko T; Okada, Sho; Minamino, Tohru; Iwanaga, Koji; Liu, Mei-Lan; Sumida, Tomokazu; Nomura, Seitaro; Sahara, Naruhiko; Mizoroki, Tatsuya; Takashima, Akihiko; Akazawa, Hiroshi; Nagai, Toshio; Shiojima, Ichiro; Komuro, Issei

    2010-06-11

    The number of patients with coronary heart disease, including myocardial infarction, is increasing and novel therapeutic strategy is awaited. Tumor suppressor protein p53 accumulates in the myocardium after myocardial infarction, causes apoptosis of cardiomyocytes, and plays an important role in the progression into heart failure. We investigated the molecular mechanisms of p53 accumulation in the heart after myocardial infarction and tested whether anti-p53 approach would be effective against myocardial infarction. Through expression screening, we found that CHIP (carboxyl terminus of Hsp70-interacting protein) is an endogenous p53 antagonist in the heart. CHIP suppressed p53 level by ubiquitinating and inducing proteasomal degradation. CHIP transcription was downregulated after hypoxic stress and restoration of CHIP protein level prevented p53 accumulation after hypoxic stress. CHIP overexpression in vivo prevented p53 accumulation and cardiomyocyte apoptosis after myocardial infarction. Promotion of CHIP function by heat shock protein (Hsp)90 inhibitor, 17-allylamino-17-demethoxy geldanamycin (17-AAG), also prevented p53 accumulation and cardiomyocyte apoptosis both in vitro and in vivo. CHIP-mediated p53 degradation was at least one of the cardioprotective effects of 17-AAG. We found that downregulation of CHIP level by hypoxia was responsible for p53 accumulation in the heart after myocardial infarction. Decreasing the amount of p53 prevented myocardial apoptosis and ameliorated ventricular remodeling after myocardial infarction. We conclude that anti-p53 approach would be effective to treat myocardial infarction.

  10. Proteasomal degradation of human CYP1B1: effect of the Asn453Ser polymorphism on the post-translational regulation of CYP1B1 expression.

    PubMed

    Bandiera, Silvio; Weidlich, Simone; Harth, Volker; Broede, Peter; Ko, Yun; Friedberg, Thomas

    2005-02-01

    Allelic variations in CYP1B1 are reported to modulate the incidence of several types of cancer. To provide a mechanistic basis for this association, we investigated the impact of nonsilent allelic changes on the intracellular levels and post-translational regulation of CYP1B1 protein. When transiently expressed in COS-1 cells, either in the presence or absence of recombinant cytochrome P450 reductase, the cellular level of the CYP1B1.4 allelic variant (containing a Ser at the amino acid position 453; Ser453) was 2-fold lower compared with the other four allelic CYP1B1 proteins (containing Asn453), as analyzed by both immunoblotting and ethoxyresorufin O-deethylase activity. This difference was caused by post-translational regulation; as in the presence of cycloheximide, the rate of degradation of immunodetectable and enzymatically active CYP1B1.4 was distinctly faster than that of CYP1B1.1. Pulse-chase analysis revealed that the half-life of CYP1B1.4 was a mere 1.6 h compared with 4.8 h for CYP1B1.1. The presence of the proteasome inhibitor MG132 [N-benzoyloxycarbonyl (Z)-Leu-Leuleucinal] increased the stability not only of immunodetectable CYP1B1, but also--unexpectedly given the size of the proteasome access channel--increased the stability of enzymatically active CYP1B1. The data presented herein also demonstrate that CYP1B1 is targeted for its polymorphism-dependent degradation by polyubiquitination but not phosphorylation. Our results importantly provide a mechanism to explain the recently reported lower incidence of endometrial cancer in individuals carrying the CYP1B1*4 compared with the CYP1B1*1 haplo-type. In addition, the mechanistic paradigms revealed herein may explain the strong overexpression of CYP1B1 in tumors compared with nondiseased tissues.

  11. Balancing Arc synthesis, mRNA decay, and proteasomal degradation: maximal protein expression triggered by rapid eye movement sleep-like bursts of muscarinic cholinergic receptor stimulation.

    PubMed

    Soulé, Jonathan; Alme, Maria; Myrum, Craig; Schubert, Manja; Kanhema, Tambudzai; Bramham, Clive R

    2012-06-22

    Cholinergic signaling induces Arc/Arg3.1, an immediate early gene crucial for synaptic plasticity. However, the molecular mechanisms that dictate Arc mRNA and protein dynamics during and after cholinergic epochs are little understood. Using human SH-SY5Y neuroblastoma cells, we show that muscarinic cholinergic receptor (mAchR) stimulation triggers Arc synthesis, whereas translation-dependent RNA decay and proteasomal degradation strictly limit the amount and duration of Arc expression. Chronic application of the mAchR agonist, carbachol (Cch), induces Arc transcription via ERK signaling and release of calcium from IP(3)-sensitive stores. Arc translation requires ERK activation, but not changes in intracellular calcium. Proteasomal degradation of Arc (half-life ∼37 min) was enhanced by thapsigargin, an inhibitor of the endoplasmic calcium-ATPase pump. Similar mechanisms of Arc protein regulation were observed in cultured rat hippocampal slices. Functionally, we studied the impact of cholinergic epoch duration and temporal pattern on Arc protein expression. Acute Cch treatment (as short as 2 min) induces transient, moderate Arc expression, whereas continuous treatment of more than 30 min induces maximal expression, followed by rapid decline. Cholinergic activity associated with rapid eye movement sleep may function to facilitate long term synaptic plasticity and memory. Employing a paradigm designed to mimic intermittent rapid eye movement sleep epochs, we show that application of Cch in a series of short bursts generates persistent and maximal Arc protein expression. The results demonstrate dynamic, multifaceted control of Arc synthesis during mAchR signaling, and implicate cholinergic epoch duration and repetition as critical determinants of Arc expression and function in synaptic plasticity and behavior.

  12. Menadione induces G2/M arrest in gastric cancer cells by down-regulation of CDC25C and proteasome mediated degradation of CDK1 and cyclin B1

    PubMed Central

    Lee, Min Ho; Cho, Yoonjung; Kim, Do Hyun; Woo, Hyun Jun; Yang, Ji Yeong; Kwon, Hye Jin; Yeon, Min Ji; Park, Min; Kim, Sa-Hyun; Moon, Cheol; Tharmalingam, Nagendran; Kim, Tae Ue; Kim, Jong-Bae

    2016-01-01

    Menadione (vitamin K3) has been reported to induce apoptotic cell death and growth inhibition in various types of cancer cells. However, involvement of menadione in cell cycle control has not been considered in gastric cancer cells yet. In the current study, we have investigated whether menadione is involved in the cell cycle regulation and suppression of growth in gastric cancer cells. In the cell cycle analysis, we found that menadione induced G2/M cell cycle arrest in AGS cells. To elucidate the underlying mechanism, we investigated the cell cycle regulatory molecules involved in the G2/M cell cycle transition. After 24 h of menadione treatment, the protein level of CDK1, CDC25C and cyclin B1 in AGS cells was decreased in a menadione dose-dependent manner. In the time course experiment, the protein level of CDC25C decreased in 6 h, and CDK1and cyclin B1 protein levels began to decrease after 18 h of menadione treatment. We found that mRNA level of CDC25C decreased by menadione treatment in 6 h. Menadione did not have an influence on mRNA level of CDK1 and cyclin B1 though the protein levels were decreased. However, the decreased protein levels of CDK1 and cyclin B1 were recovered by inhibition of proteasome. Collectively, these results suggest that menadione inhibits growth of gastric cancer cells by reducing expression of CDC25C and promoting proteasome mediated degradation of CDK1 and cyclin B1 thereby blocking transition of the cell cycle from G2 phase to M phase. PMID:28077999

  13. The Logic of the 26S Proteasome.

    PubMed

    Collins, Galen Andrew; Goldberg, Alfred L

    2017-05-18

    The ubiquitin proteasome pathway is responsible for most of the protein degradation in mammalian cells. Rates of degradation by this pathway have generally been assumed to be determined by rates of ubiquitylation. However, recent studies indicate that proteasome function is also tightly regulated and determines whether a ubiquitylated protein is destroyed or deubiquitylated and survives longer. This article reviews recent advances in our understanding of the proteasome's multistep ATP-dependent mechanism, its biochemical and structural features that ensure efficient proteolysis and ubiquitin recycling while preventing nonselective proteolysis, and the regulation of proteasome activity by interacting proteins and subunit modifications, especially phosphorylation. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Inhibition of proteasomal degradation of Mcl-1 by cobalt chloride suppresses cobalt chloride-induced apoptosis in HCT116 colorectal cancer cells.

    PubMed

    Lee, Melanie; Lapham, Abigail; Brimmell, Matthew; Wilkinson, Helen; Packham, Graham

    2008-08-01

    Cobalt promotes apoptosis in multiple cell systems, however, the molecular mechanisms that influence cobalt-induced apoptosis are not fully understood. We investigated mechanisms of cobalt chloride induced apoptosis in HCT116 colorectal cancer cells. Cobalt chloride induced dose dependent apoptosis in HCT116 cells (250-750 muM) which, at higher concentrations (500-750 muM), was associated with an increase in the expression of the Bcl-2-related Mcl-1 survival protein. Cobalt chloride caused the accumulation of higher molecular weight ubiquitin-conjugates of Mcl-1 in intact HCT116 cells and inhibited the activity of the trypsin-like site of the 20S proteasome in an in vitro assay. Although siRNA-mediated knockdown of Mcl-1 increased apoptosis in HCT116 cells, the combination of Mcl-1 siRNA and cobalt chloride induced very high levels of cell killing. Therefore, inhibition of the proteasome by cobalt chloride leads to the accumulation of Mcl-1 which acts to limit cobalt chloride induced apoptosis.

  15. Endothelial monocyte activating polypeptide-II modulates endothelial cell responses by degrading hypoxia-inducible factor-1alpha through interaction with PSMA7, a component of the proteasome

    SciTech Connect

    Tandle, Anita T.; Calvani, Maura; Uranchimeg, Badarch; Zahavi, David; Melillo, Giovanni; Libutti, Steven K.

    2009-07-01

    The majority of human tumors are angiogenesis dependent. Understanding the specific mechanisms that contribute to angiogenesis may offer the best approach to develop therapies to inhibit angiogenesis in cancer. Endothelial monocyte activating polypeptide-II (EMAP-II) is an anti-angiogenic cytokine with potent effects on endothelial cells (ECs). It inhibits EC proliferation and cord formation, and it suppresses primary and metastatic tumor growth in-vivo. However, very little is known about the molecular mechanisms behind the anti-angiogenic activity of EMAP-II. In the present study, we explored the molecular mechanism behind the anti-angiogenic activity exerted by this protein on ECs. Our results demonstrate that EMAP-II binds to the cell surface {alpha}5{beta}1 integrin receptor. The cell surface binding of EMAP-II results in its internalization into the cytoplasmic compartment where it interacts with its cytoplasmic partner PSMA7, a component of the proteasome degradation pathway. This interaction increases hypoxia-inducible factor 1-alpha (HIF-1{alpha}) degradation under hypoxic conditions. The degradation results in the inhibition of HIF-1{alpha} mediated transcriptional activity as well as HIF-1{alpha} mediated angiogenic sprouting of ECs. HIF-1{alpha} plays a critical role in angiogenesis by activating a variety of angiogenic growth factors. Our results suggest that one of the major anti-angiogenic functions of EMAP-II is exerted through its inhibition of the HIF-1{alpha} activities.

  16. WSB1 promotes tumor metastasis by inducing pVHL degradation

    PubMed Central

    Kim, Jung Jin; Lee, Seung Baek; Jang, Jinsung; Yi, Sang-Yeop; Kim, Sun-Hyun; Han, Sang-Ah; Lee, Jong-Min; Tong, Seo-Yun; Vincelette, Nicole D.; Gao, Bowen; Yin, Ping; Evans, Debra; Choi, Dong Wook; Qin, Bo; Liu, Tongzheng; Zhang, Haoxing; Deng, Min; Jen, Jin; Zhang, Jun; Wang, Liewei; Lou, Zhenkun

    2015-01-01

    The von Hippel-Lindau tumor suppressor pVHL is an E3 ligase that targets hypoxia-inducible factors (HIFs). Mutation of VHL results in HIF up-regulation and contributes to processes related to tumor progression such as invasion, metastasis, and angiogenesis. However, very little is known with regard to post-transcriptional regulation of pVHL. Here we show that WD repeat and SOCS box-containing protein 1 (WSB1) is a negative regulator of pVHL through WSB1's E3 ligase activity. Mechanistically, WSB1 promotes pVHL ubiquitination and proteasomal degradation, thereby stabilizing HIF under both normoxic and hypoxic conditions. As a consequence, WSB1 up-regulates the expression of HIF-1α’s target genes and promotes cancer invasion and metastasis through its effect on pVHL. Consistent with this, WSB1 protein level negatively correlates with pVHL level and metastasis-free survival in clinical samples. This work reveals a new mechanism of pVHL's regulation by which cancer acquires invasiveness and metastatic tendency. PMID:26545811

  17. Dual attenuation of proteasomal and autophagic BMAL1 degradation in Clock Δ19/+ mice contributes to improved glucose homeostasis.

    PubMed

    Jeong, Kwon; He, Baokun; Nohara, Kazunari; Park, Noheon; Shin, Youngmin; Kim, Seonghwa; Shimomura, Kazuhiro; Koike, Nobuya; Yoo, Seung-Hee; Chen, Zheng

    2015-07-31

    Circadian clocks orchestrate essential physiology in response to various cues, yet their mechanistic and functional plasticity remains unclear. Here, we investigated Clock(Δ19/+) heterozygous (Clk/+) mice, known to display lengthened periodicity and dampened amplitude, as a model of partially perturbed clocks. Interestingly, Clk/+ mice exhibited improved glycemic control and resistance to circadian period lengthening under high-fat diet (HFD). Furthermore, BMAL1 protein levels in Clk/+ mouse liver were upregulated compared with wild-type (WT) mice under HFD. Pharmacological and molecular studies showed that BMAL1 turnover entailed proteasomal and autophagic activities, and CLOCKΔ19 attenuated both processes. Consistent with an important role of BMAL1 in glycemic control, enhanced activation of insulin signaling was observed in Clk/+ mice relative to WT in HFD. Finally, transcriptome analysis revealed reprogramming of clock-controlled metabolic genes in Clk/+ mice. Our results demonstrate a novel role of autophagy in circadian regulation and reveal an unforeseen plasticity of circadian and metabolic networks.

  18. Necdin Promotes Ubiquitin-Dependent Degradation of PIAS1 SUMO E3 Ligase

    PubMed Central

    Gur, Ibrahim; Fujiwara, Kazushiro; Hasegawa, Koichi; Yoshikawa, Kazuaki

    2014-01-01

    Necdin, a pleiotropic protein that promotes differentiation and survival of mammalian neurons, is a member of MAGE (melanoma antigen) family proteins that share a highly conserved MAGE homology domain. Several MAGE proteins interact with ubiquitin E3 ligases and modulate their activities. However, it remains unknown whether MAGE family proteins interact with SUMO (small ubiquitin-like modifier) E3 ligases such as PIAS (protein inhibitor of activated STAT) family, Nsmce2/Mms21 and Cbx4/Pc2. In the present study, we examined whether necdin interacts with these SUMO E3 ligases. Co-immunoprecipitation analysis revealed that necdin, MAGED1, MAGEF1 and MAGEL2 bound to PIAS1 but not to Nsmce2 or Cbx4. These SUMO E3 ligases bound to MAGEA1 but failed to interact with necdin-like 2/MAGEG1. Necdin bound to PIAS1 central domains that are highly conserved among PIAS family proteins and suppressed PIAS1-dependent sumoylation of the substrates STAT1 and PML (promyelocytic leukemia protein). Remarkably, necdin promoted degradation of PIAS1 via the ubiquitin-proteasome pathway. In transfected HEK293A cells, amino- and carboxyl-terminally truncated mutants of PIAS1 bound to necdin but failed to undergo necdin-dependent ubiquitination. Both PIAS1 and necdin were associated with the nuclear matrix, where the PIAS1 terminal deletion mutants failed to localize, implying that the nuclear matrix is indispensable for necdin-dependent ubiquitination of PIAS1. Our data suggest that necdin suppresses PIAS1 both by inhibiting SUMO E3 ligase activity and by promoting ubiquitin-dependent degradation. PMID:24911587

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

    PubMed Central

    Prasanth, K. Reddisiva; Barajas, Daniel

    2014-01-01

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

  20. 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

    2015-01-01

    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.

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

    PubMed Central

    González-Prieto, Román; Cuijpers, Sabine AG; Kumar, Ramesh; Hendriks, Ivo A; Vertegaal, Alfred CO

    2015-01-01

    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

  2. The Major Immediate-Early Protein IE2 of Human Cytomegalovirus Is Sufficient to Induce Proteasomal Degradation of CD83 on Mature Dendritic Cells

    PubMed Central

    Heilingloh, Christiane S.; Grosche, Linda; Kummer, Mirko; Mühl-Zürbes, Petra; Kamm, Lisa; Scherer, Myriam; Latzko, Melanie; Stamminger, Thomas; Steinkasserer, Alexander

    2017-01-01

    Human cytomegalovirus (HCMV) is the prototypic beta-herpesvirus and widespread throughout the human population. While infection is asymptomatic in healthy individuals, it can lead to high morbidity and mortality in immunocompromised persons. Importantly, HCMV evolved multiple strategies to interfere with immune cell function in order to establish latency in infected individuals. As mature DCs (mDCs) are antigen-presenting cells able to activate naïve T cells they play a crucial role during induction of effective antiviral immune responses. Interestingly, earlier studies demonstrated that the functionally important mDC surface molecule CD83 is down-regulated upon HCMV infection resulting in a reduced T cell stimulatory capacity of the infected cells. However, the viral effector protein and the precise mechanism of HCMV-mediated CD83 reduction remain to be discovered. Using flow cytometric analyses, we observed significant down-modulation of CD83 surface expression becoming significant already 12 h after HCMV infection. Moreover, Western bot analyses revealed that, in sharp contrast to previous studies, loss of CD83 is not restricted to the membrane-bound molecule, but also occurs intracellularly. Furthermore, inhibition of the proteasome almost completely restored CD83 surface expression during HCMV infection. Results of infection kinetics and cycloheximide-actinomycin D-chase experiments, strongly suggested that an HCMV immediate early gene product is responsible for the induction of CD83 down-modulation. Consequently, we were able to identify the major immediate early protein IE2 as the viral effector protein that induces proteasomal CD83 degradation. PMID:28203230

  3. Phosphorylation of WRINKLED1 by KIN10 Results in its Proteasomal Degradation, Providing a Link Between Energy Homeostasis and Lipid Biosynthesis

    DOE PAGES

    Zhai, Zhiyang; Liu, Hui; Shanklin, John

    2017-03-17

    WRINKLED1 (WRI1), a member of the APETALA2 (AP2) class of transcription factors, positively regulates glycolysis and lipid biosynthesis in Arabidopsis thaliana. Here we identify mechanistic links between KIN10, the major SUCROSE NON-FERMENTATION-1 (SNF1)-RELATED KINASE 1 (SnRK1) involved in sugar/energy homeostasis and the posttranslational regulation of WRI1. Transient expression of WRI1 with OLEOSIN1 (OLE1) in Nicotiana benthamiana stimulates triacylglycerol (TAG) accumulation, but their coexpression with KIN10 abrogates this effect by inducing proteasomal degradation of WRI1. While WRI1 lacks canonical KIN10 target sequences, we demonstrated direct KIN10-dependent phosphorylation of WRI1 using purified E. coli-expressed components. The resulting phosphorylated WRI1 was more rapidlymore » degraded than native WRI1 in cell-free degradation assays. WRI1 phosphorylation was localized to two variants of the canonical KIN10 recognition sequence, one in each of its two AP2 DNA-binding domains. Conversion of the phosphorylation sites at T70 and S166 to Ala resulted in a loss of KIN10-dependent phosphorylation, and when coexpressed with KIN10 the WRI1 double mutant accumulated to 2-3 fold higher levels than native WRI1. In conclusion, KIN10-dependent degradation of WRI1 provides a homeostatic mechanism that favors lipid biosynthesis when intracellular sugar levels are elevated and KIN10 is inhibited; conversely, glycolysis and lipid biosynthesis are curtailed as sugar levels decrease and KIN10 regains activity.« less

  4. Residual activity and proteasomal degradation of p.Ser298Pro sulfamidase identified in patients with a mild clinical phenotype of Sanfilippo A syndrome.

    PubMed

    Muschol, Nicole; Pohl, Sandra; Meyer, Ann; Gal, Andreas; Ullrich, Kurt; Braulke, Thomas

    2011-07-01

    Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo syndrome) is a fatal inherited lysosomal storage disease accompanied by progressive neurologic degeneration. The gene underlying MPS IIIA, SGSH, encodes a lysosomal enzyme, N-sulfoglucosamine sulfohydrolase (sulfamidase). Mutational analysis of a large cohort of MPS IIIA patients showed a correlation of the missense mutation p.Ser298Pro and a slowly progressive course of the disease. We report here on the expression of the mutant p.Ser298Pro sulfamidase in BHK cells retaining low residual activity. Pulse-chase experiments showed that rapid degradation is responsible for the low steady state level of the mutant protein. Processing and secretion of p.Ser298Pro sulfamidase suggests that small amounts of the newly synthesized enzyme are transported to lysosomes. Most of the mutant sulfamidase exits the endoplasmic reticulum for proteasomal degradation. The ability to predict the clinical course of MPS IIIA in patients with the p.Ser298Pro mutation, as well as the residual enzymatic activity, and the reduced stability of the mutant sulfamidase suggest that this subgroup of patients is especially well suited to early sulfamidase replacement therapy or treatment with selective pharmacological chaperones.

  5. Inhibition of melanogenesis by jineol from Scolopendra subspinipes mutilans via MAP-Kinase mediated MITF downregulation and the proteasomal degradation of tyrosinase

    PubMed Central

    Alam, Md Badrul; Bajpai, Vivek K.; Lee, JungIn; Zhao, Peijun; Byeon, Jung-Hee; Ra, Jeong-Sic; Majumder, Rajib; Lee, Jong Sung; Yoon, Jung-In; Rather, Irfan A.; Park, Yong-Ha; Kim, Kangmin; Na, MinKyun; Lee, Sang-Han

    2017-01-01

    In this study, the authors investigated the anti-melanogenic effects of 3,8-dihydroxyquinoline (jineol) isolated from Scolopendra subspinipes mutilans, the mechanisms responsible for its inhibition of melanogenesis in melan-a cells, and its antioxidant efficacy. Mushroom tyrosinase activities and melanin contents were determined in melan-a cells, and the protein and mRNA levels of MITF, tyrosinase, TYRP-1, and TYRP-2 were assessed. Jineol exhibited significant, concentration-dependent antioxidant effects as determined by DPPH, ABTS, CUPRAC, and FRAP assays. Jineol significantly inhibited mushroom tyrosinase activity by functioning as an uncompetitive inhibitor, and markedly inhibited melanin production and intracellular tyrosinase activity in melan-a cells. In addition, jineol abolished the expressions of tyrosinase, TYRP-1, TYRP-2, and MITF, thereby blocking melanin production and interfering with the phosphorylations of ERK1/2 and p38. Furthermore, specific inhibitors of ERK1/2 and p38 prevented melanogenesis inhibition by jineol, and the proteasome inhibitor (MG-132) prevented jineol-induced reductions in cellular tyrosinase levels. Taken together, jineol was found to stimulate MAP-kinase (ERK1/2 and p38) phosphorylation and the proteolytic degradation pathway, which led to the degradations of MITF and tyrosinase, and to suppress the productions of melanin. PMID:28393917

  6. Niclosamide Prevents the Formation of Large Ubiquitin-Containing Aggregates Caused by Proteasome Inhibition

    PubMed Central

    Winget, Jason M.; Brack, Maria; Rotblat, Barak; Novoa, Carolina Arias; Balgi, Aruna D.; Sorensen, Poul H.; Roberge, Michel; Mayor, Thibault

    2010-01-01

    Background Protein aggregation is a hallmark of many neurodegenerative diseases and has been linked to the failure to degrade misfolded and damaged proteins. In the cell, aberrant proteins are degraded by the ubiquitin proteasome system that mainly targets short-lived proteins, or by the lysosomes that mostly clear long-lived and poorly soluble proteins. Both systems are interconnected and, in some instances, autophagy can redirect proteasome substrates to the lysosomes. Principal Findings To better understand the interplay between these two systems, we established a neuroblastoma cell population stably expressing the GFP-ubiquitin fusion protein. We show that inhibition of the proteasome leads to the formation of large ubiquitin-containing inclusions accompanied by lower solubility of the ubiquitin conjugates. Strikingly, the formation of the ubiquitin-containing aggregates does not require ectopic expression of disease-specific proteins. Moreover, formation of these focused inclusions caused by proteasome inhibition requires the lysine 63 (K63) of ubiquitin. We then assessed selected compounds that stimulate autophagy and found that the antihelmintic chemical niclosamide prevents large aggregate formation induced by proteasome inhibition, while the prototypical mTORC1 inhibitor rapamycin had no apparent effect. Niclosamide also precludes the accumulation of poly-ubiquitinated proteins and of p62 upon proteasome inhibition. Moreover, niclosamide induces a change in lysosome distribution in the cell that, in the absence of proteasome activity, may favor the uptake into lysosomes of ubiquitinated proteins before they form large aggregates. Conclusions Our results indicate that proteasome inhibition provokes the formation of large ubiquitin containing aggregates in tissue culture cells, even in the absence of disease specific proteins. Furthermore our study suggests that the autophagy-inducing compound niclosamide may promote the selective clearance of ubiquitinated

  7. Dissociation between AKAP3 and PKARII Promotes AKAP3 Degradation in Sperm Capacitation

    PubMed Central

    Hillman, Pnina; Ickowicz, Debby; Vizel, Ruth; Breitbart, Haim

    2013-01-01

    Ejaculated spermatozoa must undergo a series of biochemical modifications called capacitation, prior to fertilization. Protein-kinase A (PKA) mediates sperm capacitation, although its regulation is not fully understood. Sperm contain several A-kinase anchoring proteins (AKAPs), which are scaffold proteins that anchor PKA. In this study, we show that AKAP3 is degraded in bovine sperm incubated under capacitation conditions. The degradation rate is variable in sperm from different bulls and is correlated with the capacitation ability. The degradation of AKAP3 was significantly inhibited by MG-132, a proteasome inhibitor, indicating that AKAP3 degradation occurs via the proteasomal machinery. Treatment with Ca2+-ionophore induced further degradation of AKAP3; however, this effect was found to be enhanced in the absence of Ca2+ in the medium or when intracellular Ca2+ was chelated the degradation rate of AKAP3 was significantly enhanced when intracellular space was alkalized using NH4Cl, or when sperm were treated with Ht31, a peptide that contains the PKA-binding domain of AKAPs. Moreover, inhibition of PKA activity by H89, or its activation using 8Br-cAMP, increased AKAP3 degradation rate. This apparent contradiction could be explained by assuming that binding of PKA to AKAP3 protects AKAP3 from degradation. We conclude that AKAP3 degradation is regulated by intracellular alkalization and PKARII anchoring during sperm capacitation. PMID:23894359

  8. An evolutionarily conserved pathway controls proteasome homeostasis

    PubMed Central

    Rousseau, Adrien; Bertolotti, Anne

    2016-01-01

    The proteasome is essential for the selective degradation of most cellular proteins but how cells maintain adequate amounts of proteasome is unclear. Here we found an evolutionarily conserved signalling pathway controlling proteasome homeostasis. Central to this pathway is TORC1 whose inhibition induced all known yeast 19S regulatory particle assembly-chaperones (RACs) as well as proteasome subunits. Downstream of TORC1 inhibition, the yeast mitogen-activated protein kinase, Mpk1, ensured that the supply of RACs and proteasome subunits increased under challenging conditions to maintain proteasomal degradation and cell viability. This adaptive pathway was evolutionarily conserved, with mTOR and Erk5 controlling the levels of the four mammalian RACs and proteasome abundance. Thus, the central growth and stress controllers, TORC1 and Mpk1/Erk5, endow cells with a rapid and vital adaptive response to adjust proteasome abundance to the rising needs. Enhancing this pathway may be a useful therapeutic approach for diseases resulting from impaired proteasomal degradation. PMID:27462806

  9. An evolutionarily conserved pathway controls proteasome homeostasis.

    PubMed

    Rousseau, Adrien; Bertolotti, Anne

    2016-08-11

    The proteasome is essential for the selective degradation of most cellular proteins, but how cells maintain adequate amounts of proteasome is unclear. Here we show that there is an evolutionarily conserved signalling pathway controlling proteasome homeostasis. Central to this pathway is TORC1, the inhibition of which induced all known yeast 19S regulatory particle assembly-chaperones (RACs), as well as proteasome subunits. Downstream of TORC1 inhibition, the yeast mitogen-activated protein kinase, Mpk1, acts to increase the supply of RACs and proteasome subunits under challenging conditions in order to maintain proteasomal degradation and cell viability. This adaptive pathway was evolutionarily conserved, with mTOR and ERK5 controlling the levels of the four mammalian RACs and proteasome abundance. Thus, the central growth and stress controllers, TORC1 and Mpk1/ERK5, endow cells with a rapid and vital adaptive response to adjust proteasome abundance in response to the rising needs of cells. Enhancing this pathway may be a useful therapeutic approach for diseases resulting from impaired proteasomal degradation.

  10. Proteasomes: Isolation and Activity Assays

    PubMed Central

    Li, Yanjie; Tomko, Robert J.; Hochstrasser, Mark

    2015-01-01

    In eukaryotes, damaged or unneeded proteins are typically degraded by the ubiquitin-proteasome system. In this system, the protein substrate is often first covalently modified with a chain of ubiquitin polypeptides. This chain serves as a signal for delivery to the 26S proteasome, a 2.5 MDa, ATP-dependent multisubunit protease complex. The proteasome consists of a barrel-shaped 20S core particle (CP) that is capped on one or both of its ends by a 19S regulatory particle (RP). The RP is responsible for recognizing the substrate, unfolding it, and translocating it into the CP for destruction. Here we describe simple, one-step purifications scheme for isolating the 26S proteasome and its 19S RP and 20S CP subcomplexes from the yeast Saccharomyces cerevisiae, as well as assays for measuring ubiquitin-dependent and ubiquitin-independent proteolytic activity in vitro. PMID:26061243

  11. The Ubiquitin Ligase ASB4 Promotes Trophoblast Differentiation through the Degradation of ID2

    PubMed Central

    Townley-Tilson, W. H. Davin; Wu, Yaxu; Ferguson, James E.; Patterson, Cam

    2014-01-01

    Vascularization of the placenta is a critical developmental process that ensures fetal viability. Although the vascular health of the placenta affects both maternal and fetal well being, relatively little is known about the early stages of placental vascular development. The ubiquitin ligase Ankyrin repeat, SOCS box-containing 4 (ASB4) promotes embryonic stem cell differentiation to vascular lineages and is highly expressed early in placental development. The transcriptional regulator Inhibitor of DNA binding 2 (ID2) negatively regulates vascular differentiation during development and is a target of many ubiquitin ligases. Due to their overlapping spatiotemporal expression pattern in the placenta and contrasting effects on vascular differentiation, we investigated whether ASB4 regulates ID2 through its ligase activity in the placenta and whether this activity mediates vascular differentiation. In mouse placentas, ASB4 expression is restricted to a subset of cells that express both stem cell and endothelial markers. Placentas that lack Asb4 display immature vascular patterning and retain expression of placental progenitor markers, including ID2 expression. Using JAR placental cells, we determined that ASB4 ubiquitinates and represses ID2 expression in a proteasome-dependent fashion. Expression of ASB4 in JAR cells and primary isolated trophoblast stem cells promotes the expression of differentiation markers. In functional endothelial co-culture assays, JAR cells ectopically expressing ASB4 increased endothelial cell turnover and stabilized endothelial tube formation, both of which are hallmarks of vascular differentiation within the placenta. Co-transfection of a degradation-resistant Id2 mutant with Asb4 inhibits both differentiation and functional responses. Lastly, deletion of Asb4 in mice induces a pathology that phenocopies human pre-eclampsia, including hypertension and proteinuria in late-stage pregnant females. These results indicate that ASB4 mediates

  12. Dual attenuation of proteasomal and autophagic BMAL1 degradation in ClockΔ19/+ mice contributes to improved glucose homeostasis

    PubMed Central

    Jeong, Kwon; He, Baokun; Nohara, Kazunari; Park, Noheon; Shin, Youngmin; Kim, Seonghwa; Shimomura, Kazuhiro; Koike, Nobuya; Yoo, Seung-Hee; Chen, Zheng

    2015-01-01

    Circadian clocks orchestrate essential physiology in response to various cues, yet their mechanistic and functional plasticity remains unclear. Here, we investigated ClockΔ19/+ heterozygous (Clk/+) mice, known to display lengthened periodicity and dampened amplitude, as a model of partially perturbed clocks. Interestingly, Clk/+ mice exhibited improved glycemic control and resistance to circadian period lengthening under high-fat diet (HFD). Furthermore, BMAL1 protein levels in Clk/+ mouse liver were upregulated compared with wild-type (WT) mice under HFD. Pharmacological and molecular studies showed that BMAL1 turnover entailed proteasomal and autophagic activities, and CLOCKΔ19 attenuated both processes. Consistent with an important role of BMAL1 in glycemic control, enhanced activation of insulin signaling was observed in Clk/+ mice relative to WT in HFD. Finally, transcriptome analysis revealed reprogramming of clock-controlled metabolic genes in Clk/+ mice. Our results demonstrate a novel role of autophagy in circadian regulation and reveal an unforeseen plasticity of circadian and metabolic networks. PMID:26228022

  13. Increased proteasome-dependent degradation of estrogen receptor-alpha by TGF-beta1 in breast cancer cell lines.

    PubMed

    Petrel, Trevor A; Brueggemeier, Robert W

    2003-01-01

    Normal mammary epithelial cells are rapidly induced to G(1) arrest by the widely expressed cytokine, transforming growth factor beta (TGF-beta1). Studies in established breast cancer cell lines that express the estrogen receptor alpha (ERalpha) have demonstrated loss of this responsiveness. This inverse correlation suggests interpathway signaling important to cell growth and regulation. The adenocarcinoma breast cell line BT474, which was not growth arrested by TGF-beta1, was used as a model of estrogen-inducible growth to explore interpathway crosstalk. Although BT474 cells were not growth-arrested by TGF-beta1 as determined by flow cytometry analysis and 5'-bromo-3'-deoxyuridine incorporation into DNA, estrogen receptor protein levels were attenuated by 100 pM TGF-beta1 after 6 h. This decrease in ERalpha reached 50% of untreated control levels by 24 h of treatment and was further supported by a 50% decrease in estrogen-inducible DNA synthesis. Inspection of ERalpha transcripts suggested that this decrease was primarily the result of altered ERalpha protein stability or availability. Use of the proteasome inhibitor, MG132, abolished all effects on ERalpha by TGF-beta1. Collectively, this data supports a role for TGF-beta1 in regulating the growth of otherwise insensitive breast cancer cells through modulation of ERalpha stability. Copyright 2002 Wiley-Liss, Inc.

  14. Increased Proteasome-Dependent Degradation of Estrogen Receptor-Alpha by TGF-β1 in Breast Cancer Cell Lines

    PubMed Central

    Petrel, Trevor A.; Brueggemeier, Robert W.

    2008-01-01

    Normal mammary epithelial cells are rapidly induced to G1 arrest by the widely expressed cytokine, transforming growth factor beta (TGF-β1). Studies in established breast cancer cell lines that express the estrogen receptor alpha (ERα) have demonstrated loss of this responsiveness. This inverse correlation suggests interpathway signaling important to cell growth and regulation. The adenocarcinoma breast cell line BT474, which was not growth arrested by TGF-β1, was used as a model of estrogen-inducible growth to explore interpathway crosstalk. Although BT474 cells were not growth-arrested by TGF-β1 as determined by flow cytometry analysis and 5′-bromo-3′-deoxyuridine incorporation into DNA, estrogen receptor protein levels were attenuated by 100 pM TGF-β1 after 6 h. This decrease in ERα reached 50% of untreated control levels by 24 h of treatment and was further supported by a 50% decrease in estrogen-inducible DNA synthesis. Inspection of ERα transcripts suggested that this decrease was primarily the result of altered ERα protein stability or availability. Use of the proteasome inhibitor, MG132, abolished all effects on ERα by TGF-β1. Collectively, this data supports a role for TGF-β1 in regulating the growth of otherwise insensitive breast cancer cells through modulation of ERα stability. PMID:12461787

  15. Pexophagy-linked degradation of the peroxisomal membrane protein Pex3p involves the ubiquitin-proteasome system.

    PubMed

    Williams, Chris; van der Klei, Ida J

    2013-08-23

    Peroxisome autophagy, also known as pexophagy, describes the wholesale degradation of peroxisomes via the vacuole, when organelles become damaged or redundant. In the methylotrophic yeast Hansenula polymorpha, pexophagy is stimulated when cells growing on methanol are exposed to excess glucose. Degradation of the peroxisomal membrane protein Pex3p, a process that does not involve the vacuole, was shown to trigger pexophagy. In this contribution, we have characterised pexophagy-associated Pex3p degradation further. We show that Pex3p breakdown depends on ubiquitin and confirm that Pex3p is a target for ubiquitination. Furthermore, we identify a role for the peroxisomal E3 ligases Pex2p and Pex10p in Pex3p degradation, suggesting the existence of a ubiquitin-dependent pathway involved in removing proteins from the peroxisomal membrane. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Proteasome activation during cardiac hypertrophy by the chaperone H11 Kinase/Hsp22.

    PubMed

    Hedhli, Nadia; Wang, Li; Wang, Qian; Rashed, Eman; Tian, Yimin; Sui, Xiangzhen; Madura, Kiran; Depre, Christophe

    2008-02-01

    The regulation of protein degradation by the proteasome during cardiac hypertrophy remains largely unknown. Also, the proteasome translocates to the nuclear periphery in response to cellular stress in yeast, which remains unexplored in mammals. The purpose of this study was to determine the quantitative and qualitative adaptation of the proteasome during stable cardiac hypertrophy. We measured proteasome activity, expression and sub-cellular distribution in a model of chronic cardiac hypertrophy induced by the stress-response chaperone H11 Kinase/Hsp22 (Hsp22). Over-expression of Hsp22 in a transgenic (TG) mouse leads to a 30% increase in myocyte cross-sectional area compared to wild-type (WT) mice (P < 0.01). Characterization of the proteasome in hearts from TG mice vs. WT revealed an increased expression of both 19S and 20S subunits (P < 0.05), a doubling in 20S catalytic activity (P < 0.01), a redistribution of both subunits from the cytosol to the nuclear periphery, and a four-fold increase in nuclear-associated 20S catalytic activity (P < 0.001). The perinuclear proteasome co-localized and interacted with Hsp22. Inhibition of proteasome activity by epoxomicin reduced hypertrophy in TG by 50% (P < 0.05). Adeno-mediated over-expression of Hsp22 in isolated cardiac myocytes increased both cell growth and proteasome activity, and both were prevented upon inhibition of the proteasome. Similarly, stimulation of cardiac cell growth by pro-hypertrophic stimuli increased Hsp22 expression and proteasome activity, and proteasome inhibition in that setting prevented hypertrophy. Proteasome inhibitors also prevented the increase in rate of protein synthesis observed after over-expression of Hsp22 or upon addition of pro-hypertrophic stimuli. Hsp22-mediated cardiac hypertrophy promotes an increased expression and activity, and a subcellular redistribution of the proteasome. Inhibition of the proteasome reverses cardiac hypertrophy upon Hsp22 over-expression or upon

  17. Mineralocorticoid receptor degradation is promoted by Hsp90 inhibition and the ubiquitin-protein ligase CHIP.

    PubMed

    Faresse, Nourdine; Ruffieux-Daidie, Dorothée; Salamin, Mélanie; Gomez-Sanchez, Celso E; Staub, Olivier

    2010-12-01

    The mineralocorticoid receptor (MR) plays a crucial role in the regulation of Na(+) balance and blood pressure, as evidenced by gain of function mutations in the MR of hypertensive families. In the kidney, aldosterone binds to the MR, induces its nuclear translocation, and promotes a transcriptional program leading to increased transepithelial Na(+) transport via the epithelial Na(+) channel. In the unliganded state, MR is localized in the cytosol and part of a multiprotein complex, including heat shock protein 90 (Hsp90), which keeps it ligand-binding competent. 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a benzoquinone ansamycin antibiotic that binds to Hsp90 and alters its function. We investigated whether 17-AAG affects the stability and transcriptional activity of MR and consequently Na(+) reabsorption by renal cells. 17-AAG treatment lead to reduction of MR protein level in epithelial cells in vitro and in vivo, thereby interfering with aldosterone-dependent transcription. Moreover, 17-AAG inhibited aldosterone-induced Na(+) transport, possibly by interfering with MR availability for the ligand. Finally, we identified the ubiquitin-protein ligase, COOH terminus of Hsp70-interacting protein, as a novel partner of the cytosolic MR, which is responsible for its polyubiquitylation and proteasomal degradation in presence of 17-AAG. In conclusion, 17-AAG may represent a novel pharmacological tool to interfere with Na(+) reabsorption and hypertension.

  18. Parkin Protects against Oxygen-Glucose Deprivation/Reperfusion Insult by Promoting Drp1 Degradation.

    PubMed

    Tang, Jiayu; Hu, Zhiping; Tan, Jieqiong; Yang, Sonlin; Zeng, Liuwang

    2016-01-01

    Ischemic stroke results in severe brain damage and remains one of the leading causes of death and disability worldwide. Effective neuroprotective therapies are needed to reduce brain damage resulting from ischemic stroke. Mitochondria are crucial for cellular energy production and homeostasis. Modulation of mitochondrial function mediates neuroprotection against ischemic brain damage. Dynamin-related protein 1 (Drp1) and parkin play a key role in regulating mitochondrial dynamics. They are potential therapeutic targets for neuroprotection in ischemic stroke. Protective effects of parkin-Drp1 pathway on mitochondria were assessed in a cellular ischemia-reperfusion injury model. Mouse neuroblastoma Neuro2a (N2a) cells were subjected to oxygen-glucose deprivation/reperfusion (OGDR) insult. OGDR induces mitochondrial fragmentation. The expression of Drp1 protein is increased after OGDR insult, while the parkin protein level is decreased. The altered protein level of Drp1 after OGDR injury is mediated by parkin through ubiquitin proteasome system (UPS). Drp1 depletion protects against OGDR induced mitochondrial damage and apoptosis. Meanwhile, parkin overexpression protects against OGDR induced apoptosis and mitochondrial dysfunction, which is attenuated by increased expression of Drp1. Our data demonstrate that parkin protects against OGDR insult through promoting degradation of Drp1. This neuroprotective potential of parkin-Drp1 pathway against OGDR insult will pave the way for developing novel neuroprotective agents for cerebral ischemia-reperfusion related disorders.

  19. Parkin Protects against Oxygen-Glucose Deprivation/Reperfusion Insult by Promoting Drp1 Degradation

    PubMed Central

    Tang, Jiayu; Hu, Zhiping; Tan, Jieqiong; Yang, Sonlin

    2016-01-01

    Ischemic stroke results in severe brain damage and remains one of the leading causes of death and disability worldwide. Effective neuroprotective therapies are needed to reduce brain damage resulting from ischemic stroke. Mitochondria are crucial for cellular energy production and homeostasis. Modulation of mitochondrial function mediates neuroprotection against ischemic brain damage. Dynamin-related protein 1 (Drp1) and parkin play a key role in regulating mitochondrial dynamics. They are potential therapeutic targets for neuroprotection in ischemic stroke. Protective effects of parkin-Drp1 pathway on mitochondria were assessed in a cellular ischemia-reperfusion injury model. Mouse neuroblastoma Neuro2a (N2a) cells were subjected to oxygen-glucose deprivation/reperfusion (OGDR) insult. OGDR induces mitochondrial fragmentation. The expression of Drp1 protein is increased after OGDR insult, while the parkin protein level is decreased. The altered protein level of Drp1 after OGDR injury is mediated by parkin through ubiquitin proteasome system (UPS). Drp1 depletion protects against OGDR induced mitochondrial damage and apoptosis. Meanwhile, parkin overexpression protects against OGDR induced apoptosis and mitochondrial dysfunction, which is attenuated by increased expression of Drp1. Our data demonstrate that parkin protects against OGDR insult through promoting degradation of Drp1. This neuroprotective potential of parkin-Drp1 pathway against OGDR insult will pave the way for developing novel neuroprotective agents for cerebral ischemia-reperfusion related disorders. PMID:27597885

  20. Ubiquitin-independent proteasomal degradation of Fra-1 is antagonized by Erk1/2 pathway-mediated phosphorylation of a unique C-terminal destabilizer.

    PubMed

    Basbous, Jihane; Chalbos, Dany; Hipskind, Robert; Jariel-Encontre, Isabelle; Piechaczyk, Marc

    2007-06-01

    Fra-1, a transcription factor that is phylogenetically and functionally related to the proto-oncoprotein c-Fos, controls many essential cell functions. It is expressed in many cell types, albeit with differing kinetics and abundances. In cells reentering the cell cycle, Fra-1 expression is transiently stimulated albeit later than that of c-Fos and for a longer time. Moreover, Fra-1 overexpression is found in cancer cells displaying high Erk1/2 activity and has been linked to tumorigenesis. One crucial point of regulation of Fra-1 levels is controlled protein degradation, the mechanism of which remains poorly characterized. Here, we have combined genetic, pharmacological, and signaling studies to investigate this process in nontransformed cells and to elucidate how it is altered in cancer cells. We report that the intrinsic instability of Fra-1 depends on a single destabilizer contained within the C-terminal 30 to 40 amino acids. Two serines therein, S252 and S265, are phosphorylated by kinases of the Erk1/2 pathway, which compromises protein destruction upon both normal physiological induction and tumorigenic constitutive activation of this cascade. Our data also indicate that Fra-1, like c-Fos, belongs to a small group of proteins that may, under certain circumstances, undergo ubiquitin-independent degradation by the proteasome. Our work reveals both similitudes and differences between Fra-1 and c-Fos degradation mechanisms. In particular, the presence of a single destabilizer within Fra-1, instead of two that are differentially regulated in c-Fos, explains the much faster turnover of the latter when cells traverse the G(0)/G(1)-to-S-phase transition. Finally, our study offers further insights into the signaling-regulated expression of the other Fos family proteins.

  1. Nutrient-induced FNIP degradation by SCFβ-TRCP regulates FLCN complex localization and promotes renal cancer progression.

    PubMed

    Nagashima, Katsuyuki; Fukushima, Hidefumi; Shimizu, Kouhei; Yamada, Aya; Hidaka, Masumi; Hasumi, Hisashi; Ikebe, Tetsuro; Fukumoto, Satoshi; Okabe, Koji; Inuzuka, Hiroyuki

    2017-02-07

    Folliculin-interacting protein 1 and 2 (FNIP1 and FNIP2) play critical roles in preventing renal malignancy through their association with the tumor suppressor FLCN. Mutations in FLCN are associated with Birt-Hogg-Dubé (BHD) syndrome, a rare disorder with increased risk of renal cancer. Recent studies indicated that FNIP1/FNIP2 double knockout mice display enlarged polycystic kidneys and renal carcinoma, which phenocopies FLCN knockout mice, suggesting that these two proteins function together to suppress renal cancer. However, the molecular mechanism functionally linking FNIP1/FNIP2 and FLCN remains largely elusive. Here, we demonstrated that FNIP2 protein is unstable and subjected to proteasome-dependent degradation via β-TRCP and Casein Kinase 1 (CK1)-directed ubiquitination in a nutrition-dependent manner. Degradation of FNIP2 leads to lysosomal dissociation of FLCN and subsequent lysosomal association of mTOR, which in turn promotes the proliferation of renal cancer cells. These results indicate that SCFβ-TRCP negatively regulates the FLCN complex by promoting FNIP degradation and provide molecular insight into the pathogenesis of BHD-associated renal cancer.

  2. Natural small molecule FMHM inhibits lipopolysaccharide-induced inflammatory response by promoting TRAF6 degradation via K48-linked polyubiquitination.

    PubMed

    Zeng, Ke-Wu; Liao, Li-Xi; Lv, Hai-Ning; Song, Fang-Jiao; Yu, Qian; Dong, Xin; Li, Jun; Jiang, Yong; Tu, Peng-Fei

    2015-10-01

    TNF receptor-associated factor 6 (TRAF6) is a key hub protein involved in Toll-like receptor-dependent inflammatory signaling pathway, and it recruits additional proteins to form multiprotein complexes capable of activating downstream NF-κB inflammatory signaling pathway. Ubiquitin-proteasome system (UPS) plays a crucial role in various protein degradations, such as TRAF6, leading to inhibitory effects on inflammatory response and immunologic function. However, whether ubiquitination-dependent TRAF6 degradation can be used as a novel anti-inflammatory drug target still remains to be explored. FMHM, a bioactive natural small molecule compound extracted from Chinese herbal medicine Radix Polygalae, suppressed acute inflammatory response by targeting ubiquitin protein and inducing UPS-dependent TRAF6 degradation mechanism. It was found that FMHM targeted ubiquitin protein via Lys48 site directly induced Lys48 residue-linked polyubiquitination. This promoted Lys48 residue-linked polyubiquitin chain formation on TRAF6, resulting in increased TRAF6 degradation via UPS and inactivation of downstream NF-κB inflammatory pathway. Consequently, FMHM down-regulated inflammatory mediator levels in circulation, protected multiple organs against inflammatory injury in vivo, and prolong the survival of endotoxemia mouse models. Therefore, FMHM can serve as a novel lead compound for the development of TRAF6 scavenging agent via ubiquitination-dependent mode, which represents a promising strategy for treating inflammatory diseases.

  3. Mechanism of Degradation of an α-Keto-Epoxide, a Model for the Warhead for Various Proteasome Inhibitor Anticancer Agents.

    PubMed

    Phizackerley, Kirsten M; Jumaa, Mouhannad; Lopalco, Antonio; Wolfe, Bradley H; Ablan, Christopher D; Stella, Valentino J

    2016-12-20

    The anticancer agent, carfilzomib, has a unique α-keto-epoxide warhead. The model α-keto-epoxide, N-((S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)pivalamide (1), along with a few of its degradation products was synthesized and studied. The kinetics of hydrolysis and identification of some of the degradation products of 1 were performed at pH values 2, 4, 5, 7, and 8 at 25°C, 40°C, and 60°C and followed by HPLC and liquid chromatography-mass spectroscopy, respectively. 1 degraded independent of pH between pH values 4-7 but showed some acid catalysis at pH 2 and base catalysis at pH 8. Energy of activation, Ea, values progressed from 16.8 ± 0.1 at pH 2 to 20.3 ± 0.1 kcal/mole at pH 8. The major initial degradation products in the pH range 4-5 were the S,R diol (hydrolysis of the epoxide), and S,R chlorohydrin (in the presence of chloride ions). At pH 7-8, the major products were the R,R diastereomer and the S,R and R,R diols. At pH 2, additional unidentified products were seen with relative retention times of 0.28, 0.30, 0.33, and 0.35 and masses equivalent to the diols. The study of 1 provides insight into the degradation of future drugs that use an α-keto-epoxide functional group.

  4. The Proteasome Subunit Rpn8 Interacts with the Small Nucleolar RNA Protein (snoRNP) Assembly Protein Pih1 and Mediates Its Ubiquitin-independent Degradation in Saccharomyces cerevisiae.

    PubMed

    Paci, Alexandr; Liu, Peter X H; Zhang, Lingjie; Zhao, Rongmin

    2016-05-27

    Pih1 is a scaffold protein of the Rvb1-Rvb2-Tah1-Pih1 (R2TP) protein complex, which is conserved in fungi and animals. The chaperone-like activity of the R2TP complex has been implicated in the assembly of multiple protein complexes, such as the small nucleolar RNA protein complex. However, the mechanism of the R2TP complex activity in vivo and the assembly of the complex itself are still largely unknown. Pih1 is an unstable protein and tends to aggregate when expressed alone. The C-terminal fragment of Pih1 contains multiple destabilization factors and acts as a degron when fused to other proteins. In this study, we investigated Pih1 interactors and identified a specific interaction between Pih1 and the proteasome subunit Rpn8 in yeast Saccharomyces cerevisiae when HSP90 co-chaperone Tah1 is depleted. By analyzing truncation mutants, we identified that the C-terminal 30 amino acids of Rpn8 are sufficient for the binding to Pih1 C terminus. With in vitro and in vivo degradation assays, we showed that the Pih1 C-terminal fragment Pih1(282-344) is able to induce a ubiquitin-independent degradation of GFP. Additionally, we demonstrated that truncation of the Rpn8 C-terminal disordered region does not affect proteasome assembly but specifically inhibits the degradation of the GFP-Pih1(282-344) fusion protein in vivo and Pih1 in vitro We propose that Pih1 is a ubiquitin-independent proteasome substrate, and the direct interaction with Rpn8 C terminus mediates its proteasomal degradation.

  5. Proteasome Activation by Small Molecules.

    PubMed

    Leestemaker, Yves; de Jong, Annemieke; Witting, Katharina F; Penning, Renske; Schuurman, Karianne; Rodenko, Boris; Zaal, Esther A; van de Kooij, Bert; Laufer, Stefan; Heck, Albert J R; Borst, Jannie; Scheper, Wiep; Berkers, Celia R; Ovaa, Huib

    2017-06-22

    Drugs that increase 26S proteasome activity have potential therapeutic applications in the treatment of neurodegenerative diseases. A chemical genetics screen of over 2,750 compounds using a proteasome activity probe as a readout in a high-throughput live-cell fluorescence-activated cell sorting-based assay revealed more than ten compounds that increase proteasome activity, with the p38 MAPK inhibitor PD169316 being one of the most potent ones. Genetic and chemical inhibition of either p38 MAPK, its upstream regulators, ASK1 and MKK6, and downstream target, MK2, enhance proteasome activity. Chemical activation of the 26S proteasome increases PROTAC-mediated and ubiquitin-dependent protein degradation and decreases the levels of both overexpressed and endogenous α-synuclein, without affecting the overall protein turnover. In addition, survival of cells overexpressing toxic α-synuclein assemblies is increased in the presence of p38 MAPK inhibitors. These findings highlight the potential of activation of 26S proteasome activity and that this can be achieved through multiple mechanisms by distinct molecules. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Tubeimoside-1 suppresses tumor angiogenesis by stimulation of proteasomal VEGFR2 and Tie2 degradation in a non-small cell lung cancer xenograft model

    PubMed Central

    Gu, Yuan; Körbel, Christina; Scheuer, Claudia; Nenicu, Anca; Menger, Michael D.; Laschke, Matthias W.

    2016-01-01

    Tubeimoside-1 (TBMS1) is a potent anti-tumor phytochemical. Its functional and molecular mode of action, however, remains elusive so far. Since angiogenesis is essential for tumor progression and metastasis, we herein investigated the anti-angiogenic effects of the compound. In a non-small cell lung cancer (NSCLC) xenograft model we found that treatment of CD1 nu/nu mice with TBMS1 (5mg/kg) significantly suppressed the growth and vascularization of NCI-H460 flank tumors. Moreover, TBMS1 dose-dependently reduced vascular sprouting in a rat aortic ring assay. In vitro, TBMS1 induced endothelial cell apoptosis without decreasing the viability of NSCLC tumor cells and inhibited the migration of endothelial cells by disturbing their actin filament organization. TBMS1 further stimulated the proteasomal degradation of vascular endothelial growth factor receptor-2 (VEGFR2) and Tie2 in endothelial cells, which down-regulated AKT/mTOR signaling. These findings indicate that TBMS1 represents a novel phytochemical for anti-angiogenic treatment of cancer and other angiogenesis-related diseases. PMID:26701724

  7. The influenza A virus matrix protein 2 undergoes retrograde transport from the endoplasmic reticulum into the cytoplasm and bypasses cytoplasmic proteasomal degradation.

    PubMed

    Bhowmick, Sanchari; Chakravarty, Chandrani; Sellathamby, Shanmugaapriya; Lal, Sunil K

    2017-04-01

    The matrix protein 2 (M2) is a spliced product of segment 7 genome of influenza A virus. Previous studies indicate its role in uncoating of the viral ribonucleoprotein complex during viral entry and in membrane scission while budding. Despite its crucial role in the viral life cycle, little is known about its subcellular distribution and dynamics. In this study, we have shown that the M2 protein is translocated from the membrane to the cytoplasm by a retrograde route via endosomes and the Golgi network. It utilizes retromer cargo while moving from the endosome to the trans-Golgi network and prevents endosome fusion with the lysosome. Further, M2 interacts with the endoplasmic-reticulum-resident AAA-ATPase p97 for its release into the cytoplasm. Our study also revealed that the M2 protein in the cellular milieu does not undergo ubiquitin-mediated proteasomal degradation. The migration of M2 through this pathway inside the infected cell suggests possible new roles that the M2 protein may have in the host cytoplasm, apart from its previously described functions.

  8. FANCJ protein is important for the stability of FANCD2/FANCI proteins and protects them from proteasome and caspase-3 dependent degradation.

    PubMed

    Clark, David W; Tripathi, Kaushlendra; Dorsman, Josephine C; Palle, Komaraiah

    2015-10-06

    Fanconi anemia (FA) is a rare genome instability syndrome with progressive bone marrow failure and cancer susceptibility. FANCJ is one of 17 genes mutated in FA-patients, comprises a DNA helicase that is vital for properly maintaining genomic stability and is known to function in the FA-BRCA DNA repair pathway. While exact role(s) of FANCJ in this repair process is yet to be determined, it is known to interact with primary effector FANCD2. However, FANCJ is not required for FANCD2 activation but is important for its ability to fully respond to DNA damage. In this report, we determined that transient depletion of FANCJ adversely affects stability of FANCD2 and its co-regulator FANCI in multiple cell lines. Loss of FANCJ does not significantly alter cell cycle progression or FANCD2 transcription. However, in the absence of FANCJ, the majority of FANCD2 is degraded by both the proteasome and Caspase-3 dependent mechanism. FANCJ is capable of complexing with and stabilizing FANCD2 even in the absence of a functional helicase domain. Furthermore, our data demonstrate that FANCJ is important for FANCD2 stability and proper activation of DNA damage responses to replication blocks induced by hydroxyurea.

  9. FANCJ protein is important for the stability of FANCD2/FANCI proteins and protects them from proteasome and caspase-3 dependent degradation

    PubMed Central

    Clark, David W.; Tripathi, Kaushlendra; Dorsman, Josephine C.; Palle, Komaraiah

    2015-01-01

    Fanconi anemia (FA) is a rare genome instability syndrome with progressive bone marrow failure and cancer susceptibility. FANCJ is one of 17 genes mutated in FA-patients, comprises a DNA helicase that is vital for properly maintaining genomic stability and is known to function in the FA-BRCA DNA repair pathway. While exact role(s) of FANCJ in this repair process is yet to be determined, it is known to interact with primary effector FANCD2. However, FANCJ is not required for FANCD2 activation but is important for its ability to fully respond to DNA damage. In this report, we determined that transient depletion of FANCJ adversely affects stability of FANCD2 and its co-regulator FANCI in multiple cell lines. Loss of FANCJ does not significantly alter cell cycle progression or FANCD2 transcription. However, in the absence of FANCJ, the majority of FANCD2 is degraded by both the proteasome and Caspase-3 dependent mechanism. FANCJ is capable of complexing with and stabilizing FANCD2 even in the absence of a functional helicase domain. Furthermore, our data demonstrate that FANCJ is important for FANCD2 stability and proper activation of DNA damage responses to replication blocks induced by hydroxyurea. PMID:26336824

  10. Hedgehog-dependent E3-ligase Midline1 regulates ubiquitin-mediated proteasomal degradation of Pax6 during visual system development

    PubMed Central

    Pfirrmann, Thorsten; Jandt, Enrico; Ranft, Swantje; Lokapally, Ashwin; Neuhaus, Herbert; Perron, Muriel

    2016-01-01

    Pax6 is a key transcription factor involved in eye, brain, and pancreas development. Although pax6 is expressed in the whole prospective retinal field, subsequently its expression becomes restricted to the optic cup by reciprocal transcriptional repression of pax6 and pax2. However, it remains unclear how Pax6 protein is removed from the eyestalk territory on time. Here, we report that Mid1, a member of the RBCC/TRIM E3 ligase family, which was first identified in patients with the X-chromosome–linked Opitz BBB/G (OS) syndrome, interacts with Pax6. We found that the forming eyestalk is a major domain of mid1 expression, controlled by the morphogen Sonic hedgehog (Shh). Here, Mid1 regulates the ubiquitination and proteasomal degradation of Pax6 protein. Accordantly, when Mid1 levels are knocked down, Pax6 expression is expanded and eyes are enlarged. Our findings indicate that remaining or misaddressed Pax6 protein is cleared from the eyestalk region to properly set the border between the eyestalk territory and the retina via Mid1. Thus, we identified a posttranslational mechanism, regulated by Sonic hedgehog, which is important to suppress Pax6 activity and thus breaks pax6 autoregulation at defined steps during the formation of the visual system. PMID:27555585

  11. SYVN1, NEDD8, and FBXO2 Proteins Regulate ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Ubiquitin-mediated Proteasomal Degradation.

    PubMed

    Ramachandran, Shyam; Osterhaus, Samantha R; Parekh, Kalpaj R; Jacobi, Ashley M; Behlke, Mark A; McCray, Paul B

    2016-12-02

    We previously reported that delivery of a microRNA-138 mimic or siRNA against SIN3A to cultured cystic fibrosis (ΔF508/ΔF508) airway epithelia partially restored ΔF508-cystic fibrosis transmembrane conductance regulator (CFTR)-mediated cAMP-stimulated Cl(-) conductance. We hypothesized that dissecting this microRNA-138/SIN3A-regulated gene network would identify individual proteins contributing to the rescue of ΔF508-CFTR function. Among the genes in the network, we rigorously validated candidates using functional CFTR maturation and electrolyte transport assays in polarized airway epithelia. We found that depletion of the ubiquitin ligase SYVN1, the ubiquitin/proteasome system regulator NEDD8, or the F-box protein FBXO2 partially restored ΔF508-CFTR-mediated Cl(-) transport in primary cultures of human cystic fibrosis airway epithelia. Moreover, knockdown of SYVN1, NEDD8, or FBXO2 in combination with corrector compound 18 further potentiated rescue of ΔF508-CFTR-mediated Cl(-) conductance. This study provides new knowledge of the CFTR biosynthetic pathway. It suggests that SYVN1 and FBXO2 represent two distinct multiprotein complexes that may degrade ΔF508-CFTR in airway epithelia and identifies a new role for NEDD8 in regulating ΔF508-CFTR ubiquitination.

  12. Parkin Promotes Degradation of the Mitochondrial Pro-Apoptotic ARTS Protein

    PubMed Central

    Kemeny, Stav; Dery, Dikla; Loboda, Yelena; Rovner, Marshall; Lev, Tali; Zuri, Dotan; Finberg, John P. M.; Larisch, Sarit

    2012-01-01

    Parkinson’s disease (PD) is associated with excessive cell death causing selective loss of dopaminergic neurons. Dysfunction of the Ubiquitin Proteasome System (UPS) is associated with the pathophysiology of PD. Mutations in Parkin which impair its E3-ligase activity play a major role in the pathogenesis of inherited PD. ARTS (Sept4_i2) is a mitochondrial protein, which initiates caspase activation upstream of cytochrome c release in the mitochondrial apoptotic pathway. Here we show that Parkin serves as an E3-ubiquitin ligase to restrict the levels of ARTS through UPS-mediated degradation. Though Parkin binds equally to ARTS and Sept4_i1 (H5/PNUTL2), the non-apoptotic splice variant of Sept4, Parkin ubiquitinates and degrades only ARTS. Thus, the effect of Parkin on ARTS is specific and probably related to its pro-apoptotic function. High levels of ARTS are sufficient to promote apoptosis in cultured neuronal cells, and rat brains treated with 6-OHDA reveal high levels of ARTS. However, over-expression of Parkin can protect cells from ARTS-induced apoptosis. Furthermore, Parkin loss-of-function experiments reveal that reduction of Parkin causes increased levels of ARTS and apoptosis. We propose that in brain cells in which the E3-ligase activity of Parkin is compromised, ARTS levels increase and facilitate apoptosis. Thus, ARTS is a novel substrate of Parkin. These observations link Parkin directly to a pro-apoptotic protein and reveal a novel connection between Parkin, apoptosis, and PD. PMID:22792159

  13. Hydroxylamine Promoted Goethite Surface Fenton Degradation of Organic Pollutants.

    PubMed

    Hou, Xiaojing; Huang, Xiaopeng; Jia, Falong; Ai, Zhihui; Zhao, Jincai; Zhang, Lizhi

    2017-03-30

    In this study, we construct a surface Fenton system with hydroxylamine (NH2OH), goethite (α-FeOOH), and H2O2 (α-FeOOH-HA/H2O2) to degrade various organic pollutants including dyes (methyl orange, methylene blue, and rhodamine B), pesticides (pentachlorophenol, alachlor, and atrazine), and antibiotics (tetracycline, chloramphenicol, and lincomycin) at pH 5.0. In this surface Fenton system, the presence of NH2OH could greatly promote the H2O2 decomposition on the α-FeOOH surface to produce •OH without releasing any detectable iron ions during the alachlor degradation, which was different from some previously reported heterogeneous Fenton counterparts. Moreover, the •OH generation rate constant of this surface Fenton system was 102 - 104 times those of previous heterogeneous Fenton processes. The interaction between α-FeOOH and NH2OH was investigated with using attenuated total reflectance Fourier transform infrared spectroscopy and density functional theory calculations. The effective degradation of organic pollutants in this surface Fenton system was ascribed to the efficient Fe(III)/Fe(II) cycle on the α-FeOOH surface promoted by NH2OH, which was confirmed by X-ray photoelectron spectroscopy analysis. The degradation intermediates and mineralization of alachlor in this surface Fenton system were then systematically investigated using total organic carbon and ion chromatography, liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. This study offers a new strategy to degrade organic pollutants, and also sheds light on the environmental effects of goethite.

  14. A High-Throughput Screening Assay Using a Photoconvertable Protein for Identifying Inhibitors of Transcription, Translation, or Proteasomal Degradation.

    PubMed

    Heidary, David K; Fox, Ashley; Richards, Chris I; Glazer, Edith C

    2017-04-01

    Dysregulated transcription, translation, and protein degradation are common features of cancer cells, regardless of specific genetic profiles. Several clinical anticancer agents take advantage of this characteristic vulnerability and interfere with the processes of transcription and translation or inhibit protein degradation. However, traditional assays that follow the process of protein production and removal require multistep processing and are not easily amenable to high-throughput screening. The use of recombinant fluorescent proteins provides a convenient solution to this problem, and moreover, photoconvertable fluorescent proteins allow for ratiometric detection of both new protein production and removal of existing proteins. Here, the photoconvertable protein Dendra2 is used in the development of in-cell assays of protein production and degradation that are optimized and validated for high-throughput screening. Conversion from the green to red emissive form can be achieved using a high-intensity light-emitting diode array, producing a stable pool of the red fluorescent form of Dendra2. This allows for rates of protein production or removal to be quantified in a plate reader or by fluorescence microscopy, providing a means to measure the potencies of inhibitors that affect these key processes.

  15. Hsp70 and antifibrillogenic peptides promote degradation and inhibit intracellular aggregation of amyloidogenic light chains.

    SciTech Connect

    Dul, J. L.; Davis, D. P.; Williamson, E. K.; Stevens, F. J.; Argon, Y.; Univ. of Chicago

    2001-02-19

    In light chain (LC) amyloidosis an immunoglobulin LC assembles into fibrils that are deposited in various tissues. Little is known about how these fibrils form in vivo. We previously showed that a known amyloidogenic LC, SMA, can give rise to amyloid fibrils in vitro when a segment of one of its {beta} sheets undergoes a conformational change, exposing an Hsp70 binding site. To examine SMA aggregation in vivo, we expressed it and its wild-type counterpart, LEN, in COS cells. While LEN is rapidly oxidized and subsequently secreted, newly synthesized SMA remains in the reduced state. Most SMA molecules are dislocated out of the ER into the cytosol, where they are ubiquitinylated and degraded by proteasomes. A parallel pathway for molecules that are not degraded is condensation into perinuclear aggresomes that are surrounded by vimentin-containing intermediate filaments and are dependent upon intact microtubules. Inhibition of proteasome activity shifts the balance toward aggresome formation. Intracellular aggregation is decreased and targeting to proteasomes improved by overexpression of the cytosolic chaperone Hsp70. Importantly, transduction into the cell of an Hsp70 target peptide, derived from the LC sequence, also reduces aggresome formation and increases SMA degradation. These results demonstrate that an amyloidogenic LC can aggregate intracellularly despite the common presentation of extracellular aggregates, and that a similar molecular surface mediates both in vitro fibril formation and in vivo aggregation. Furthermore, rationally designed peptides can be used to suppress this aggregation and may provide a feasible therapeutic approach.

  16. Discovery of Marinopyrrole A (Maritoclax) as a Selective Mcl-1 Antagonist that Overcomes ABT-737 Resistance by Binding to and Targeting Mcl-1 for Proteasomal Degradation*

    PubMed Central

    Doi, Kenichiro; Li, Rongshi; Sung, Shen-Shu; Wu, Hongwei; Liu, Yan; Manieri, Wanda; Krishnegowda, Gowdahalli; Awwad, Andy; Dewey, Alden; Liu, Xin; Amin, Shantu; Cheng, Chunwei; Qin, Yong; Schonbrunn, Ernst; Daughdrill, Gary; Loughran, Thomas P.; Sebti, Said; Wang, Hong-Gang

    2012-01-01

    The anti-apoptotic Bcl-2 family of proteins, including Bcl-2, Bcl-XL and Mcl-1, are well-validated drug targets for cancer treatment. Several small molecules have been designed to interfere with Bcl-2 and its fellow pro-survival family members. While ABT-737 and its orally active analog ABT-263 are the most potent and specific inhibitors to date that bind Bcl-2 and Bcl-XL with high affinity but have a much lower affinity for Mcl-1, they are not very effective as single agents in certain cancer types because of elevated levels of Mcl-1. Accordingly, compounds that specifically target Mcl-1 may overcome this resistance. In this study, we identified and characterized the natural product marinopyrrole A as a novel Mcl-1-specific inhibitor and named it maritoclax. We found that maritoclax binds to Mcl-1, but not Bcl-XL, and is able to disrupt the interaction between Bim and Mcl-1. Moreover, maritoclax induces Mcl-1 degradation via the proteasome system, which is associated with the pro-apoptotic activity of maritoclax. Importantly, maritoclax selectively kills Mcl-1-dependent, but not Bcl-2- or Bcl-XL-dependent, leukemia cells and markedly enhances the efficacy of ABT-737 against hematologic malignancies, including K562, Raji, and multidrug-resistant HL60/VCR, by ∼60- to 2000-fold at 1–2 μm. Taken together, these results suggest that maritoclax represents a new class of Mcl-1 inhibitors, which antagonizes Mcl-1 and overcomes ABT-737 resistance by targeting Mcl-1 for degradation. PMID:22311987

  17. Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE: The Case for a Combined Approach.

    PubMed

    Panfair, Dilrajkaur; Kusmierczyk, Andrew R

    2016-12-17

    Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is not completely understood. All proteasomes contain a structurally conserved core particle (CP), or 20S proteasome, containing two heptameric β subunit rings sandwiched between two heptameric α subunit rings. Archaeal 20S proteasomes are compositionally simpler compared to their eukaryotic counterparts, yet they both share a common assembly mechanism. Consequently, archaeal 20S proteasomes continue to be important models for eukaryotic proteasome assembly. Specifically, recombinant expression of archaeal 20S proteasomes coupled with nondenaturing polyacrylamide gel electrophoresis (PAGE) has yielded many important insights into proteasome biogenesis. Here, we discuss a means to improve upon the usual strategy of coexpression of archaeal proteasome α and β subunits prior to nondenaturing PAGE. We demonstrate that although rapid and efficient, a coexpression approach alone can miss key assembly intermediates. In the case of the proteasome, coexpression may not allow detection of the half-proteasome, an intermediate containing one complete α-ring and one complete β-ring. However, this intermediate is readily detected via lysate mixing. We suggest that combining coexpression with lysate mixing yields an approach that is more thorough in analyzing assembly, yet remains labor nonintensive. This approach may be useful for the study of other recombinant multiprotein complexes.

  18. Transcription Factor AP-2α Is Preferentially Cleaved by Caspase 6 and Degraded by Proteasome during Tumor Necrosis Factor Alpha-Induced Apoptosis in Breast Cancer Cells

    PubMed Central

    Nyormoi, Okot; Wang, Zhi; Doan, Dao; Ruiz, Maribelis; McConkey, David; Bar-Eli, Menashe

    2001-01-01

    Several reports have linked activating protein 2α (AP-2α) to apoptosis, leading us to hypothesize that AP-2α is a substrate for caspases. We tested this hypothesis by examining the effects of tumor necrosis factor alpha (TNF-α) on the expression of AP-2 in breast cancer cells. Here, we provide evidence that TNF-α downregulates AP-2α and AP-2γ expression posttranscriptionally during TNF-α-induced apoptosis. Both a general caspase antagonist (zVADfmk) and a caspase 6-preferred antagonist (zVEIDfmk) inhibited TNF-α-induced apoptosis and AP-2α downregulation. In vivo tests showed that AP-2α was cleaved by caspases ahead of the DNA fragmentation phase of apoptosis. Recombinant caspase 6 cleaved AP-2α preferentially, although caspases 1 and 3 also cleaved it, albeit at 50-fold or higher concentrations. Activated caspase 6 was detected in TNF-α-treated cells, thus confirming its involvement in AP-2α cleavage. All three caspases cleaved AP-2α at asp19 of the sequence asp-arg-his-asp (DRHD19). Mutating D19 to A19 abrogated AP-2α cleavage by all three caspases. TNF-α-induced cleavage of AP-2α in vivo led to AP-2α degradation and loss of DNA-binding activity, both of which were prevented by pretreatment with zVEIDfmk. AP-2α degradation but not cleavage was inhibited in vivo by PS-431 (a proteasome antagonist), suggesting that AP-2α is degraded subsequent to cleavage by caspase 6 or caspase 6-like enzymes. Cells transfected with green fluorescent protein-tagged mutant AP-2α are resistant to TNF-α-induced apoptosis, further demonstrating the link between caspase-mediated cleavage of AP-2α and apoptosis. This is the first report to demonstrate that degradation of AP-2α is a critical event in TNF-α-induced apoptosis. Since the DRHD sequence in vertebrate AP-2 is widely conserved, its cleavage by caspases may represent an important mechanism for regulating cell survival, proliferation, differentiation, and apoptosis. PMID:11438643

  19. Role of Ubiquitin-Mediated Degradation System in Plant Biology.

    PubMed

    Sharma, Bhaskar; Joshi, Deepti; Yadav, Pawan K; Gupta, Aditya K; Bhatt, Tarun K

    2016-01-01

    Ubiquitin-mediated proteasomal degradation is an important mechanism to control protein load in the cells. Ubiquitin binds to a protein on lysine residue and usually promotes its degradation through 26S proteasome system. Abnormal proteins and regulators of many processes, are targeted for degradation by the ubiquitin-proteasome system. It allows cells to maintain the response to cellular level signals and altered environmental conditions. The ubiquitin-mediated proteasomal degradation system plays a key role in the plant biology, including abiotic stress, immunity, and hormonal signaling by interfering with key components of these pathways. The involvement of the ubiquitin system in many vital processes led scientists to explore more about the ubiquitin machinery and most importantly its targets. In this review, we have summarized recent discoveries of the plant ubiquitin system and its involvement in critical processes of plant biology.

  20. Proteasome functional insufficiency in cardiac pathogenesis

    PubMed Central

    Li, Jie; Zheng, Hanqiao; Su, Huabo; Powell, Saul R.

    2011-01-01

    The ubiquitin-proteasome system (UPS) is responsible for the degradation of most cellular proteins. Alterations in cardiac UPS, including changes in the degradation of regulatory proteins and proteasome functional insufficiency, are observed in many forms of heart disease and have been shown to play an important role in cardiac pathogenesis. In the past several years, remarkable progress in understanding the mechanisms that regulate UPS-mediated protein degradation has been achieved. A transgenic mouse model of benign enhancement of cardiac proteasome proteolytic function has been created. This has led to the first demonstration of the necessity of proteasome functional insufficiency in the genesis of important pathological processes. Cardiomyocyte-restricted enhancement of proteasome proteolytic function by overexpression of proteasome activator 28α protects against cardiac proteinopathy and myocardial ischemia-reperfusion injury. Additionally, exciting advances have recently been achieved in the search for a pharmacological agent to activate the proteasome. These breakthroughs are expected to serve as an impetus to further investigation into the involvement of UPS dysfunction in molecular pathogenesis and to the development of new therapeutic strategies for combating heart disease. An interplay between the UPS and macroautophagy is increasingly suggested in noncardiac systems but is not well understood in the cardiac system. Further investigations into the interplay are expected to provide a more comprehensive picture of cardiac protein quality control and degradation. PMID:21949118

  1. Anaphase promoting complex-dependent degradation of transcriptional repressors Nrm1 and Yhp1 in Saccharomyces cerevisiae.

    PubMed

    Ostapenko, Denis; Solomon, Mark J

    2011-07-01

    The anaphase-promoting complex/cyclosome (APC/C) is an essential ubiquitin ligase that targets cell cycle proteins for proteasome-mediated degradation in mitosis and G1. The APC regulates a number of cell cycle processes, including spindle assembly, mitotic exit, and cytokinesis, but the full range of its functions is still unknown. To better understand cellular pathways controlled by the APC, we performed a proteomic screen to identify additional APC substrates. We analyzed cell cycle-regulated proteins whose expression peaked during the period when other APC substrates were expressed. Subsequent analysis identified several proteins, including the transcriptional repressors Nrm1 and Yhp1, as authentic APC substrates. We found that APC(Cdh1) targeted Nrm1 and Yhp1 for degradation in early G1 through Destruction-box motifs and that the degradation of these repressors coincided with transcriptional activation of MBF and Mcm1 target genes, respectively. In addition, Nrm1 was stabilized by phosphorylation, most likely by the budding yeast cyclin-dependent protein kinase, Cdc28. We found that expression of stabilized forms of Nrm1 and Yhp1 resulted in reduced cell fitness, due at least in part to incomplete activation of G1-specific genes. Therefore, in addition to its known functions, APC-mediated targeting of Nrm1 and Yhp1 coordinates transcription of multiple genes in G1 with other cell cycle events.

  2. The 11S Proteasomal Activator REGγ Impacts Polyglutamine-Expanded Androgen Receptor Aggregation and Motor Neuron Viability through Distinct Mechanisms

    PubMed Central

    Yersak, Jill M.; Montie, Heather L.; Chevalier-Larsen, Erica S.; Liu, Yuhong; Huang, Lan; Rechsteiner, Martin; Merry, Diane E.

    2017-01-01

    Spinal and bulbar muscular atrophy (SBMA) is caused by expression of a polyglutamine (polyQ)-expanded androgen receptor (AR). The inefficient nuclear proteasomal degradation of the mutant AR results in the formation of nuclear inclusions containing amino-terminal fragments of the mutant AR. PA28γ (also referred to as REGγ) is a nuclear 11S-proteasomal activator with limited proteasome activation capabilities compared to its cytoplasmic 11S (PA28α, PA28β) counterparts. To clarify the role of REGγ in polyQ-expanded AR metabolism, we carried out genetic and biochemical studies in cell models of SBMA. Overexpression of REGγ in a PC12 cell model of SBMA increased polyQ-expanded AR aggregation and contributed to polyQ-expanded AR toxicity in the presence of dihydrotestosterone (DHT). These effects of REGγ were independent of its association with the proteasome and may be due, in part, to the decreased binding of polyQ-expanded AR by the E3 ubiquitin-ligase MDM2. Unlike its effects in PC12 cells, REGγ overexpression rescued transgenic SBMA motor neurons from DHT-induced toxicity in a proteasome binding-dependent manner, suggesting that the degradation of a specific 11S proteasome substrate or substrates promotes motor neuron viability. One potential substrate that we found to play a role in mutant AR toxicity is the splicing factor SC35. These studies reveal that, depending on the cellular context, two biological roles for REGγ impact cell viability in the face of polyQ-expanded AR; a proteasome binding-independent mechanism directly promotes mutant AR aggregation while a proteasome binding-dependent mechanism promotes cell viability. The balance between these functions likely determines REGγ effects on polyQ-expanded AR-expressing cells. PMID:28596723

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

    PubMed

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

    2015-03-01

    RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92(pol) replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role

  4. Modeling proteasome dynamics in Parkinson's disease

    NASA Astrophysics Data System (ADS)

    Sneppen, Kim; Lizana, Ludvig; Jensen, Mogens H.; Pigolotti, Simone; Otzen, Daniel

    2009-09-01

    In Parkinson's disease (PD), there is evidence that α-synuclein (αSN) aggregation is coupled to dysfunctional or overburdened protein quality control systems, in particular the ubiquitin-proteasome system. Here, we develop a simple dynamical model for the on-going conflict between αSN aggregation and the maintenance of a functional proteasome in the healthy cell, based on the premise that proteasomal activity can be titrated out by mature αSN fibrils and their protofilament precursors. In the presence of excess proteasomes the cell easily maintains homeostasis. However, when the ratio between the available proteasome and the αSN protofilaments is reduced below a threshold level, we predict a collapse of homeostasis and onset of oscillations in the proteasome concentration. Depleted proteasome opens for accumulation of oligomers. Our analysis suggests that the onset of PD is associated with a proteasome population that becomes occupied in periodic degradation of aggregates. This behavior is found to be the general state of a proteasome/chaperone system under pressure, and suggests new interpretations of other diseases where protein aggregation could stress elements of the protein quality control system.

  5. Proteasome Modulates Mitochondrial Function During Cellular Senescence

    PubMed Central

    Torres, Claudio A.; Perez, Viviana I.

    2009-01-01

    Proteasome plays fundamental roles in the removal of oxidized proteins and in the normal degradation of short-lived proteins. Previously we have provided evidences that the impairment in proteasome observed during the replicative senescence of human fibroblasts has significant effects on MAPK signaling, proliferation, life span, senescent phenotype and protein oxidative status. These studies have demonstrated that proteasome inhibition and replicative senescence caused accumulation of intracellular protein carbonyl content. In this study, we have investigated the mechanisms by which proteasome dysfunction modulates protein oxidation during cellular senescence. The results indicate that proteasome inhibition during replicative senescence have significant effects on the intra and extracellular ROS production in vitro. The data also show that ROS impaired the proteasome function, which is partially reversible by antioxidants. Increases in ROS after proteasome inhibition correlated with a significant negative effect on the activity of most mitochondrial electron transporters. We propose that failures in proteasome during cellular senescence lead to mitochondrial dysfunction, ROS production and oxidative stress. Furthermore, it is likely that changes in proteasome dynamics could generate a pro-oxidative condition at the immediate extracellular microenvironment that could cause tissue injury during aging, in vivo. PMID:17976388

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

    PubMed

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

    2009-01-01

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

  7. HSV-2 glycoprotein gD targets the CC domain of tetherin and promotes tetherin degradation via lysosomal pathway.

    PubMed

    Liu, Yalan; Li, Mei; Zhang, Di; Zhang, Mudan; Hu, Qinxue

    2016-09-15

    HSV-2 is the major cause of genital herpes. We previously demonstrated that the host viral restriction factor tetherin restricts HSV-2 release and is antagonized by several HSV-2 glycoproteins. However, the mechanisms underlying HSV-2 glycoproteins mediated counteraction of tetherin remain unclear. In this study, we investigated whether tetherin restricts the cell-to-cell spread of HSV-2 and the mechanisms underlying HSV-2 gD mediated antagonism of tetherin. Infectious center assays were used to test whether tetherin could affect cell-to-cell spread of HSV-2. Coimmunoprecipitation assays were performed to map the tetherin domains required for HSV-2 gD-mediated downregulation. Immunoflurence assays were performed to detect the accumulation of tetherin in lysosomes or proteasomes. All experiments were repeated for at least three times and the data were performed statistical analysis. 1) Tetherin restricts cell-to-cell spread of HSV-2; 2) HSV-2 gD specifically interacts with the CC domain of tetherin; 3) HSV-2 gD promotes tetherin to the lysosomal degradation pathway. Tetherin not only restricts HSV-2 release but also its cell-to-cell spread. In turn, HSV-2 gD targets the CC domain of tetherin and promotes its degradation in the lysosome. Findings in this study have increased our understanding of tetherin restriction and viral countermeasures.

  8. Differential regulation of the REGγ–proteasome pathway by p53/TGF-β signalling and mutant p53 in cancer cells

    PubMed Central

    Ali, Amjad; Wang, Zhuo; Fu, Junjiang; Ji, Lei; Liu, Jiang; Li, Lei; Wang, Hui; Chen, Jiwu; Caulin, Carlos; Myers, Jeffrey N.; Zhang, Pei; Xiao, Jianru; Zhang, Bianhong; Li, Xiaotao

    2013-01-01

    Proteasome activity is frequently enhanced in cancer to accelerate metastasis and tumorigenesis. REGγ, a proteasome activator known to promote p53/p21/p16 degradation, is often overexpressed in cancer cells. Here we show that p53/TGF-β signalling inhibits the REGγ–20S proteasome pathway by repressing REGγ expression. Smad3 and p53 interact on the REGγ promoter via the p53RE/SBE region. Conversely, mutant p53 binds to the REGγ promoter and recruits p300. Importantly, mutant p53 prevents Smad3/N-CoR complex formation on the REGγ promoter, which enhances the activity of the REGγ–20S proteasome pathway and contributes to mutant p53 gain of function. Depletion of REGγ alters the cellular response to p53/TGF-β signalling in drug resistance, proliferation, cell cycle progression and proteasome activity. Moreover, p53 mutations show a positive correlation with REGγ expression in cancer samples. These findings suggest that targeting REGγ–20S proteasome for cancer therapy may be applicable to human tumours with abnormal p53/Smad protein status. Furthermore, this study demonstrates a link between p53/TGF-β signalling and the REGγ–20S proteasome pathway, and provides insight into the REGγ/p53 feedback loop. PMID:24157709

  9. Rab2 promotes autophagic and endocytic lysosomal degradation.

    PubMed

    Lőrincz, Péter; Tóth, Sarolta; Benkő, Péter; Lakatos, Zsolt; Boda, Attila; Glatz, Gábor; Zobel, Martina; Bisi, Sara; Hegedűs, Krisztina; Takáts, Szabolcs; Scita, Giorgio; Juhász, Gábor

    2017-07-03

    Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes. © 2017 Lőrincz et al.

  10. Green tea polyphenol EGCG suppresses Wnt/β-catenin signaling by promoting GSK-3β- and PP2A-independent β-catenin phosphorylation/degradation

    PubMed Central

    Oh, Sangtaek; Gwak, Jungsug; Park, Seoyoung; Yang, Chung S.

    2014-01-01

    (−)-Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been reported to inhibit the Wnt/β-catenin pathway, which is aberrantly up-regulated in colorectal cancers, but its precise mechanism of action remains unclear. Here, we used a sensitive cell-based system to demonstrate that EGCG suppresses β-catenin response transcription (CRT), activated by Wnt3a-conditioned medium (Wnt3a-CM), by promoting the degradation of intracellular β-catenin. EGCG induced β-catenin N-terminal phosphorylation at the Ser33/37 residues and subsequently promoted its degradation; however, this effect was not observed for oncogenic forms of β-catenin. Pharmacological inhibition or depletion of glycogen synthase kinase-3β (GSK-3β) did not abrogate the EGCG-mediated β-catenin degradation. EGCG did not affect the activity and expression of protein phosphatase 2A (PP2A). Consistently, the phosphorylation and degradation of β-catenin was found in adenomatous polyposis coli (APC) mutated colon cancer cells after EGCG treatment. EGCG repressed the expression of cyclin D1 and c-myc, which are β-catenin/T-cell factor-dependent genes, and inhibited the proliferation of colon cancer cells. Our findings suggest that EGCG exerts its cancer-preventive or anticancer activity against colon cancer cells by promoting the phosphorylation and proteasomal degradation of β-catenin through a mechanism independent of the GSK-3β and PP2A. PMID:25352148

  11. The recognition of ubiquitinated proteins by the proteasome.

    PubMed

    Grice, Guinevere L; Nathan, James A

    2016-09-01

    The ability of ubiquitin to form up to eight different polyubiquitin chain linkages generates complexity within the ubiquitin proteasome system, and accounts for the diverse roles of ubiquitination within the cell. Understanding how each type of ubiquitin linkage is correctly interpreted by ubiquitin binding proteins provides important insights into the link between chain recognition and cellular fate. A major function of ubiquitination is to signal degradation of intracellular proteins by the 26S proteasome. Lysine-48 (K48) linked polyubiquitin chains are well established as the canonical signal for proteasomal degradation, but recent studies show a role for other ubiquitin linked chains in facilitating degradation by the 26S proteasome. Here, we review how different types of polyubiquitin linkage bind to ubiquitin receptors on the 26S proteasome, how they signal degradation and discuss the implications of ubiquitin chain linkage in regulating protein breakdown by the proteasome.

  12. Regulation of proteasome activity in health and disease

    PubMed Central

    Schmidt, Marion; Finley, Daniel

    2013-01-01

    The ubiquitin-proteasome system (UPS) is the primary selective degradation system in the nuclei and cytoplasm of eukaryotic cells, required for the turnover of myriad soluble proteins. The hundreds of factors that comprise the UPS include an enzymatic cascade that tags proteins for degradation via the covalent attachment of a poly-ubiquitin chain, and a large multimeric enzyme that degrades ubiquitinated proteins, the proteasome. Protein degradation by the UPS regulates many pathways and is a crucial component of the cellular proteostasis network. Dysfunction of the ubiquitination machinery or the proteolytic activity of the proteasome is associated with numerous human diseases. In this review we discuss the contributions of the proteasome to human pathology, describe mechanisms that regulate the proteolytic capacity of the proteasome, and discuss strategies to modulate proteasome function as a therapeutic approach to ameliorate diseases associated with altered UPS function. PMID:23994620

  13. Interferon-γ-induced p27KIP1 binds to and targets MYC for proteasome-mediated degradation

    PubMed Central

    Zakaria, Siti Mariam; Frings, Oliver; Fahlén, Sara; Nilsson, Helén; Goodwin, Jacob; von der Lehr, Natalie; Su, Yingtao; Lüscher, Bernhard; Castell, Alina; Larsson, Lars-Gunnar

    2016-01-01

    The Myc oncoprotein is tightly regulated at multiple levels including ubiquitin-mediated protein turnover. We recently demonstrated that inhibition of Cdk2-mediated phosphorylation of Myc at Ser-62 pharmacologically or through interferon (IFN)-γ-induced expression of p27Kip1 (p27) repressed Myc's activity to suppress cellular senescence and differentiation. In this study we identified an additional activity of p27 to interfere with Myc independent of Ser-62 phosphorylation. p27 is required and sufficient for IFN-γ-induced turnover of Myc. p27 interacted with Myc in the nucleus involving the C-termini of the two proteins, including Myc box 4 of Myc. The C-terminus but not the Cdk2 binding fragment of p27 was sufficient for inducing Myc degradation. Protein expression data of The Cancer Genome Atlas breast invasive carcinoma set revealed significantly lower Myc protein levels in tumors with highly expressed p27 lacking phosphorylation at Thr-157 - a marker for active p27 localized in the nucleus. Further, these conditions correlated with favorable tumor stage and patient outcome. This novel regulation of Myc by IFN-γ/p27KIP1 potentially offers new possibilities for therapeutic intervention in tumors with deregulated Myc. PMID:26701207

  14. CHIP promotes Runx2 degradation and negatively regulates osteoblast differentiation

    PubMed Central

    Li, Xueni; Huang, Mei; Zheng, Huiling; Wang, Yinyin; Ren, Fangli; Shang, Yu; Zhai, Yonggong; Irwin, David M.; Shi, Yuguang; Chen, Di; Chang, Zhijie

    2008-01-01

    Runx2, an essential transactivator for osteoblast differentiation, is tightly regulated at both the transcriptional and posttranslational levels. In this paper, we report that CHIP (C terminus of Hsc70-interacting protein)/STUB1 regulates Runx2 protein stability via a ubiquitination-degradation mechanism. CHIP interacts with Runx2 in vitro and in vivo. In the presence of increased Runx2 protein levels, CHIP expression decreases, whereas the expression of other E3 ligases involved in Runx2 degradation, such as Smurf1 or WWP1, remains constant or increases during osteoblast differentiation. Depletion of CHIP results in the stabilization of Runx2, enhances Runx2-mediated transcriptional activation, and promotes osteoblast differentiation in primary calvarial cells. In contrast, CHIP overexpression in preosteoblasts causes Runx2 degradation, inhibits osteoblast differentiation, and instead enhances adipogenesis. Our data suggest that negative regulation of the Runx2 protein by CHIP is critical in the commitment of precursor cells to differentiate into the osteoblast lineage. PMID:18541707

  15. High glucose promotes Aβ production by inhibiting APP degradation.

    PubMed

    Yang, Yi; Wu, Yili; Zhang, Shuting; Song, Weihong

    2013-01-01

    Abnormal deposition of neuriticplaques is the uniqueneuropathological hallmark of Alzheimer's disease (AD).Amyloid β protein (Aβ), the major component of plaques, is generated from sequential cleavage of amyloidβ precursor protein (APP) by β-secretase and γ-secretase complex. Patients with diabetes mellitus (DM), characterized by chronic hyperglycemia,have increased risk of AD development.However, the role of high blood glucose in APP processing and Aβ generation remains elusive. In this study, we investigated the effect of high glucose on APP metabolism and Aβ generation in cultured human cells. We found that high glucose treatment significantly increased