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Sample records for 26s proteasome subunits

  1. Localization of the regulatory particle subunit Sem1 in the 26S proteasome

    SciTech Connect

    Bohn, Stefan; Sakata, Eri; Beck, Florian; Pathare, Ganesh R.; Schnitger, Jérôme; Nágy, Istvan; Baumeister, Wolfgang Förster, Friedrich

    2013-05-31

    Highlights: •26S proteasome subunit Sem1 was mapped using cryo-EM and cross-linking data. •C-terminal helix of Sem1 located near winged helix motif of Rpn7. •N-terminal part of Sem1 tethers Rpn7, Rpn3 and lid helical bundle. •Sem1 binds differently to PCI-domains of proteasome subunit Rpn7 and TREX-2 subunit Thp1. -- Abstract: The ubiquitin–proteasome system is responsible for regulated protein degradation in the cell with the 26S proteasome acting as its executive arm. The molecular architecture of this 2.5 MDa complex has been established recently, with the notable exception of the small acidic subunit Sem1. Here, we localize the C-terminal helix of Sem1 binding to the PCI domain of the subunit Rpn7 using cryo-electron microscopy single particle reconstruction of proteasomes purified from yeast cells with sem1 deletion. The approximate position of the N-terminal region of Sem1 bridging the cleft between Rpn7 and Rpn3 was inferred based on site-specific cross-linking data of the 26S proteasome. Our structural studies indicate that Sem1 can assume different conformations in different contexts, which supports the idea that Sem1 functions as a molecular glue stabilizing the Rpn3/Rpn7 heterodimer.

  2. Phosphorylation of 20S proteasome alpha subunit C8 (alpha7) stabilizes the 26S proteasome and plays a role in the regulation of proteasome complexes by gamma-interferon.

    PubMed Central

    Bose, Suchira; Stratford, Fiona L L; Broadfoot, Kerry I; Mason, Grant G F; Rivett, A Jennifer

    2004-01-01

    In animal cells there are several regulatory complexes which interact with 20S proteasomes and give rise to functionally distinct proteasome complexes. gamma-Interferon upregulates three immuno beta catalytic subunits of the 20S proteasome and the PA28 regulator, and decreases the level of 26S proteasomes. It also decreases the level of phosphorylation of two proteasome alpha subunits, C8 (alpha7) and C9 (alpha3). In the present study we have investigated the role of phosphorylation of C8 by protein kinase CK2 in the formation and stability of 26S proteasomes. An epitope-tagged C8 subunit expressed in mammalian cells was efficiently incorporated into both 20S proteasomes and 26S proteasomes. Investigation of mutants of C8 at the two known CK2 phosphorylation sites demonstrated that these are the two phosphorylation sites of C8 in animal cells. Although phosphorylation of C8 was not absolutely essential for the formation of 26S proteasomes, it did have a substantial effect on their stability. Also, when cells were treated with gamma-interferon, there was a marked decrease in phosphorylation of C8, a decrease in the level of 26S proteasomes, and an increase in immunoproteasomes and PA28 complexes. These results suggest that the down-regulation of 26S proteasomes after gamma-interferon treatment results from the destabilization that occurs after dephosphorylation of the C8 subunit. PMID:14583091

  3. Mouse homologue of yeast Prp19 interacts with mouse SUG1, the regulatory subunit of 26S proteasome

    SciTech Connect

    Sihn, Choong-Ryoul; Cho, Si Young; Lee, Jeong Ho; Lee, Tae Ryong; Kim, Sang Hoon . E-mail: shkim@khu.ac.kr

    2007-04-27

    Yeast Prp19 has been shown to involve in pre-mRNA splicing and DNA repair as well as being an ubiquitin ligase. Mammalian homologue of yeast Prp19 also plays on similar functional activities in cells. In the present study, we isolated mouse SUG1 (mSUG1) as binding partner of mouse Prp19 (mPrp19) by the yeast two-hybrid system. We confirmed the interaction of mPrp9 with mSUG1 by GST pull-down assay and co-immunoprecipitation assay. The N-terminus of mPrp19 including U-box domain was associated with the C-terminus of mSUG1. Although, mSUG1 is a regulatory subunit of 26S proteasome, mPrp19 was not degraded in the proteasome-dependent pathway. Interestingly, GFP-mPrp19 fusion protein was co-localized with mSUG1 protein in cytoplasm as the formation of the speckle-like structures in the presence of a proteasome inhibitor MG132. In addition, the activity of proteasome was increased in cells transfected with mPrp19. Taken together, these results suggest that mPrp19 involves the regulation of protein turnover and may transport its substrates to 26S proteasome through mSUG1 protein.

  4. N-Terminal Coiled-Coil Structure of ATPase Subunits of 26S Proteasome Is Crucial for Proteasome Function

    PubMed Central

    Inobe, Tomonao; Genmei, Reiko

    2015-01-01

    The proteasome is an essential proteolytic machine in eukaryotic cells, where it removes damaged proteins and regulates many cellular activities by degrading ubiquitinated proteins. Its heterohexameric AAA+ ATPase Rpt subunits play a central role in proteasome activity by the engagement of substrate unfolding and translocation for degradation; however, its detailed mechanism remains poorly understood. In contrast to AAA+ ATPase domains, their N-terminal regions of Rpt subunits substantially differ from each other. Here, to investigate the requirements and roles of the N-terminal regions of six Rpt subunits derived from Saccharomyces cerevisiae, we performed systematic mutational analysis using conditional knockdown yeast strains for each Rpt subunit and bacterial heterologous expression system of the base subcomplex. We showed that the formation of the coiled-coil structure was the most important for the N-terminal region of Rpt subunits. The primary role of coiled-coil structure would be the maintenance of the ring structure with the defined order. However, the coiled-coil region would be also be involved in substrate recognition and an interaction between lid and base subcomplexes. PMID:26208326

  5. Tomato 26S Proteasome subunit RPT4a regulates ToLCNDV transcription and activates hypersensitive response in tomato.

    PubMed

    Sahu, Pranav Pankaj; Sharma, Namisha; Puranik, Swati; Chakraborty, Supriya; Prasad, Manoj

    2016-01-01

    Involvement of 26S proteasomal subunits in plant pathogen-interactions, and the roles of each subunit in independently modulating the activity of many intra- and inter-cellular regulators controlling physiological and defense responses of a plant were well reported. In this regard, we aimed to functionally characterize a Solanum lycopersicum 26S proteasomal subunit RPT4a (SlRPT4) gene, which was differentially expressed after Tomato leaf curl New Delhi virus (ToLCNDV) infection in tolerant cultivar H-88-78-1. Molecular analysis revealed that SlRPT4 protein has an active ATPase activity. SlRPT4 could specifically bind to the stem-loop structure of intergenic region (IR), present in both DNA-A and DNA-B molecule of the bipartite viral genome. Lack of secondary structure in replication-associated gene fragment prevented formation of DNA-protein complex suggesting that binding of SlRPT4 with DNA is secondary structure specific. Interestingly, binding of SlRPT4 to IR inhibited the function of RNA Pol-II and subsequently reduced the bi-directional transcription of ToLCNDV genome. Virus-induced gene silencing of SlRPT4 gene incited conversion of tolerant attributes of cultivar H-88-78-1 into susceptibility. Furthermore, transient overexpression of SlRPT4 resulted in activation of programmed cell death and antioxidant enzymes system. Overall, present study highlights non-proteolytic function of SlRPT4 and their participation in defense pathway against virus infection in tomato. PMID:27252084

  6. Tomato 26S Proteasome subunit RPT4a regulates ToLCNDV transcription and activates hypersensitive response in tomato

    PubMed Central

    Sahu, Pranav Pankaj; Sharma, Namisha; Puranik, Swati; Chakraborty, Supriya; Prasad, Manoj

    2016-01-01

    Involvement of 26S proteasomal subunits in plant pathogen-interactions, and the roles of each subunit in independently modulating the activity of many intra- and inter-cellular regulators controlling physiological and defense responses of a plant were well reported. In this regard, we aimed to functionally characterize a Solanum lycopersicum 26S proteasomal subunit RPT4a (SlRPT4) gene, which was differentially expressed after Tomato leaf curl New Delhi virus (ToLCNDV) infection in tolerant cultivar H-88-78-1. Molecular analysis revealed that SlRPT4 protein has an active ATPase activity. SlRPT4 could specifically bind to the stem-loop structure of intergenic region (IR), present in both DNA-A and DNA-B molecule of the bipartite viral genome. Lack of secondary structure in replication-associated gene fragment prevented formation of DNA-protein complex suggesting that binding of SlRPT4 with DNA is secondary structure specific. Interestingly, binding of SlRPT4 to IR inhibited the function of RNA Pol-II and subsequently reduced the bi-directional transcription of ToLCNDV genome. Virus-induced gene silencing of SlRPT4 gene incited conversion of tolerant attributes of cultivar H-88-78-1 into susceptibility. Furthermore, transient overexpression of SlRPT4 resulted in activation of programmed cell death and antioxidant enzymes system. Overall, present study highlights non-proteolytic function of SlRPT4 and their participation in defense pathway against virus infection in tomato. PMID:27252084

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

    PubMed Central

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

    2015-01-01

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

  8. Glucocorticoids regulate mRNA levels for subunits of the 19 S regulatory complex of the 26 S proteasome in fast-twitch skeletal muscles.

    PubMed Central

    Combaret, Lydie; Taillandier, Daniel; Dardevet, Dominique; Béchet, Daniel; Rallière, Cécile; Claustre, Agnès; Grizard, Jean; Attaix, Didier

    2004-01-01

    Circulating levels of glucocorticoids are increased in many traumatic and muscle-wasting conditions that include insulin-dependent diabetes, acidosis, infection, and starvation. On the basis of indirect findings, it appeared that these catabolic hormones are required to stimulate Ub (ubiquitin)-proteasome-dependent proteolysis in skeletal muscles in such conditions. The present studies were performed to provide conclusive evidence for an activation of Ub-proteasome-dependent proteolysis after glucocorticoid treatment. In atrophying fast-twitch muscles from rats treated with dexamethasone for 6 days, compared with pair-fed controls, we found (i) increased MG132-inhibitable proteasome-dependent proteolysis, (ii) an enhanced rate of substrate ubiquitination, (iii) increased chymotrypsin-like proteasomal activity of the proteasome, and (iv) a co-ordinate increase in the mRNA expression of several ATPase (S4, S6, S7 and S8) and non-ATPase (S1, S5a and S14) subunits of the 19 S regulatory complex, which regulates the peptidase and the proteolytic activities of the 26 S proteasome. These studies provide conclusive evidence that glucocorticoids activate Ub-proteasome-dependent proteolysis and the first in vivo evidence for a hormonal regulation of the expression of subunits of the 19 S complex. The results suggest that adaptations in gene expression of regulatory subunits of the 19 S complex by glucocorticoids are crucial in the regulation of the 26 S muscle proteasome. PMID:14636157

  9. The RPT2 subunit of the 26S proteasome directs complex assembly, histone dynamics, and gametophyte and sporophyte development in Arabidopsis.

    PubMed

    Lee, Kwang-Hee; Minami, Atsushi; Marshall, Richard S; Book, Adam J; Farmer, Lisa M; Walker, Joseph M; Vierstra, Richard D

    2011-12-01

    The regulatory particle (RP) of the 26S proteasome contains a heterohexameric ring of AAA-ATPases (RPT1-6) that unfolds and inserts substrates into the core protease (CP) for degradation. Through genetic analysis of the Arabidopsis thaliana gene pair encoding RPT2, we show that this subunit plays a critical role in 26S proteasome assembly, histone dynamics, and plant development. rpt2a rpt2b double null mutants are blocked in both male and female gamete transmission, demonstrating that the subunit is essential. Whereas rpt2b mutants are phenotypically normal, rpt2a mutants display a range of defects, including impaired leaf, root, trichome, and pollen development, delayed flowering, stem fasciation, hypersensitivity to mitomycin C and amino acid analogs, hyposensitivity to the proteasome inhibitor MG132, and decreased 26S complex stability. The rpt2a phenotype can be rescued by both RPT2a and RPT2b, indicative of functional redundancy, but not by RPT2a mutants altered in ATP binding/hydrolysis or missing the C-terminal hydrophobic sequence that docks the RPT ring onto the CP. Many rpt2a phenotypes are shared with mutants lacking the chromatin assembly factor complex CAF1. Like caf1 mutants, plants missing RPT2a or reduced in other RP subunits contain less histones, thus implicating RPT2 specifically, and the 26S proteasome generally, in plant nucleosome assembly. PMID:22158466

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

    SciTech Connect

    Lee, Boo-Ja; Kwon, Sun Jae; Kim, Sung-Kyu; Kim, Ki-Jeong; Park, Chang-Jin; Kim, Young-Jin; Park, Ohkmae K.; Paek, Kyung-Hee . E-mail: khpaek95@korea.ac.kr

    2006-12-15

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

  11. Gene Expression Analysis of the 26S Proteasome Subunit PSMB4 Reveals Significant Upregulation, Different Expression and Association with Proliferation in Human Pulmonary Neuroendocrine Tumours

    PubMed Central

    Mairinger, Fabian Dominik; Walter, Robert Fred Henry; Theegarten, Dirk; Hager, Thomas; Vollbrecht, Claudia; Christoph, Daniel Christian; Worm, Karl; Ting, Saskia; Werner, Robert; Stamatis, Georgios; Mairinger, Thomas; Baba, Hideo; Zarogoulidis, Konstantinos; Huang, Haidong; Li, Qiang; Tsakiridis, Kosmas; Zarogoulidis, Paul; Schmid, Kurt Werner; Wohlschlaeger, Jeremias

    2014-01-01

    Background: Proteasomal subunit PSMB4 was suggested to be a survival gene in an animal model of hepatocellular carcinoma and in glioblastoma cell lines. In pulmonary adenocarcinoma, a high expression of these genes was found to be associated with poor differentiation and survival. This study investigates the gene expression levels of 26S proteasome subunits in human pulmonary neuroendocrine tumours including typical (TC) and atypical (AC) carcinoid tumours as well as small cell (SCLC) and large cell (LCNEC) neuroendocrine carcinomas. Material and methods: Gene expression levels of proteasomal subunits (PSMA1, PSMA5, PSMB4, PSMB5 and PSMD1) were investigated in 80 neuroendocrine pulmonary tumours (each 20 TC, AC, LCNLC and SCLC) and compared to controls. mRNA levels were determined by using TaqMan assays. Immunohistochemistry on tissue microarrays (TMA) was performed to determine the expression of ki67, cleaved caspase 3 and PSMB4. Results: All proteasomal subunit gene expressions were significantly upregulated in TC, AC, SCLC and LCNEC compared to controls. PSMB4 mRNA is differently expressed between all neuroendocrine tumour subtypes demonstrating the highest expression and greatest range in LCNEC (p=0.043), and is significantly associated with proliferative activity (p=0.039). Conclusion: In line with other 26S proteasomal subunits PSMB4 is significantly increased, but differently expressed between pulmonary neuroendocrine tumours and is associated with the proliferative activity. Unlike in pulmonary adenocarcinomas, no association with biological behaviour was observed, suggesting that increased proteasomal subunit gene expression is a common and probably early event in the tumorigenesis of pulmonary neuroendocrine tumours regardless of their differentiation. PMID:25157275

  12. Nin1p, a regulatory subunit of the 26S proteasome, is necessary for activation of Cdc28p kinase of Saccharomyces cerevisiae.

    PubMed Central

    Kominami, K; DeMartino, G N; Moomaw, C R; Slaughter, C A; Shimbara, N; Fujimuro, M; Yokosawa, H; Hisamatsu, H; Tanahashi, N; Shimizu, Y

    1995-01-01

    The nin1-1 mutant of Saccharomyces cerevisiae cannot perform the G1/S and G2/M transitions at restrictive temperatures. At such temperatures, nin1-1 strains fail to activate histone H1 kinase after release from alpha factor-imposed G1 block and after release from hydroxyurea-imposed S block. The nin1-1 mutation shows synthetic lethality with certain cdc28 mutant alleles such as cdc28-IN. Two lines of evidence indicate that Nin1p is a component of the 26S proteasome complex: (i) Nin1p, as well as the known component of the 26S proteasome, shifted to the 26S proteasome peak in the glycerol density gradient after preincubation of crude extract with ATP-Mg2+, and (ii) nin1-1 cells accumulated polyubiquitinated proteins under restrictive conditions. These results suggest that activation of Cdc28p kinase requires proteolysis. We have cloned a human cDNA encoding a regulatory subunit of the 26S proteasome, p31, which was found to be a homolog of Nin1p. Images PMID:7621825

  13. Expression of the 26S proteasome subunit RPN10 is upregulated by salt stress in Dunaliella viridis.

    PubMed

    Sun, Xiaobin; Meng, Xiangzong; Xu, Zhengkai; Song, Rentao

    2010-08-15

    Green algae of the genus Dunaliella can adapt to hypersaline environments and are considered model organisms for salinity tolerance. In an EST analysis in Dunaliella viridis under salt stress, we isolated a salt-inducible cDNA coding for the 26S proteasome subunit RPN10, designated DvRPN10. The DvRPN10 cDNA is 1472 bp and encodes a polypeptide of 377 amino acids. The DvRPN10 protein shares a high similarity to orthologs from other species. The function of DvRPN10 was confirmed by complementation of the yeast Deltarpn10 mutant. Q-PCR analysis of D. viridis cells grown in different salinities revealed that the transcript level of DvRPN10 increased in proportion to the external salinity within a range of 0.5-3 M NaCl, but decreased significantly at extremely high salinities (4-5 M NaCl). When a salinity shock of 1-3 M NaCl was applied to D. viridis cells, DvRPN10 mRNA levels remained steady during the first 36 h, and then gradually elevated to the level observed at 3 M NaCl. The gene structure of DvRPN10 was revealed by sequencing of a BAC clone containing this gene. Possible transcription factor binding sites related to stress tolerance were found in the promoter region of DvRPN10. The expression of DvRPN10 in response to the external salinity suggests that RPN10-mediated protein degradation plays a role in the salinity tolerance of D. viridis. PMID:20430475

  14. CDNA cloning of p112, the largest regulatory subunit of the human 26s proteasome, and functional analysis of its yeast homologue, sen3p.

    PubMed Central

    Yokota, K; Kagawa, S; Shimizu, Y; Akioka, H; Tsurumi, C; Noda, C; Fujimuro, M; Yokosawa, H; Fujiwara, T; Takahashi, E; Ohba, M; Yamasaki, M; DeMartino, G N; Slaughter, C A; Toh-e, A; Tanaka, K

    1996-01-01

    The 26S proteasome is a large multisubunit protease complex, the largest regulatory subunit of which is a component named p112. Molecular cloning of cDNA encoding human p112 revealed a polypeptide predicted to have 953 amino acid residues and a molecular mass of 105,865. The human p112 gene was mapped to the q37.1-q37.2 region of chromosome 2. Computer analysis showed that p112 has strong similarity to the Saccharomyces cerevisiae Sen3p, which has been listed in a gene bank as a factor affecting tRNA splicing endonuclease. The SEN3 also was identified in a synthetic lethal screen with the nin1-1 mutant, a temperature-sensitive mutant of NIN1. NIN1 encodes p31, another regulatory subunit of the 26S proteasome, which is necessary for activation of Cdc28p kinase. Disruption of the SEN3 did not affect cell viability, but led to temperature-sensitive growth. The human p112 cDNA suppressed the growth defect at high temperature in a SEN3 disruptant, indicating that p112 is a functional homologue of the yeast Sen3p. Maintenance of SEN3 disruptant cells at the restrictive temperature resulted in a variety of cellular dysfunctions, including defects in proteolysis mediated by the ubiquitin pathway, in the N-end rule system, in the stress response upon cadmium exposure, and in nuclear protein transportation. The functional abnormality induced by SEN3 disruption differs considerably from various phenotypes shown by the nin1-1 mutation, suggesting that these two regulatory subunits of the 26S proteasome play distinct roles in the various processes mediated by the 26S proteasome. Images PMID:8816993

  15. Induction of 26S proteasome subunit PSMB5 by the bifunctional inducer 3-methylcholanthrene through the Nrf2-ARE, but not the AhR/Arnt-XRE, pathway

    SciTech Connect

    Kwak, Mi-Kyoung . E-mail: mkwak@yumail.ac.kr; Kensler, Thomas W.

    2006-07-14

    The 26S proteasome is responsible for degradation of abnormal intracellular proteins, including oxidatively damaged proteins and may play a role as a component of a cellular antioxidative system. However, little is known about regulation of proteasome expression. In the present study, regulation of proteasome expression by the bifunctional enzyme inducer and a specific signaling pathway for this regulation were investigated in murine neuroblastoma cells. Expression of catalytic core subunits including PSMB5 and peptidase activities of the proteasome were elevated following incubation with 3-methylcholanthrene (3-MC). Studies using reporter genes containing the murine Psmb5 promoter showed that transcriptional activity of this gene was enhanced by 3-MC. Overexpression of AhR/Arnt did not affect activation of the Pmsb5 promoter by 3-MC and deletion of the xenobiotic response elements (XREs) from this promoter exerted modest effects on inducibility in response to 3-MC. However, mutation of the proximal AREs of the Psmb5 promoter largely abrogated its inducibility by 3-MC. In addition, this promoter showed a blunted response toward 3-MC in the absence of nrf2; 3-MC incubation increased nuclear levels of Nrf2 only in wild-type cells. Collectively, these results indicate that expression of proteasome subunit PSMB5 is modulated by bifunctional enzyme inducers in a manner independent of the AhR/Arnt-XRE pathway but dependent upon the Nrf2-ARE pathway.

  16. Characterization of the 26S proteasome network in Plasmodium falciparum

    PubMed Central

    Wang, Lihui; Delahunty, Claire; Fritz-Wolf, Karin; Rahlfs, Stefan; Helena Prieto, Judith; Yates, John R.; Becker, Katja

    2015-01-01

    In eukaryotic cells, the ubiquitin-proteasome system as a key regulator of protein quality control is an excellent drug target. We therefore aimed to analyze the 26S proteasome complex in the malaria parasite Plasmodium falciparum, which still threatens almost half of the world’s population. First, we established an affinity purification protocol allowing for the isolation of functional 26S proteasome complexes from the parasite. Subunit composition of the proteasome and component stoichiometry were studied and physiologic interacting partners were identified via in situ protein crosslinking. Furthermore, intrinsic ubiquitin receptors of the plasmodial proteasome were determined and their roles in proteasomal substrate recognition were analyzed. Notably, PfUSP14 was characterized as a proteasome-associated deubiquitinase resulting in the concept that targeting proteasomal deubiquitinating activity in P. falciparum may represent a promising antimalarial strategy. The data provide insights into a profound network orchestrated by the plasmodial proteasome and identified novel drug target candidates in the ubiquitin-proteasome system. PMID:26639022

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

    PubMed Central

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

    2000-01-01

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

  18. Structure-Driven Developments of 26S Proteasome Inhibitors.

    PubMed

    Śledź, Paweł; Baumeister, Wolfgang

    2016-01-01

    The 26S proteasome is a 2.5-MDa complex, and it operates at the executive end of the ubiquitin-proteasome pathway. It is a proven target for therapeutic agents for the treatment of some cancers and autoimmune diseases, and moreover, it has potential as a target of antibacterial agents. Most inhibitors, including all molecules approved for clinical use, target the 20S proteolytic core complex; its structure was determined two decades ago. Hitherto, efforts to develop inhibitors targeting the 19S regulatory particle subunits have been less successful. This is, in part, because the molecular architecture of this subcomplex has been, until recently, poorly understood, and high-resolution structures have been available only for a few subunits. In this review, we describe, from a structural perspective, the development of inhibitory molecules that target both the 20S and 19S subunits of the proteasome. We highlight the recent progress achieved in structure-based drug-discovery approaches, and we discuss the prospects for further improvement. PMID:26738474

  19. Inherent Asymmetry in the 26S Proteasome Is Defined by the Ubiquitin Receptor RPN13*

    PubMed Central

    Berko, Dikla; Herkon, Ora; Braunstein, Ilana; Isakov, Elada; David, Yael; Ziv, Tamar; Navon, Ami; Stanhill, Ariel

    2014-01-01

    The 26S double-capped proteasome is assembled in a hierarchic event that is orchestrated by dedicated set of chaperons. To date, all stoichiometric subunits are considered to be present in equal ratios, thus providing symmetry to the double-capped complex. Here, we show that although the vast majority (if not all) of the double-capped 26S proteasomes, both 19S complexes, contain the ubiquitin receptor Rpn10/S5a, only one of these 19S particles contains the additional ubiquitin receptor Rpn13, thereby defining asymmetry in the 26S proteasome. These results were validated in yeast and mammals, utilizing biochemical and unbiased AQUA-MS methodologies. Thus, the double-capped 26S proteasomes are asymmetric in their polyubiquitin binding capacity. Our data point to a potential new role for ubiquitin receptors as directionality factors that may participate in the prevention of simultaneous substrates translocation into the 20S from both 19S caps. PMID:24429290

  20. D1 dopamine receptor stimulation impairs striatal proteasome activity in Parkinsonism through 26S proteasome disassembly.

    PubMed

    Barroso-Chinea, Pedro; Thiolat, Marie-Laure; Bido, Simone; Martinez, Audrey; Doudnikoff, Evelyne; Baufreton, Jérôme; Bourdenx, Mathieu; Bloch, Bertrand; Bezard, Erwan; Martin-Negrier, Marie-Laure

    2015-06-01

    Among the mechanisms underlying the development of L-dopa-induced dyskinesia (LID) in Parkinson's disease, complex alterations in dopamine signaling in D1 receptor (D1R)-expressing medium spiny striatal neurons have been unraveled such as, but not limited to, dysregulation of D1R expression, lateral diffusion, intraneuronal trafficking, subcellular localization and desensitization, leading to a pathological anchorage of D1R at the plasma membrane. Such anchorage is partly due to a decreased proteasomal activity that is specific of the L-dopa-exposed dopamine-depleted striatum, results from D1R activation and feeds-back the D1R exaggerated cell surface abundance. The precise mechanisms by which L-dopa affects striatal proteasome activity remained however unknown. We here show, in a series of in vitro ex vivo and in vivo models, that such rapid modulation of striatal proteasome activity intervenes through D1R-mediated disassembly of the 26S proteasome rather than change in transcription or translation of proteasome or proteasome subunits intraneuronal relocalization. PMID:25766677

  1. Structure of the human 26S proteasome at a resolution of 3.9 Å.

    PubMed

    Schweitzer, Andreas; Aufderheide, Antje; Rudack, Till; Beck, Florian; Pfeifer, Günter; Plitzko, Jürgen M; Sakata, Eri; Schulten, Klaus; Förster, Friedrich; Baumeister, Wolfgang

    2016-07-12

    Protein degradation in eukaryotic cells is performed by the Ubiquitin-Proteasome System (UPS). The 26S proteasome holocomplex consists of a core particle (CP) that proteolytically degrades polyubiquitylated proteins, and a regulatory particle (RP) containing the AAA-ATPase module. This module controls access to the proteolytic chamber inside the CP and is surrounded by non-ATPase subunits (Rpns) that recognize substrates and deubiquitylate them before unfolding and degradation. The architecture of the 26S holocomplex is highly conserved between yeast and humans. The structure of the human 26S holocomplex described here reveals previously unidentified features of the AAA-ATPase heterohexamer. One subunit, Rpt6, has ADP bound, whereas the other five have ATP in their binding pockets. Rpt6 is structurally distinct from the other five Rpt subunits, most notably in its pore loop region. For Rpns, the map reveals two main, previously undetected, features: the C terminus of Rpn3 protrudes into the mouth of the ATPase ring; and Rpn1 and Rpn2, the largest proteasome subunits, are linked by an extended connection. The structural features of the 26S proteasome observed in this study are likely to be important for coordinating the proteasomal subunits during substrate processing. PMID:27342858

  2. Structure of the human 26S proteasome at a resolution of 3.9 Å

    PubMed Central

    Schweitzer, Andreas; Aufderheide, Antje; Rudack, Till; Beck, Florian; Pfeifer, Günter; Plitzko, Jürgen M.; Sakata, Eri; Schulten, Klaus; Förster, Friedrich; Baumeister, Wolfgang

    2016-01-01

    Protein degradation in eukaryotic cells is performed by the Ubiquitin-Proteasome System (UPS). The 26S proteasome holocomplex consists of a core particle (CP) that proteolytically degrades polyubiquitylated proteins, and a regulatory particle (RP) containing the AAA-ATPase module. This module controls access to the proteolytic chamber inside the CP and is surrounded by non-ATPase subunits (Rpns) that recognize substrates and deubiquitylate them before unfolding and degradation. The architecture of the 26S holocomplex is highly conserved between yeast and humans. The structure of the human 26S holocomplex described here reveals previously unidentified features of the AAA-ATPase heterohexamer. One subunit, Rpt6, has ADP bound, whereas the other five have ATP in their binding pockets. Rpt6 is structurally distinct from the other five Rpt subunits, most notably in its pore loop region. For Rpns, the map reveals two main, previously undetected, features: the C terminus of Rpn3 protrudes into the mouth of the ATPase ring; and Rpn1 and Rpn2, the largest proteasome subunits, are linked by an extended connection. The structural features of the 26S proteasome observed in this study are likely to be important for coordinating the proteasomal subunits during substrate processing. PMID:27342858

  3. 26S Proteasome: Hunter and Prey in Auxin Signaling.

    PubMed

    Kong, Xiangpei; Zhang, Liangran; Ding, Zhaojun

    2016-07-01

    Auxin binds to TRANSPORT INHIBITOR RESPONSE 1 and AUXIN SIGNALLING F-BOX proteins (TIR1/AFBs) and promotes the degradation of Aux/IAA transcriptional repressors. The proteasome regulator PROTEASOME REGULATOR1 (PTRE1) has now been shown to be required for auxin-mediated repression of 26S proteasome activity, thus providing new insights into the fine-tuning of the homoeostasis of Aux/IAA proteins and auxin signaling. PMID:27246455

  4. Autoubiquitination of the 26S proteasome on Rpn13 regulates breakdown of ubiquitin conjugates.

    PubMed

    Besche, Henrike C; Sha, Zhe; Kukushkin, Nikolay V; Peth, Andreas; Hock, Eva-Maria; Kim, Woong; Gygi, Steven; Gutierrez, Juan A; Liao, Hua; Dick, Lawrence; Goldberg, Alfred L

    2014-05-16

    Degradation rates of most proteins in eukaryotic cells are determined by their rates of ubiquitination. However, possible regulation of the proteasome's capacity to degrade ubiquitinated proteins has received little attention, although proteasome inhibitors are widely used in research and cancer treatment. We show here that mammalian 26S proteasomes have five associated ubiquitin ligases and that multiple proteasome subunits are ubiquitinated in cells, especially the ubiquitin receptor subunit, Rpn13. When proteolysis is even partially inhibited in cells or purified 26S proteasomes with various inhibitors, Rpn13 becomes extensively and selectively poly-ubiquitinated by the proteasome-associated ubiquitin ligase, Ube3c/Hul5. This modification also occurs in cells during heat-shock or arsenite treatment, when poly-ubiquitinated proteins accumulate. Rpn13 ubiquitination strongly decreases the proteasome's ability to bind and degrade ubiquitin-conjugated proteins, but not its activity against peptide substrates. This autoinhibitory mechanism presumably evolved to prevent binding of ubiquitin conjugates to defective or stalled proteasomes, but this modification may also be useful as a biomarker indicating the presence of proteotoxic stress and reduced proteasomal capacity in cells or patients. PMID:24811749

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

    PubMed

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

    2015-06-18

    Autophagic turnover of intracellular constituents is critical for cellular housekeeping, nutrient recycling, and various aspects of growth and development in eukaryotes. Here we show that autophagy impacts the other major degradative route involving the ubiquitin-proteasome system by eliminating 26S proteasomes, a process we termed proteaphagy. Using Arabidopsis proteasomes tagged with GFP, we observed their deposition into vacuoles via a route requiring components of the autophagy machinery. This transport can be initiated separately by nitrogen starvation and chemical or genetic inhibition of the proteasome, implying distinct induction mechanisms. Proteasome inhibition stimulates comprehensive ubiquitylation of the complex, with the ensuing proteaphagy requiring the proteasome subunit RPN10, which can simultaneously bind both ATG8 and ubiquitin. Collectively, we propose that Arabidopsis RPN10 acts as a selective autophagy receptor that targets inactive 26S proteasomes by concurrent interactions with ubiquitylated proteasome subunits/targets and lipidated ATG8 lining the enveloping autophagic membranes. PMID:26004230

  6. Structure of an endogenous yeast 26S proteasome reveals two major conformational states

    PubMed Central

    Luan, Bai; Huang, Xiuliang; Wu, Jianping; Mei, Ziqing; Wang, Yiwei; Xue, Xiaobin; Yan, Chuangye; Wang, Jiawei; Finley, Daniel J.; Shi, Yigong; Wang, Feng

    2016-01-01

    The eukaryotic proteasome mediates degradation of polyubiquitinated proteins. Here we report the single-particle cryoelectron microscopy (cryo-EM) structures of the endogenous 26S proteasome from Saccharomyces cerevisiae at 4.6- to 6.3-Å resolution. The fine features of the cryo-EM maps allow modeling of 18 subunits in the regulatory particle and 28 in the core particle. The proteasome exhibits two distinct conformational states, designated M1 and M2, which correspond to those reported previously for the proteasome purified in the presence of ATP-γS and ATP, respectively. These conformations also correspond to those of the proteasome in the presence and absence of exogenous substrate. Structure-guided biochemical analysis reveals enhanced deubiquitylating enzyme activity of Rpn11 upon assembly of the lid. Our structures serve as a molecular basis for mechanistic understanding of proteasome function. PMID:26929360

  7. The murine cardiac 26S proteasome: an organelle awaiting exploration.

    PubMed

    Gomes, Aldrin V; Zong, Chenggong; Edmondson, Ricky D; Berhane, Beniam T; Wang, Guang-Wu; Le, Steven; Young, Glen; Zhang, Jun; Vondriska, Thomas M; Whitelegge, Julian P; Jones, Richard C; Joshua, Irving G; Thyparambil, Sheeno; Pantaleon, Dawn; Qiao, Joe; Loo, Joseph; Ping, Peipei

    2005-06-01

    Multiprotein complexes have been increasingly recognized as essential functional units for a variety of cellular processes, including the protein degradation system. Selective degradation of proteins in eukaryotes is primarily conducted by the ubiquitin proteasome system. The current knowledge base, pertaining to the proteasome complexes in mammalian cells, relies largely upon information gained in the yeast system, where the 26S proteasome is hypothesized to contain a 20S multiprotein core complex and one or two 19S regulatory complexes. To date, the molecular structure of the proteasome system, the proteomic composition of the entire 26S multiprotein complexes, and the specific designated function of individual components within this essential protein degradation system in the heart remain virtually unknown. A functional proteomic approach, employing multidimensional chromatography purification combined with liquid chromatography tandem mass spectrometry and protein chemistry, was utilized to explore the murine cardiac 26S proteasome system. This article presents an overview on the subject of protein degradation in mammalian cells. In addition, this review shares the limited information that has been garnered thus far pertaining to the molecular composition, function, and regulation of this important organelle in the cardiac cells. PMID:16093497

  8. The 26S Proteasome and Initiation of Gene Transcription

    PubMed Central

    Durairaj, Geetha; Kaiser, Peter

    2014-01-01

    Transcription activation is the foremost step of gene expression and is modulated by various factors that act in synergy. Misregulation of this process and its associated factors has severe effects and hence requires strong regulatory control. In recent years, growing evidence has highlighted the 26S proteasome as an important contributor to the regulation of transcription initiation. Well known for its role in protein destruction, its contribution to protein synthesis was initially viewed with skepticism. However, studies over the past several years have established the proteasome as an important component of transcription initiation through proteolytic and non-proteolytic activities. In this review, we discuss findings made so far in understanding the connections between transcription initiation and the 26S proteasome complex. PMID:25211636

  9. Structural characterization of the interaction of Ubp6 with the 26S proteasome.

    PubMed

    Aufderheide, Antje; Beck, Florian; Stengel, Florian; Hartwig, Michaela; Schweitzer, Andreas; Pfeifer, Günter; Goldberg, Alfred L; Sakata, Eri; Baumeister, Wolfgang; Förster, Friedrich

    2015-07-14

    In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde. To this end we have used single-particle electron cryomicroscopy in combination with cross-linking and mass spectrometry. Ubp6 binds to the regulatory particle non-ATPase (Rpn) 1 via its N-terminal ubiquitin-like domain, whereas its catalytic USP domain is positioned variably. Addition of ubiquitin aldehyde stabilizes the binding of the USP domain in a position where it bridges the proteasome subunits Rpn1 and the regulatory particle triple-A ATPase (Rpt) 1. The USP domain binds to Rpt1 in the immediate vicinity of the Ubp6 active site, which may effect its activation. The catalytic triad is positioned in proximity to the mouth of the ATPase module and to the deubiquitylating enzyme Rpn11, strongly implying their functional linkage. On the proteasome side, binding of Ubp6 favors conformational switching of the 26S proteasome into an intermediate-energy conformational state, in particular upon the addition of ubiquitin aldehyde. This modulation of the conformational space of the 26S proteasome by Ubp6 explains the effects of Ubp6 on the kinetics of proteasomal degradation. PMID:26130806

  10. Structural characterization of the interaction of Ubp6 with the 26S proteasome

    PubMed Central

    Aufderheide, Antje; Beck, Florian; Stengel, Florian; Hartwig, Michaela; Schweitzer, Andreas; Pfeifer, Günter; Goldberg, Alfred L.; Sakata, Eri; Baumeister, Wolfgang; Förster, Friedrich

    2015-01-01

    In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde. To this end we have used single-particle electron cryomicroscopy in combination with cross-linking and mass spectrometry. Ubp6 binds to the regulatory particle non-ATPase (Rpn) 1 via its N-terminal ubiquitin-like domain, whereas its catalytic USP domain is positioned variably. Addition of ubiquitin aldehyde stabilizes the binding of the USP domain in a position where it bridges the proteasome subunits Rpn1 and the regulatory particle triple-A ATPase (Rpt) 1. The USP domain binds to Rpt1 in the immediate vicinity of the Ubp6 active site, which may effect its activation. The catalytic triad is positioned in proximity to the mouth of the ATPase module and to the deubiquitylating enzyme Rpn11, strongly implying their functional linkage. On the proteasome side, binding of Ubp6 favors conformational switching of the 26S proteasome into an intermediate-energy conformational state, in particular upon the addition of ubiquitin aldehyde. This modulation of the conformational space of the 26S proteasome by Ubp6 explains the effects of Ubp6 on the kinetics of proteasomal degradation. PMID:26130806

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

    PubMed

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

    2016-06-01

    Baculoviruses are large DNA viruses that infect insect species such as Lepidoptera and are used in biotechnology for protein production and in agriculture as insecticides against crop pests. Baculoviruses require activity of host proteasomes for efficient reproduction, but how they control the cellular proteome and interact with the ubiquitin proteasome system (UPS) of infected cells remains unknown. In this report, we analyzed possible changes in the subunit composition of 26S proteasomes of the fall armyworm, Spodoptera frugiperda (Sf9), cells in the course of infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). 26S proteasomes were purified from Sf9 cells by an immune affinity method and subjected to 2D gel electrophoresis followed by MALDI-TOF mass spectrometry and Mascot search in bioinformatics databases. A total of 34 homologues of 26S proteasome subunits of eukaryotic species were identified including 14 subunits of the 20S core particle (7 α and 7 β subunits) and 20 subunits of the 19S regulatory particle (RP). The RP contained homologues of 11 of RPN-type and 6 of RPT-type subunits, 2 deubiquitinating enzymes (UCH-14/UBP6 and UCH-L5/UCH37), and thioredoxin. Similar 2D-gel maps of 26S proteasomes purified from uninfected and AcMNPV-infected cells at 48hpi confirmed the structural integrity of the 26S proteasome in insect cells during baculovirus infection. However, subtle changes in minor forms of some proteasome subunits were detected. A portion of the α5(zeta) cellular pool that presumably was not associated with the proteasome underwent partial proteolysis at a late stage in infection. PMID:26945516

  12. Degradation of oxidized proteins by the proteasome: Distinguishing between the 20S, 26S, and immunoproteasome proteolytic pathways.

    PubMed

    Raynes, Rachel; Pomatto, Laura C D; Davies, Kelvin J A

    2016-08-01

    The proteasome is a ubiquitous and highly plastic multi-subunit protease with multi-catalytic activity that is conserved in all eukaryotes. The most widely known function of the proteasome is protein degradation through the 26S ubiquitin-proteasome system, responsible for the vast majority of protein degradation during homeostasis. However, the proteasome also plays an important role in adaptive immune responses and adaptation to oxidative stress. The unbound 20S proteasome, the core common to all proteasome conformations, is the main protease responsible for degrading oxidized proteins. During periods of acute stress, the 19S regulatory cap of the 26S proteasome disassociates from the proteolytic core, allowing for immediate ATP/ubiquitin-independent protein degradation by the 20S proteasome. Despite the abundance of unbound 20S proteasome compared to other proteasomal conformations, many publications fail to distinguish between the two proteolytic systems and often regard the 26S proteasome as the dominant protease. Further confounding the issue are the differential roles these two proteolytic systems have in adaptation and aging. In this review, we will summarize the increasing evidence that the 20S core proteasome constitutes the major conformation of the proteasome system and that it is far from a latent protease requiring activation by binding regulators. PMID:27155164

  13. The 26S proteasome in Schistosoma mansoni: bioinformatics analysis, developmental expression, and RNA interference (RNAi) studies.

    PubMed

    Nabhan, Joseph F; El-Shehabi, Fouad; Patocka, Nicholas; Ribeiro, Paula

    2007-11-01

    The 26S proteasome is a proteolytic complex responsible for the degradation of the vast majority of eukaryotic proteins. Regulated proteolysis by the proteasome is thought to influence cell cycle progression, transcriptional control, and other critical cellular processes. Here, we used a bioinformatics approach to identify the proteasomal constituents of the parasitic trematode Schistosoma mansoni. A detailed search of the S. mansoni genome database identified a total of 31 putative proteasomal subunits, including 17 subunits of the regulatory (19S) complex and 14 predicted catalytic (20S) subunits. A quantitative real-time RT-PCR analysis of subunit expression levels revealed that the S. mansoni proteasome components are differentially expressed among cercaria, schistosomula, and adult worms. In particular, the data suggest that the proteasome may be downregulated during the early stages of schistosomula development and is subsequently upregulated as the parasite matures to the adult stage. To test for biological relevance, we developed a transfection-based RNA interference method to knockdown the expression of the proteasome subunit, SmRPN11/POH1. Transfection of in vitro transformed S. mansoni schistosomula with specific short-interfering RNAs (siRNAs) diminished SmRPN11/POH1 expression nearly 80%, as determined by quantitative RT-PCR analysis, and also decreased parasite viability 78%, whereas no significant effect could be seen after treatment with the same amount of an irrelevant siRNA. These results indicate that the subunit SmRPN11/POH1 is an essential gene in schistosomes and further suggest an important role for the proteasome in parasite development and survival. PMID:17892869

  14. Near-atomic resolution structural model of the yeast 26S proteasome

    PubMed Central

    Beck, Florian; Unverdorben, Pia; Bohn, Stefan; Schweitzer, Andreas; Pfeifer, Günter; Sakata, Eri; Nickell, Stephan; Plitzko, Jürgen M.; Villa, Elizabeth; Baumeister, Wolfgang; Förster, Friedrich

    2012-01-01

    The 26S proteasome operates at the executive end of the ubiquitin-proteasome pathway. Here, we present a cryo-EM structure of the Saccharomyces cerevisiae 26S proteasome at a resolution of 7.4 Å or 6.7 Å (Fourier-Shell Correlation of 0.5 or 0.3, respectively). We used this map in conjunction with molecular dynamics-based flexible fitting to build a near-atomic resolution model of the holocomplex. The quality of the map allowed us to assign α-helices, the predominant secondary structure element of the regulatory particle subunits, throughout the entire map. We were able to determine the architecture of the Rpn8/Rpn11 heterodimer, which had hitherto remained elusive. The MPN domain of Rpn11 is positioned directly above the AAA-ATPase N-ring suggesting that Rpn11 deubiquitylates substrates immediately following commitment and prior to their unfolding by the AAA-ATPase module. The MPN domain of Rpn11 dimerizes with that of Rpn8 and the C-termini of both subunits form long helices, which are integral parts of a coiled-coil module. Together with the C-terminal helices of the six PCI-domain subunits they form a very large coiled-coil bundle, which appears to serve as a flexible anchoring device for all the lid subunits. PMID:22927375

  15. Thiostrepton interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates

    PubMed Central

    Sandu, Cristinel; Chandramouli, Nagaranjan; Glickman, Joseph Fraser; Molina, Henrik; Kuo, Chueh-Ling; Kukushkin, Nikolay; Goldberg, Alfred L; Steller, Hermann

    2015-01-01

    Here, we report a novel mechanism of proteasome inhibition mediated by Thiostrepton (Thsp), which interacts covalently with Rpt subunits of the 19S proteasome and proteasome substrates. We identified Thsp in a cell-based high-throughput screen using a fluorescent reporter sensitive to degradation by the ubiquitin–proteasome pathway. Thiostrepton behaves as a proteasome inhibitor in several paradigms, including cell-based reporters, detection of global ubiquitination status, and proteasome-mediated labile protein degradation. In vitro, Thsp does not block the chymotrypsin activity of the 26S proteasome. In a cell-based IκBα degradation assay, Thsp is a slow inhibitor and 4 hrs of treatment achieves the same effects as MG-132 at 30 min. We show that Thsp forms covalent adducts with proteins in human cells and demonstrate their nature by mass spectrometry. Furthermore, the ability of Thsp to interact covalently with the cysteine residues is essential for its proteasome inhibitory function. We further show that a Thsp modified peptide cannot be degraded by proteasomes in vitro. Importantly, we demonstrate that Thsp binds covalently to Rpt subunits of the 19S regulatory particle and forms bridges with a proteasome substrate. Taken together, our results uncover an important role of Thsp in 19S proteasome inhibition. PMID:26033448

  16. Atomic structure of the 26S proteasome lid reveals the mechanism of deubiquitinase inhibition

    PubMed Central

    Dambacher, Corey M; Worden, Evan J; Herzik, Mark A; Martin, Andreas; Lander, Gabriel C

    2016-01-01

    The 26S proteasome is responsible for the selective, ATP-dependent degradation of polyubiquitinated cellular proteins. Removal of ubiquitin chains from targeted substrates at the proteasome is a prerequisite for substrate processing and is accomplished by Rpn11, a deubiquitinase within the ‘lid’ sub-complex. Prior to the lid’s incorporation into the proteasome, Rpn11 deubiquitinase activity is inhibited to prevent unwarranted deubiquitination of polyubiquitinated proteins. Here we present the atomic model of the isolated lid sub-complex, as determined by cryo-electron microscopy at 3.5 Å resolution, revealing how Rpn11 is inhibited through its interaction with a neighboring lid subunit, Rpn5. Through mutagenesis of specific residues, we describe the network of interactions that are required to stabilize this inhibited state. These results provide significant insight into the intricate mechanisms of proteasome assembly, outlining the substantial conformational rearrangements that occur during incorporation of the lid into the 26S holoenzyme, which ultimately activates the deubiquitinase for substrate degradation. DOI: http://dx.doi.org/10.7554/eLife.13027.001 PMID:26744777

  17. Regulation of Sperm Capacitation by the 26S Proteasome: An Emerging New Paradigm in Spermatology.

    PubMed

    Kerns, Karl; Morales, Patricio; Sutovsky, Peter

    2016-05-01

    The ubiquitin proteasome system (UPS) participates in many biological processes ranging from cell cycle and antigen processing to cellular defense and signaling. Work of the last decade has made it evident that the UPS is involved in many sperm-related processes leading up to and as part of fertilization. The current knowledge of UPS involvement and changes during sperm capacitation are reviewed together with a list of known proteasome-associated sperm proteins and a discussion of the relationships between these proteins and the proteasome. Proteasomal inhibitors such as MG-132 and epoxomicin significantly alter capacitation and prevent acrosome reaction. The 26S proteasome degrades AKAP3, an A-kinase anchoring protein, partially regulating the release of protein-kinase A (PKA), a vital component necessary for the steps leading up to capacitation. Further, changes occur in 20S core subunit localization and abundance throughout capacitation. Proteasome-interacting valosine-containing protein (VCP) undergoes tyrosine phosphorylation; however, its physiological roles in capacitation and fertilization remain unknown. The E1-type ubiquitin-activating enzyme (UBA1) inhibitor PYR-41 also alters acrosomal membrane remodeling during capacitation. Furthermore, after capacitation, the acrosomal proteasomes facilitate the degradation of zona pellucida glycoproteins leading up to fertilization. Methods to modulate the sperm proteasome activity during sperm storage and capacitation may translate to increased reproductive efficiency in livestock animals. Human male infertility diagnostics may benefit from incorporation of research outcomes built upon relationships between UPS and capacitation. Altogether, the studies reviewed here support the involvement of UPS in sperm capacitation and present opportunities for new discoveries. PMID:27053366

  18. An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome.

    PubMed

    Förster, Friedrich; Lasker, Keren; Beck, Florian; Nickell, Stephan; Sali, Andrej; Baumeister, Wolfgang

    2009-10-16

    The 26S proteasome is the most downstream element of the ubiquitin-proteasome pathway of protein degradation. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The RP consists of 6 AAA-ATPases and at least 13 non-ATPase subunits. Based on a cryo-EM map of the 26S proteasome, structures of homologs, and physical protein-protein interactions we derive an atomic model of the AAA-ATPase-CP sub-complex. The ATPase order in our model (Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5) is in excellent agreement with the recently identified base-precursor complexes formed during the assembly of the RP. Furthermore, the atomic CP-AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP-RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners. PMID:19653995

  19. An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome

    SciTech Connect

    Foerster, Friedrich; Lasker, Keren; Beck, Florian; Nickell, Stephan; Sali, Andrej; Baumeister, Wolfgang

    2009-10-16

    The 26S proteasome is the most downstream element of the ubiquitin-proteasome pathway of protein degradation. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The RP consists of 6 AAA-ATPases and at least 13 non-ATPase subunits. Based on a cryo-EM map of the 26S proteasome, structures of homologs, and physical protein-protein interactions we derive an atomic model of the AAA-ATPase-CP sub-complex. The ATPase order in our model (Rpt1/Rpt2/Rpt6/Rpt3/Rpt4/Rpt5) is in excellent agreement with the recently identified base-precursor complexes formed during the assembly of the RP. Furthermore, the atomic CP-AAA-ATPase model suggests that the assembly chaperone Nas6 facilitates CP-RP association by enhancing the shape complementarity between Rpt3 and its binding CP alpha subunits partners.

  20. The ATP costs and time required to degrade ubiquitinated proteins by the 26 S proteasome.

    PubMed

    Peth, Andreas; Nathan, James A; Goldberg, Alfred L

    2013-10-01

    The degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis. To investigate if the six proteasomal ATPases function independently or in a cyclic manner, as proposed recently, we used yeast mutants that prevent ATP binding to Rpt3, Rpt5, or Rpt6. Although proteasomes contain six ATPase subunits, each of these single mutations caused a 66% reduction in basal ATP hydrolysis, and each blocked completely the 2-3-fold stimulation of ATPase activity induced by ubiquitinated substrates. Therefore, the ATPase subunits must function in a ordered manner, in which each is required for the stimulation of ATPase activity by substrates. Although ATP is essential for multiple steps in proteasome function, when the rate of ATP hydrolysis was reduced incrementally, the degradation of Ub5-DHFR (where Ub is ubiquitin and DHFR is dihydrofolate reductase) decreased exactly in parallel. This direct proportionality implies that a specific number of ATPs is consumed in degrading a ubiquitinated protein. When the ubiquitinated DHFR was more tightly folded (upon addition of the ligand folate), the rate of ATP hydrolysis was unchanged, but the time to degrade a Ub5-DHFR molecule (∼13 s) and the energy expenditure (50-80 ATPs/Ub5-DHFR) both increased by 2-fold. With a mutation in the ATPase C terminus that reduced gate opening into the 20 S proteasome, the energy costs and time required for conjugate degradation also increased. Thus, different ubiquitin conjugates activate similarly the ATPase subunit cycle that drives proteolysis, but polypeptide structure determines the time required for degradation and thus the energy cost. PMID:23965995

  1. Uncommon functional properties of the first piscine 26S proteasome from the Antarctic notothenioid Trematomus bernacchii

    PubMed Central

    Gogliettino, Marta; Balestrieri, Marco; Riccio, Alessia; Facchiano, Angelo; Fusco, Carmela; Palazzo, Vincenzo Cecere; Rossi, Mosè; Cocca, Ennio; Palmieri, Gianna

    2016-01-01

    Protein homoeostasis is a fundamental process allowing the preservation of functional proteins and it has a great impact on the life of the Antarctic organisms. However, the effect of low temperatures on protein turnover is poorly understood and the cold-adaptation of the degradation machinery remains an unresolved issue. As the 26S proteasome represents the main proteolytic system devoted to the controlled degradation of intracellular proteins, the purpose of the present study was to investigate the functions of this complex in the notothenioid Trematomus bernacchii, in order to better understand its role in the physiology of Antarctic fish. To this aim, we purified and characterized the 26S proteasome from T. bernacchii and isolated the cDNAs codifying seven of the 14 subunits belonging to the proteasome 20S core particle. Results provided evidences of the high resistance of the piscine 26S proteasome to oxidative agents and of its ‘uncommon’ ability to efficiently hydrolyse oxidized bovine serum albumin (BSA), suggesting that this enzymatic complex could play a key role in the antioxidant defense systems in fish inhabiting permanently cold marine environments. These unique properties were also reflected by the 3D model analysis, which revealed a higher structural stability of the piscine complex respect to the murine template. Finally, a comparative analysis, performed in a variety of tissues collected from T. bernacchii and the temperate fish Dicentrarchus labrax, showed a lower protein retention in the cold-adapted fish, possibly due to a better efficiency of its degradation machinery. PMID:26933238

  2. Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome

    PubMed Central

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

    2014-01-01

    The 26S proteasome is a 2.5 MDa molecular machine that executes the degradation of substrates of the ubiquitin–proteasome pathway. The molecular architecture of the 26S proteasome was recently established by cryo-EM approaches. For a detailed understanding of the sequence of events from the initial binding of polyubiquitylated substrates to the translocation into the proteolytic core complex, it is necessary to move beyond static structures and characterize the conformational landscape of the 26S proteasome. To this end we have subjected a large cryo-EM dataset acquired in the presence of ATP and ATP-γS to a deep classification procedure, which deconvolutes coexisting conformational states. Highly variable regions, such as the density assigned to the largest subunit, Rpn1, are now well resolved and rendered interpretable. Our analysis reveals the existence of three major conformations: in addition to the previously described ATP-hydrolyzing (ATPh) and ATP-γS conformations, an intermediate state has been found. Its AAA-ATPase module adopts essentially the same topology that is observed in the ATPh conformation, whereas the lid is more similar to the ATP-γS bound state. Based on the conformational ensemble of the 26S proteasome in solution, we propose a mechanistic model for substrate recognition, commitment, deubiquitylation, and translocation into the core particle. PMID:24706844

  3. Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome.

    PubMed

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

    2014-04-15

    The 26S proteasome is a 2.5 MDa molecular machine that executes the degradation of substrates of the ubiquitin-proteasome pathway. The molecular architecture of the 26S proteasome was recently established by cryo-EM approaches. For a detailed understanding of the sequence of events from the initial binding of polyubiquitylated substrates to the translocation into the proteolytic core complex, it is necessary to move beyond static structures and characterize the conformational landscape of the 26S proteasome. To this end we have subjected a large cryo-EM dataset acquired in the presence of ATP and ATP-γS to a deep classification procedure, which deconvolutes coexisting conformational states. Highly variable regions, such as the density assigned to the largest subunit, Rpn1, are now well resolved and rendered interpretable. Our analysis reveals the existence of three major conformations: in addition to the previously described ATP-hydrolyzing (ATPh) and ATP-γS conformations, an intermediate state has been found. Its AAA-ATPase module adopts essentially the same topology that is observed in the ATPh conformation, whereas the lid is more similar to the ATP-γS bound state. Based on the conformational ensemble of the 26S proteasome in solution, we propose a mechanistic model for substrate recognition, commitment, deubiquitylation, and translocation into the core particle. PMID:24706844

  4. [Structures and functions of the 26S proteasome Rpn10 family].

    PubMed

    Kawahara, Hiroyuki

    2002-09-01

    The ubiquitin-dependent proteolytic pathway is thought to be one of the vital systems for cellular regulations, including control of the cell cycle, differentiation and apoptosis. In this pathway, poly-ubiquitinated proteins are selectively degraded by the 26S proteasome, a multisubunit proteolytic machinery. Recognition of the poly-ubiquitin chain by the 26S proteasome should be a key step leading to the selective degradation of target proteins, and the Rpn10 subunit of the 26S proteasome has been shown to preferentially bind the poly-ubiquitin chain in vitro. We previously reported that the mouse Rpn10 mRNA family is generated from a single gene by developmentally regulated, alternative splicing. To determine whether such alternative splicing mechanisms occur in organisms other than the mouse, we searched for Rpn10 isoforms in various species. Here we summarize the gene organization of the Rpn10 in lower species and provide evidence that the competence for generating all distinct forms of Rpn10 alternative splicing has expanded through evolution. Some of the Rpn10 family genes were found to be expressed in distinct developmental stages, suggesting that they have distinct functions during embryogenesis. For example, Rpn10c and Rpn10e were exclusively expressed at specific developmental stages and in specific tissues, while Rpn10a was expressed constitutively. Our experimental results indicate that the respective Rpn10 proteins possess distinct roles in the progression of development. Furthermore, some of the Rpn10 variants specifically interacted with important developmental regulators. PMID:12235853

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-01-26

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

  7. HSP70 Mediates Dissociation and Reassociation of the 26S Proteasome During Adaptation to Oxidative Stress

    PubMed Central

    Grune, Tilman; Catalgol, Betül; Licht, Anke; Ermak, Gennady; Pickering, Andrew; Ngo, Jenny K.; Davies, Kelvin J. A.

    2011-01-01

    We report an entirely new role for the HSP70 chaperone in dissociating 26S proteasome complexes (into free 20S proteasomes and bound 19S regulators), preserving 19S regulators, and reconstituting 26S proteasomes in the first 1-3 hours following mild oxidative stress. These responses, coupled with direct 20S proteasome activation by poly-ADP ribose polymerase in the nucleus and by PA28αβ in the cytoplasm, instantly provides cells with increased capacity to degrade oxidatively damaged proteins and to survive the initial effects of stress exposure. Subsequent adaptive (hormetic) processes (3-24 hours following stress exposure), mediated by several signal transduction pathways and involving increased transcription/translation of 20S proteasomes, immunoproteasomes, and PA28αβ, abrogate the need for 26S proteasome dissociation. During this adaptive period, HSP70 releases its bound 19S regulators, 26S proteasomes are reconstituted, and ATP-stimulated proteolysis is restored. The 26S proteasome-dependent, and ATP-stimulated, turnover of ubiquitinylated proteins is essential for normal cell metabolism, and its restoration is required for successful stress-adaptation. PMID:21767633

  8. The Lysine 48 and Lysine 63 Ubiquitin Conjugates Are Processed Differently by the 26 S Proteasome*

    PubMed Central

    Jacobson, Andrew D.; Zhang, Nan-Yan; Xu, Ping; Han, Ke-Jun; Noone, Seth; Peng, Junmin; Liu, Chang-Wei

    2009-01-01

    The role of Lys-63 ubiquitin chains in targeting proteins for proteasomal degradation is still obscure. We systematically compared proteasomal processing of Lys-63 ubiquitin chains with that of the canonical proteolytic signal, Lys-48 ubiquitin chains. Quantitative mass spectrometric analysis of ubiquitin chains in HeLa cells determines that the levels of Lys-63 ubiquitin chains are insensitive to short-time proteasome inhibition. Also, the Lys-48/Lys-63 ratio in the 26 S proteasome-bound fraction is 1.7-fold more than that in the cell lysates, likely because some cellular Lys-63 ubiquitin conjugates are sequestered by Lys-63 chain-specific binding proteins. In vitro, Lys-48 and Lys-63 ubiquitin chains bind the 26 S proteasome comparably, whereas Lys-63 chains are deubiquitinated 6-fold faster than Lys-48 chains. Also, Lys-63 tetraubiquitin-conjugated UbcH10 is rapidly deubiquitinated into the monoubiquitinated form, whereas Lys-48 tetraubiquitin targets UbcH10 for degradation. Furthermore, we found that both the ubiquitin aldehyde- and 1,10-phenanthroline-sensitive deubiquitinating activities of the 26 S proteasome contribute to Lys-48- and Lys-63-linkage deubiquitination, albeit the inhibitory extents are different. Together, our findings suggest that compared with Lys-48 chains, cellular Lys-63 chains have less proteasomal accessibility, and proteasome-bound Lys-63 chains are more rapidly deubiquitinated, which could cause inefficient degradation of Lys-63 conjugates. PMID:19858201

  9. DNA damage modulates interactions between microRNAs and the 26S proteasome

    PubMed Central

    Tsimokha, Anna S; Kulichkova, Valentina A.; Karpova, Elena V.; Zaykova, Julia J.; Aksenov, Nikolai D; Vasilishina, Anastasia A.; Kropotov, Andrei V.; Antonov, Alexey; Barlev, Nikolai A.

    2014-01-01

    26S proteasomes are known as major non-lysosomal cellular machines for coordinated and specific destruction of ubiquitinylated proteins. The proteolytic activities of proteasomes are controlled by various post-translational modifications in response to environmental cues, including DNA damage. Besides proteolysis, proteasomes also associate with RNA hydrolysis and splicing. Here, we extend the functional diversity of proteasomes by showing that they also dynamically associate with microRNAs (miRNAs) both in the nucleus and cytoplasm of cells. Moreover, DNA damage induced by an anti-cancer drug, doxorubicin, alters the repertoire of proteasome-associated miRNAs, enriching the population of miRNAs that target cell cycle checkpoint regulators and DNA repair proteins. Collectively, these data uncover yet another potential mode of action for proteasomes in the cell via their dynamic association with microRNAs. PMID:25004448

  10. Disulfide Engineering to Map Subunit Interactions in the Proteasome and Other Macromolecular Complexes

    PubMed Central

    Hochstrasser, Mark; Funakoshi, Minoru

    2013-01-01

    Summary In studies of protein complexes for which high-resolution structural data are unavailable, it is often still possible to determine both nearest-neighbor relationships between subunits and atomic-resolution details of these interactions. The eukaryotic 26S proteasome, a ~2.5 MDa protein complex with at least 33 different subunits, is a prime example. Important information about quaternary organization and assembly of proteasomes has been gained using a combination of sequence alignments with related proteins of known tertiary structure, molecular modeling, and disulfide engineering to allow oxidative crosslinking between predicted polypeptide neighbors. Here we provide detailed protocols for engineered cysteine crosslinking of yeast proteasome subunits in whole cell extracts, in active 26S proteasome complexes first isolated by native polyacrylamide gel electrophoresis, and in subcomplexes that function as potential assembly intermediates. PMID:22350897

  11. Tau-driven 26S proteasome impairment and cognitive dysfunction can be prevented early in disease by activating cAMP-PKA signaling

    PubMed Central

    Myeku, Natura; Clelland, Catherine L; Emrani, Sheina; Kukushkin, Nikolay V; Yu, Wai Haung; Goldberg, Alfred L; Duff, Karen E

    2016-01-01

    The ubiquitin proteasome system (UPS) degrades misfolded proteins including those implicated in neurodegenerative diseases. We investigated the effects of tau accumulation on proteasome function in a mouse model of tauopathy and in a cross to a UPS reporter mouse (line Ub-G76V-GFP). Accumulation of insoluble tau was associated with a decrease in the peptidase activity of brain 26S proteasomes, higher levels of ubiquitinated proteins and undegraded Ub-G76V-GFP. 26S proteasomes from mice with tauopathy were physically associated with tau and were less active in hydrolyzing ubiquitinated proteins, small peptides and ATP. 26S proteasomes from normal mice incubated with recombinant oligomers or fibrils also showed lower hydrolyzing capacity in the same assays, implicating tau as a proteotoxin. Administration of an agent that activates cAMP–protein kinase A (PKA) signaling led to attenuation of proteasome dysfunction, probably through proteasome subunit phosphorylation. In vivo, this led to lower levels of aggregated tau and improvements in cognitive performance. PMID:26692334

  12. Indirect inhibition of 26S proteasome activity in a cellular model of Huntington’s disease

    PubMed Central

    Hipp, Mark S.; Patel, Chetan N.; Bersuker, Kirill; Riley, Brigit E.; Kaiser, Stephen E.; Shaler, Thomas A.; Brandeis, Michael

    2012-01-01

    Pathognomonic accumulation of ubiquitin (Ub) conjugates in human neurodegenerative diseases, such as Huntington’s disease, suggests that highly aggregated proteins interfere with 26S proteasome activity. In this paper, we examine possible mechanisms by which an N-terminal fragment of mutant huntingtin (htt; N-htt) inhibits 26S function. We show that ubiquitinated N-htt—whether aggregated or not—did not choke or clog the proteasome. Both Ub-dependent and Ub-independent proteasome reporters accumulated when the concentration of mutant N-htt exceeded a solubility threshold, indicating that stabilization of 26S substrates is not linked to impaired Ub conjugation. Above this solubility threshold, mutant N-htt was rapidly recruited to cytoplasmic inclusions that were initially devoid of Ub. Although synthetically polyubiquitinated N-htt competed with other Ub conjugates for access to the proteasome, the vast majority of mutant N-htt in cells was not Ub conjugated. Our data confirm that proteasomes are not directly impaired by aggregated N-terminal fragments of htt; instead, our data suggest that Ub accumulation is linked to impaired function of the cellular proteostasis network. PMID:22371559

  13. 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

  14. Studies on the activation by ATP of the 26 S proteasome complex from rat skeletal muscle.

    PubMed Central

    Dahlmann, B; Kuehn, L; Reinauer, H

    1995-01-01

    The 26 S proteasome complex is thought to catalyse the breakdown of ubiquitinated proteins within eukaryotic cells. In addition it has been found that the complex also degrades short-lived proteins such as ornithine decarboxylase in a ubiquitin-independent manner. Both proteolytic processes are paralleled by the hydrolysis of ATP. Here we show that ATP also affects the hydrolytic activity towards fluorigenic peptide substrates by the 26 S proteasome complex from rat skeletal muscle tissue. Low concentrations of ATP (about 25 microM) optimally activate the so-called chymotryptic and tryptic activity by increasing the rate of peptide hydrolysis but not peptidylglutamylpeptide hydrolysis. Activation of the enzyme by ATP is transient but this effect can be enhanced and prolonged by including in the assay an ATP-regenerating system, indicating that ATP is hydrolysed by the 26 S proteasome complex. Although ATP cannot be substituted for by adenosine 5'-[beta,gamma-methylene]triphosphate or AMP, hydrolysis of the phosphoanhydride bond of ATP seems not to be necessary for the activation process of the proteasome complex, a conclusion drawn from the findings that ATP analogues such as adenosine 5'-[beta,gamma-imido]triphosphate, adenosine 5'-O-[gamma-thio]triphosphate, adenosine 5'-O-[beta-thio]-diphosphate and adenosine 5'-[alpha,beta-methylene]triphosphate give the same effect as ATP, and vanadate does not prevent ATP activation. These effects are independent of the presence of Mg2+. Thus, ATP and other nucleotides may act as allosteric activators of peptide-hydrolysing activities of the 26 S proteasome complex as has also been found with the lon protease from Escherichia coli. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 PMID:7619056

  15. Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

    PubMed Central

    Lasker, Keren; Förster, Friedrich; Bohn, Stefan; Walzthoeni, Thomas; Villa, Elizabeth; Unverdorben, Pia; Beck, Florian; Aebersold, Ruedi; Sali, Andrej; Baumeister, Wolfgang

    2012-01-01

    The 26S proteasome is at the executive end of the ubiquitin-proteasome pathway for the controlled degradation of intracellular proteins. While the structure of its 20S core particle (CP) has been determined by X-ray crystallography, the structure of the 19S regulatory particle (RP), which recruits substrates, unfolds them, and translocates them to the CP for degradation, has remained elusive. Here, we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. The “lid” of the RP (consisting of Rpn3/5/6/7/8/9/11/12) is organized in a modular fashion. Rpn3/5/6/7/9/12 form a horseshoe-shaped heterohexamer, which connects to the CP and roofs the AAA-ATPase module, positioning the Rpn8/Rpn11 heterodimer close to its mouth. Rpn2 is rigid, supporting the lid, while Rpn1 is conformationally variable, positioned at the periphery of the ATPase ring. The ubiquitin receptors Rpn10 and Rpn13 are located in the distal part of the RP, indicating that they were recruited to the complex late in its evolution. The modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates. PMID:22307589

  16. Bufalin derivative BF211 inhibits proteasome activity in human lung cancer cells in vitro by inhibiting β1 subunit expression and disrupting proteasome assembly

    PubMed Central

    Sun, Peng; Feng, Li-xing; Zhang, Dong-mei; Liu, Miao; Liu, Wang; Mi, Tian; Wu, Wan-ying; Jiang, Bao-hong; Yang, Min; Hu, Li-hong; Guo, De-an; Liu, Xuan

    2016-01-01

    Aim: Bufalin is one of the active components in the traditional Chinese medicine ChanSu that is used to treat arrhythmia, inflammation and cancer. BF211 is a bufalin derivative with stronger cytotoxic activity in cancer cells. The aim of this study was to identify the putative target proteins of BF211 and the signaling pathways in cancer cells. Methods: A549 human lung cancer cells were treated with BF211. A SILAC-based proteomic analysis was used to detect the protein expression profiles of BF211-treated A549 cells. Cellular proteasome activities were examined using fluorogenic peptide substrates, and the binding affinities of BF211 to recombinant proteasome subunit proteins were evaluated using the Biacore assay. The expression levels of proteasome subunits were determined using RT-PCR and Western blotting, and the levels of the integral 26S proteasome were evaluated using native PAGE analysis. Results: The proteomic analysis revealed that 1282 proteins were differentially expressed in BF211-treated A549 cells, and the putative target proteins of BF211 were associated with various cellular functions, including transcription, translation, mRNA splicing, ribosomal protein synthesis and proteasome function. In A549 cells, BF211 (5, 10, and 20 nmol/L) dose-dependently inhibited the enzymatic activities of proteasome. But BF211 displayed a moderate affinity in binding to proteasome β1 subunit and no binding affinity to the β2 and β5 subunits. Moreover, BF211 (0.1, 1, and 10 nmol/L) did not inhibit the proteasome activities in the cell lysates. BF211 (5, 10, and 20 nmol/L) significantly decreased the expression level of proteasome β1 subunit and the levels of integral 26S proteasome in A549 cells. Similarly, knockdown of the β1 subunit with siRNA in A549 cells significantly decreased integral 26S proteasome and proteasome activity. Conclusion: BF211 inhibits proteasome activity in A549 cells by decreasing β1 subunit expression and disrupting proteasome assembly

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

    PubMed Central

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

    2016-01-01

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

  18. Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells.

    PubMed Central

    Gardner, R C; Assinder, S J; Christie, G; Mason, G G; Markwell, R; Wadsworth, H; McLaughlin, M; King, R; Chabot-Fletcher, M C; Breton, J J; Allsop, D; Rivett, A J

    2000-01-01

    Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The K(i) values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3xK(i)). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFkappaB activation with IC(50) values comparable to their K(i) values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells. PMID:10677365

  19. COP9 Signalosome- and 26S Proteasome-dependent Regulation of SCFTIR1 Accumulation in Arabidopsis*S⃞

    PubMed Central

    Stuttmann, Johannes; Lechner, Esther; Guérois, Raphael; Parker, Jane E.; Nussaume, Laurent; Genschik, Pascal; Noël, Laurent D.

    2009-01-01

    Ubiquitination and proteasome-mediated degradation of proteins are crucial for eukaryotic physiology and development. The largest class of E3 ubiquitin ligases is made up of the cullin-RING ligases (CRLs), which themselves are positively regulated through conjugation of the ubiquitin-like peptide RUB/NEDD8 to cullins. RUB modification is antagonized by the COP9 signalosome (CSN), an evolutionarily conserved eight-subunit complex that is essential in most eukaryotes and cleaves RUB from cullins. The CSN behaves genetically as an activator of CRLs, although it abolishes CRL activity in vitro. This apparent paradox was recently reconciled in different organisms, as the CSN was shown to prevent autocatalytic degradation of several CRL substrate adaptors. We tested for such a mechanism in the model plant Arabidopsis by measuring the impact of a newly identified viable csn2 mutant on the activity and stability of SCFTIR1, a receptor to the phytohormone auxin and probably the best characterized plant CRL. Our analysis reveals that not only the F-box protein TIR1 but also relevant cullins are destabilized in csn2 and other Arabidopsis csn mutants. These results provide an explanation for the auxin resistance of csn mutants. We further observed in vivo a post-translational modification of TIR1 dependent on the proteasome inhibitor MG-132 and provide evidence for proteasome-mediated degradation of TIR1, CUL1, and ASK1 (Arabidopsis SKP1 homolog). These results are consistent with CSN-dependent protection of Arabidopsis CRLs from autocatalytic degradation, as observed in other eukaryotes, and provide evidence for antagonist roles of the CSN and 26S proteasome in modulating accumulation of the plant CRL SCFTIR1. PMID:19147500

  20. Cytokine induced changes in proteasome subunit composition are concentration dependent.

    PubMed

    Stohwasser, R; Kloetzel, P M

    1996-09-01

    In eukaryotes, 20S proteasome subunit composition is controlled by the cytokine interferon-gamma (IFN-gamma). IFN-gamma induces the synthesis of the beta-subunits LMP2, LMP7 and MECL-1, which in consequence replace their constitutive subunit homologs delta, MB1 and MC14/Z in the 20S complex. By pulse labeling mouse RMA cells and immunoprecipitation of proteasome complexes with the antibody MP3, we have analysed the effect of different IFN-gamma concentrations on proteasomal subunit composition. Our experiments show that IFN-gamma concentrations as low as 5 U/ml induce subunit substitutions and that overall proteasomal subunit composition is dependent on the cytokine concentration used. An IFN-gamma concentration of 50 U/ml is sufficient for complete replacement of subunit delta by LMP2. In contrast, IFN-gamma treatment never induces a complete replacement of subunit MC14 by MECL-1. These subunits are present at an approximate 1:1 molar ratio, suggesting that both subunits coexist in the same 20S proteasome complex. Furthermore, different regulatory mechanisms have to be postulated for the synthesis and incorporation of the three IFN-gamma inducible proteasome subunits. Both IFN-gamma as well as IL-2 also seem to influence the modification state of the alpha subunit C8. Since the subunit composition is dependent on the cytokine concentration used and strongly influences the proteolytic properties of the 20S proteasome complex, our experiments represent a caveat for experiments in which IFN-gamma dependent proteasomal enzyme characteristics have been analysed without monitoring the subunit composition. PMID:9067255

  1. Nucleotide excision repair and the 26S proteasome function together to promote trinucleotide repeat expansions.

    PubMed

    Concannon, Claire; Lahue, Robert S

    2014-01-01

    Trinucleotide repeat (TNR) expansion underpins a number of inheritable neurological human disorders. Multiple mechanisms are thought to contribute to the expansion process. The incorrect processing of the repeat tract by DNA repair proteins can drive this mutation process forward, as expansions are suppressed following ablation of certain repair factors in mouse models and cell models of disease. Nucleotide excision repair (NER) is one repair pathway implicated in TNR instability, although most previous work focussed on TNR contractions, not expansions. Here we investigated the role of NER in modulating expansions of threshold-length (CTG·CAG) repeats in yeast. We show that both the global genome and transcription-coupled repair subpathways promote expansions of threshold-length TNRs. Furthermore, NER works with the 26S proteasome to drive expansions, based on analysis of double mutants defective in both pathways, and of Rad23, a protein involved in both NER and the shuttling of ubiquitinated proteins to the proteasome. This work provides the first evidence that both subpathways of NER can promote threshold-length TNR expansions and that NER interacts with the proteasome to drive expansions. PMID:24359926

  2. An assay for 26S proteasome activity based on fluorescence anisotropy measurements of dye-labeled protein substrates.

    PubMed

    Bhattacharyya, Sucharita; Renn, Jonathan P; Yu, Houqing; Marko, John F; Matouschek, Andreas

    2016-09-15

    The 26S proteasome is the molecular machine at the center of the ubiquitin proteasome system and is responsible for adjusting the concentrations of many cellular proteins. It is a drug target in several human diseases, and assays for the characterization of modulators of its activity are valuable. The 26S proteasome consists of two components: a core particle, which contains the proteolytic sites, and regulatory caps, which contain substrate receptors and substrate processing enzymes, including six ATPases. Current high-throughput assays of proteasome activity use synthetic fluorogenic peptide substrates that report directly on the proteolytic activity of the proteasome, but not on the activities of the proteasome caps that are responsible for protein recognition and unfolding. Here, we describe a simple and robust assay for the activity of the entire 26S proteasome using fluorescence anisotropy to follow the degradation of fluorescently labeled protein substrates. We describe two implementations of the assay in a high-throughput format and show that it meets the expected requirement of ATP hydrolysis and the presence of a canonical degradation signal or degron in the target protein. PMID:27296635

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

    PubMed Central

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

    2013-01-01

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

  4. The life cycle of the 26S proteasome: from birth, through regulation and function, and onto its death

    PubMed Central

    Livneh, Ido; Cohen-Kaplan, Victoria; Cohen-Rosenzweig, Chen; Avni, Noa; Ciechanover, Aaron

    2016-01-01

    The 26S proteasome is a large, ∼2.5 MDa, multi-catalytic ATP-dependent protease complex that serves as the degrading arm of the ubiquitin system, which is the major pathway for regulated degradation of cytosolic, nuclear and membrane proteins in all eukaryotic organisms. PMID:27444871

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

    PubMed Central

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

    2012-01-01

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

  6. Complete subunit architecture of the proteasome regulatory particle

    PubMed Central

    Lander, Gabriel C.; Estrin, Eric; Matyskiela, Mary E.; Bashore, Charlene; Nogales, Eva; Martin, Andreas

    2011-01-01

    The proteasome is the major ATP-dependent protease in eukaryotic cells, but limited structural information strongly restricts a mechanistic understanding of its activities. The proteasome regulatory particle, consisting of the lid and base subcomplexes, recognizes and processes poly-ubiquitinated substrates. We used electron microscopy and a newly-developed heterologous expression system for the lid to delineate the complete subunit architecture of the regulatory particle. Our studies reveal the spatial arrangement of ubiquitin receptors, deubiquitinating enzymes, and the protein unfolding machinery at subnanometer resolution, outlining the substrate’s path to degradation. Unexpectedly, the ATPase subunits within the base unfoldase are arranged in a spiral staircase, providing insight into potential mechanisms for substrate translocation through the central pore. Large conformational rearrangements of the lid upon holoenzyme formation suggest allosteric regulation of deubiquitination. We provide a structural basis for the ability of the proteasome to degrade a diverse set of substrates and thus regulate vital cellular processes. PMID:22237024

  7. The 26S proteasome is a multifaceted target for anti-cancer therapies.

    PubMed

    Grigoreva, Tatyana A; Tribulovich, Vyacheslav G; Garabadzhiu, Alexander V; Melino, Gerry; Barlev, Nickolai A

    2015-09-22

    Proteasomes play a critical role in the fate of proteins that are involved in major cellular processes, including signal transduction, gene expression, cell cycle, replication, differentiation, immune response, cellular response to stress, etc. In contrast to non-specific degradation by lysosomes, proteasomes are highly selective and destroy only the proteins that are covalently labelled with small proteins, called ubiquitins. Importantly, many diseases, including neurodegenerative diseases and cancers, are intimately connected to the activity of proteasomes making them an important pharmacological target. Currently, the vast majority of inhibitors are aimed at blunting the proteolytic activities of proteasomes. However, recent achievements in solving structures of proteasomes at very high resolution provided opportunities to design new classes of small molecules that target other physiologically-important enzymatic activities of proteasomes, including the de-ubiquitinating one. This review attempts to catalog the information available to date about novel classes of proteasome inhibitors that may have important pharmacological ramifications. PMID:26295307

  8. The 26S proteasome is a multifaceted target for anti-cancer therapies

    PubMed Central

    Grigoreva, Tatyana A; Tribulovich, Vyacheslav G.; Garabadzhiu, Alexander V.; Melino, Gerry; Barlev, Nickolai A.

    2015-01-01

    Proteasomes play a critical role in the fate of proteins that are involved in major cellular processes, including signal transduction, gene expression, cell cycle, replication, differentiation, immune response, cellular response to stress, etc. In contrast to non-specific degradation by lysosomes, proteasomes are highly selective and destroy only the proteins that are covalently labelled with small proteins, called ubiquitins. Importantly, many diseases, including neurodegenerative diseases and cancers, are intimately connected to the activity of proteasomes making them an important pharmacological target. Currently, the vast majority of inhibitors are aimed at blunting the proteolytic activities of proteasomes. However, recent achievements in solving structures of proteasomes at very high resolution provided opportunities to design new classes of small molecules that target other physiologically-important enzymatic activities of proteasomes, including the de-ubiquitinating one. This review attempts to catalog the information available to date about novel classes of proteasome inhibitors that may have important pharmacological ramifications. PMID:26295307

  9. Subunit specific inhibitors of proteasomes and their potential for immunomodulation

    PubMed Central

    Kisselev, Alexei F; Groettrup, Marcus

    2015-01-01

    Specialized variants of the constitutive 20S proteasome in the immune system like the immunoproteasomes and the thymoproteasome contain active site-bearing subunits which differ in their cleavage priorities and substrate binding pockets. The immunoproteasome plays a crucial role in antigen processing and for the differentiation of pro-inflammatory T helper cells which are involved in the pathogenesis of autoimmunity. Selective inhibitors of the immunoproteasome and constitutive proteasome have recently been generated which interfere with the development and progression of autoimmune diseases. Here we describe these inhibitors and their therapeutic potential as predicted from preclinical models. PMID:25217863

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

    PubMed

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

    2016-02-01

    The COP9 signalosome (CSN) and the proteasomal LID are conserved macromolecular complexes composed of at least eight subunits with molecular weights of approximately 350 kDa. CSN and LID are part of the ubiquitin–proteasome pathway and cleave isopeptide linkages of lysine side chains on target proteins. CSN cleaves the isopeptide bond of ubiquitin-like protein Nedd8 from cullins, whereas the LID cleaves ubiquitin from target proteins sentenced for degradation. CSN and LID are structurally and functionally similar but the order of the assembly pathway seems to be different. The assembly differs in at least the last subunit joining the pre-assembled subcomplex. This review addresses the similarities and differences in structure, function and assembly of CSN and LID. PMID:26497135

  11. Immunoaffinity purification of the functional 20S proteasome from human cells via transient overexpression of specific proteasome subunits.

    PubMed

    Livinskaya, Veronika A; Barlev, Nickolai A; Nikiforov, Andrey A

    2014-05-01

    The proteasome is a multi-subunit proteolytic complex that plays a central role in protein degradation in all eukaryotic cells. It regulates many vital cellular processes therefore its dysfunction can lead to various pathologies including cancer and neurodegeneration. Isolation of enzymatically active proteasomes is a key step to the successful study of the proteasome regulation and functions. Here we describe a simple and efficient protocol for immunoaffinity purification of the functional 20S proteasomes from human HEK 293T cells after transient overexpression of specific proteasome subunits tagged with 3xFLAG. To construct 3xFLAG-fusion proteins, DNA sequences encoding the 20S proteasome subunits PSMB5, PSMA5, and PSMA3 were cloned into mammalian expression vector pIRES-hrGFP-1a. The corresponding recombinant proteins PSMB5-3xFLAG, PSMA5-3xFLAG, or PSMA3-3xFLAG were transiently overexpressed in human HEK 293T cells and were shown to be partially incorporated into the intact proteasome complexes. 20S proteasomes were immunoprecipitated from HEK 293T cell extracts under mild conditions using antibodies against FLAG peptide. Isolation of highly purified 20S proteasomes were confirmed by SDS-PAGE and Western blotting using antibodies against different proteasome subunits. Affinity purified 20S proteasomes were shown to possess chymotrypsin- and trypsin-like peptidase activities confirming their functionality. This simple single-step affinity method of the 20S proteasome purification can be instrumental to subsequent functional studies of proteasomes in human cells. PMID:24583181

  12. Identification of proteasome subunit beta type 6 (PSMB6) associated with deltamethrin resistance in mosquitoes by proteomic and bioassay analyses.

    PubMed

    Sun, Linchun; Ye, Yuting; Sun, Haibo; Yu, Jing; Zhang, Li; Sun, Yan; Zhang, Donghui; Ma, Lei; Shen, Bo; Zhu, Changliang

    2013-01-01

    Deltamethrin (DM) insecticides are currently being promoted worldwide for mosquito control, because of the high efficacy, low mammalian toxicity and less environmental impact. Widespread and improper use of insecticides induced resistance, which has become a major obstacle for the insect-borne disease management. Resistance development is a complex and dynamic process involving many genes. To better understand the possible molecular mechanisms involved in DM resistance, a proteomic approach was employed for screening of differentially expressed proteins in DM-susceptible and -resistant mosquito cells. Twenty-seven differentially expressed proteins were identified by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). Four members of the ubiquitin-proteasome system were significantly elevated in DM-resistant cells, suggesting that the ubiquitin-proteasome pathway may play an important role in DM resistance. Proteasome subunit beta type 6 (PSMB6) is a member of 20S proteasomal subunit family, which forms the proteolytic core of 26S proteasome. We used pharmaceutical inhibitor and molecular approaches to study the contributions of PSMB6 in DM resistance: the proteasome inhibitor MG-132 and bortezomib were used to suppress the proteasomal activity and siRNA was designed to block the function of PSMB6. The results revealed that both MG-132 and bortezomib increased the susceptibility in DM-resistant cells and resistance larvae. Moreover, PSMB6 knockdown decreased cellular viability under DM treatment. Taken together, our study indicated that PSMB6 is associated with DM resistance in mosquitoes and that proteasome inhibitors such as MG-132 or bortezomib are suitable for use as a DM synergist for vector control. PMID:23762443

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

    PubMed

    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

    2016-08-01

    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

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

    PubMed

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

    2013-01-01

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

  15. Inhibition of the 26S proteasome as a possible mechanism for toxicity of heavy metal species.

    PubMed

    Tomco, Dajena; Schmitt, Sara; Heeg, Mary Jane; Dou, Q Ping; Verani, Cláudio N

    2014-03-01

    In this paper we report on the synthesis of five metal complexes coordinated to the [NN'O] ligand HL(iodo) (2,4-diiodo-6-((pyridine-2-ylmethylamino)methyl)phenol), namely [Al(III)(L(iodo))2]ClO4 (1), [Cd(II)(L(iodo))Cl]·H2O (2), [Hg(II)(L(iodo))2]·4DMSO (3), [Pb(II)(L(iodo))NO3] (4), and [Sn(IV)(L(iodo))Cl3] (5). Species 1-5 are thoroughly characterized by spectroscopic and spectrometric methods, as well as by elemental analysis. X-ray crystallography results for complex 3 indicate the presence of Hg(II) ion hexacoordinated to two facially oriented [NN'O] ligands, whereas for complex 5 an Sn(IV) ion chelates to one deprotonated ligand and three chlorido coligands. The toxicity of species 1-5 is tested against transformed human prostate epithelial cells CRL2221 and we observe that the five complexes demonstrate high levels of cell growth inhibition in a dose-dependent manner. In order to evaluate the relationship between these species and the proteasome, we test 1-5 against purified 20S, CRL2221 cell extracts, and intact cells, followed by the measurement of the percent chymotrypsin-like activity inhibition levels. Results suggest a good correlation between the toxicity of [Hg(II)(L(iodo))2]·4DMSO (3) and proteasome inhibition. PMID:24452142

  16. 26S Proteasome regulation of Ankrd1/CARP in adult rat ventricular myocytes and human microvascular endothelial cells

    SciTech Connect

    Samaras, Susan E.; Chen, Billy; Koch, Stephen R.; Sawyer, Douglas B.; Lim, Chee Chew; Davidson, Jeffrey M.

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer The 26S proteasome regulates Ankrd1 levels in cardiomyocytes and endothelial cells. Black-Right-Pointing-Pointer Ankrd1 protein degrades 60-fold faster in endothelial cells than cardiomyocytes. Black-Right-Pointing-Pointer Differential degradation appears related to nuclear vs. sarcolemmal localization. Black-Right-Pointing-Pointer Endothelial cell density shows uncoupling of Ankrd1 mRNA and protein levels. -- Abstract: Ankyrin repeat domain 1 protein (Ankrd1), also known as cardiac ankyrin repeat protein (CARP), increases dramatically after tissue injury, and its overexpression improves aspects of wound healing. Reports that Ankrd1/CARP protein stability may affect cardiovascular organization, together with our findings that the protein is crucial to stability of the cardiomyocyte sarcomere and increased in wound healing, led us to compare the contribution of Ankrd1/CARP stability to its abundance. We found that the 26S proteasome is the dominant regulator of Ankrd1/CARP degradation, and that Ankrd1/CARP half-life is significantly longer in cardiomyocytes (h) than endothelial cells (min). In addition, higher endothelial cell density decreased the abundance of the protein without affecting steady state mRNA levels. Taken together, our data and that of others indicate that Ankrd1/CARP is highly regulated at multiple levels of its expression. The striking difference in protein half-life between a muscle and a non-muscle cell type suggests that post-translational proteolysis is correlated with the predominantly structural versus regulatory role of the protein in the two cell types.

  17. Autophagic Turnover of Inactive 26S Proteasomes in Yeast Is Directed by the Ubiquitin Receptor Cue5 and the Hsp42 Chaperone.

    PubMed

    Marshall, Richard S; McLoughlin, Fionn; Vierstra, Richard D

    2016-08-01

    The autophagic clearance of 26S proteasomes (proteaphagy) is an important homeostatic mechanism within the ubiquitin system that modulates proteolytic capacity and eliminates damaged particles. Here, we define two proteaphagy routes in yeast that respond to either nitrogen starvation or particle inactivation. Whereas the core autophagic machineries required for Atg8 lipidation and vesiculation are essential for both routes, the upstream Atg1 kinase participates only in starvation-induced proteaphagy. Following inactivation, 26S proteasomes become extensively modified with ubiquitin. Although prior studies with Arabidopsis implicated RPN10 in tethering ubiquitylated proteasomes to ATG8 lining the autophagic membranes, yeast proteaphagy employs the evolutionarily distinct receptor Cue5, which simultaneously binds ubiquitin and Atg8. Proteaphagy of inactivated proteasomes also requires the oligomeric Hsp42 chaperone, suggesting that ubiquitylated proteasomes are directed by Hsp42 to insoluble protein deposit (IPOD)-type structures before encapsulation. Together, Cue5 and Hsp42 provide a quality control checkpoint in yeast directed at recycling dysfunctional 26S proteasomes. PMID:27477278

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

    PubMed Central

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

    2015-01-01

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

  19. TOPORS, a Dual E3 Ubiquitin and Sumo1 Ligase, Interacts with 26 S Protease Regulatory Subunit 4, Encoded by the PSMC1 Gene

    PubMed Central

    Czub, Barbara; Shah, Amna Z.; Alfano, Giovanna; Kruczek, Przemysław M.; Chakarova, Christina F.; Bhattacharya, Shomi S.

    2016-01-01

    The significance of the ubiquitin-proteasome system (UPS) for protein degradation has been highlighted in the context of neurodegenerative diseases, including retinal dystrophies. TOPORS, a dual E3 ubiquitin and SUMO1 ligase, forms a component of the UPS and selected substrates for its enzymatic activities, such as DJ-1/PARK7 and APOBEC2, are important for neuronal as well as retinal homeostasis, respectively. TOPORS is ubiquitously expressed, yet its mutations are only known to result in autosomal dominant retinitis pigmentosa. We performed a yeast two-hybrid (Y2H) screen of a human retinal cDNA library in order to identify interacting protein partners of TOPORS from the retina, and thus begin delineating the putative disease mechanism(s) associated with the retina-specific phenotype resulting from mutations in TOPORS. The screen led to isolation of the 26 S protease regulatory subunit 4 (P26s4/ PSMC1), an ATPase indispensable for correct functioning of UPS-mediated proteostasis. The interaction between endogenous TOPORS and P26s4 proteins was validated by co-immuno-precipitation from mammalian cell extracts and further characterised by immunofluorescent co-localisation studies in cell lines and retinal sections. Findings from hTERT-RPE1 and 661W cells demonstrated that TOPORS and P26s4 co-localise at the centrosome in cultured cells. Immunofluorescent staining of mouse retinae revealed a strong P26s4 reactivity at the interface between retinal pigmented epithelium (RPE) layer and the photoreceptors outer segments (OS). This finding leads us to speculate that P26s4, along with TOPORS, may have a role(s) in RPE phagocytosis, in addition to contributing to the overall photoreceptor and retinal homeostasis via the UPS. PMID:26872363

  20. Molecular characterization of NbPAF encoding the alpha6 subunit of the 20S proteasome in Nicotiana benthamiana.

    PubMed

    Kim, Moonil; Yang, Kyoung-Sil; Kim, Yu-Kyung; Paek, Kyung-Hee; Pai, Hyun-Sook

    2003-02-28

    The 26S proteasome involved in degradation of proteins covalently modified with polyubiquitin consists of the 20S proteasome and 19S regulatory complex. The NbPAF gene encoding the alpha6 subunit of the 20S proteasome was identified from Nicotiana benthamiana. NbPAF exhibits high sequence homology with the corresponding genes from Arabidopsis, human and yeast. The deduced amino acid sequence of NbPAF reveals that this protein contains the proteasome alpha-type subunits signature and nuclear localization signal at the N-terminus. The genomic Southern blot analysis suggests that the N. benthamiana genome contains one copy of NbPAF. The NbPAF mRNA was detected abundantly in flowers and weakly in roots and stems, but it was almost undetectable in mature leaves. In response to stresses, accumulation of the NbPAF mRNA was stimulated by methyl jasmonate, NaCl and salicylic acid, but not by abscisic acid and cold treatment in leaves. The NbPAF-GFP fusion protein was localized in the cytoplasm and nucleus. PMID:12661772

  1. Gel-based chemical cross-linking analysis of 20S proteasome subunit-subunit interactions in breast cancer.

    PubMed

    Song, Hai; Xiong, Hua; Che, Jing; Xi, Qing-Song; Huang, Liu; Xiong, Hui-Hua; Zhang, Peng

    2016-08-01

    The ubiquitin-proteasome system plays a pivotal role in breast tumorigenesis by controlling transcription factors, thus promoting cell cycle growth, and degradation of tumor suppressor proteins. However, breast cancer patients have failed to benefit from proteasome inhibitor treatment partially due to proteasome heterogeneity, which is poorly understood in malignant breast neoplasm. Chemical crosslinking is an increasingly important tool for mapping protein three-dimensional structures and proteinprotein interactions. In the present study, two cross-linkers, bis (sulfosuccinimidyl) suberate (BS(3)) and its water-insoluble analog disuccinimidyl suberate (DSS), were used to map the subunit-subunit interactions in 20S proteasome core particle (CP) from MDA-MB-231 cells. Different types of gel electrophoresis technologies were used. In combination with chemical cross-linking and mass spectrometry, we applied these gel electrophoresis technologies to the study of the noncovalent interactions among 20S proteasome subunits. Firstly, the CP subunit isoforms were profiled. Subsequently, using native/SDSPAGE, it was observed that 0.5 mmol/L BS(3) was a relatively optimal cross-linking concentration for CP subunit-subunit interaction study. 2-DE analysis of the cross-linked CP revealed that α1 might preinteract with α2, and α3 might pre-interact with α4. Moreover, there were different subtypes of α1α2 and α3α4 due to proteasome heterogeneity. There was no significant difference in cross-linking pattern for CP subunits between BS(3) and DSS. Taken together, the gel-based characterization in combination with chemical cross-linking could serve as a tool for the study of subunit interactions within a multi-subunit protein complex. The heterogeneity of 20S proteasome subunit observed in breast cancer cells may provide some key information for proteasome inhibition strategy. PMID:27465334

  2. ARS5 is a component of the 26S proteasome complex, and negatively regulates thiol biosynthesis and arsenic tolerance in Arabidopsis.

    PubMed

    Sung, Dong-Yul; Kim, Tae-Houn; Komives, Elizabeth A; Mendoza-Cózatl, David G; Schroeder, Julian I

    2009-09-01

    A forward-genetic screen in Arabidopsis led to the isolation of several arsenic tolerance mutants. ars5 was the strongest arsenate- and arsenite-resistant mutant identified in this genetic screen. Here, we report the characterization and cloning of the ars5 mutant gene. ars5 is shown to exhibit an increased accumulation of arsenic and thiol compounds during arsenic stress. Rough mapping together with microarray-based expression mapping identified the ars5 mutation in the alpha subunit F (PAF1) of the 26S proteasome complex. Characterization of an independent paf1 T-DNA insertion allele and complementation by PAF1 confirmed that paf1 mutation is responsible for the enhanced thiol accumulation and arsenic tolerance phenotypes. Arsenic tolerance was not observed in a knock-out mutant of the highly homologous PAF2 gene. However, genetic complementation of ars5 by the overexpression of PAF2 suggests that the PAF2 protein is functionally equivalent to PAF1 when expressed at high levels. No detectible difference was observed in total ubiquitinylated protein profiles between ars5 and wild-type (WT) Arabidopsis, suggesting that the arsenic tolerance observed in ars5 is not derived from a general impairment in proteasome-mediated protein degradation. Quantitative RT-PCR showed that arsenic induces the enhanced transcriptional activation of several key genes that function in glutathione and phytochelatin biosynthesis in the WT, and this arsenic induction of gene expression is more dramatic in ars5. The enhanced transcriptional response to arsenic and the increased accumulation of thiol compounds in ars5, compared with WT, suggest the presence of a positive regulation pathway for thiol biosynthesis that is enhanced in the ars5 background. PMID:19453443

  3. ARS5 is a component of the 26S proteasome complex and negatively regulates thiol biosynthesis and arsenic tolerance in Arabidopsis

    PubMed Central

    Sung, Dong-Yul; Kim, Tae-Houn; Komives, Elizabeth A.; Mendoza-Cózatl, David G.; Schroeder, Julian I.

    2010-01-01

    Summary A forward genetic screen in Arabidopsis led to the isolation of several arsenic tolerance mutants. ars5 is the strongest arsenate and arsenite resistant mutant identified in this genetic screen. Here, we report the characterization and cloning of the ars5 mutant gene. ars5 is shown to exhibit an increased accumulation of arsenic and thiol compounds during arsenic stress. Rough mapping together with microarray-based expression mapping identified the ars5 mutation in the alpha subunit F (PAF1) of the 26S proteasome complex. Characterization of an independent paf1 T-DNA insertion allele and complementation by PAF1 confirmed that paf1 mutation is responsible for the enhanced thiol accumulation and the arsenic tolerance phenotypes. Arsenic tolerance was not observed in a knockout mutant of the highly homologous PAF2 gene. However, genetic complementation of ars5 by over expression of PAF2 suggests that the PAF2 protein is functionally equivalent to PAF1 when expressed at high levels. No detectible difference was observed in total ubiquitinylated protein profiles between ars5 and wild type Arabidopsis, suggesting that the arsenic tolerance observed in ars5 is not derived from a general impairment in proteasome-mediated protein degradation. Quantitative RT-PCR showed that arsenic induces enhanced transcriptional activation of several key genes that function in glutathione and phytochelatin biosynthesis in wild type and this arsenic-induction of gene expression is more dramatic in ars5. The enhanced transcriptional response to arsenic and the increased accumulation of thiol compounds in ars5 compared to WT suggest the presence of a positive regulation pathway for thiol biosynthesis that is enhanced in the ars5 background. PMID:19453443

  4. Use of missense proteasome subunits for conditional lethality in the tephritid fruit flies Anastrepha suspensa and Ceratitis capitata

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteasomes play a critical role in eukaryote development by regulating protein degradation. In Drosophila, mis-sense mutations in the 20S proteasome subunit lead to the production of dominant temperature-sensitive (DTS) "poison subunits" or antimorphs that disrupt proteasome function. DTS5 and DTS...

  5. Phosphorylation of the C-terminal tail of proteasome subunit α7 is required for binding of the proteasome quality control factor Ecm29

    PubMed Central

    Wani, Prashant S.; Suppahia, Anjana; Capalla, Xavier; Ondracek, Alex; Roelofs, Jeroen

    2016-01-01

    The proteasome degrades many short-lived proteins that are labeled with an ubiquitin chain. The identification of phosphorylation sites on the proteasome subunits suggests that degradation of these substrates can also be regulated at the proteasome. In yeast and humans, the unstructured C-terminal region of α7 contains an acidic patch with serine residues that are phosphorylated. Although these were identified more than a decade ago, the molecular implications of α7 phosphorylation have remained unknown. Here, we showed that yeast Ecm29, a protein involved in proteasome quality control, requires the phosphorylated tail of α7 for its association with proteasomes. This is the first example of proteasome phosphorylation dependent binding of a proteasome regulatory factor. Ecm29 is known to inhibit proteasomes and is often found enriched on mutant proteasomes. We showed that the ability of Ecm29 to bind to mutant proteasomes requires the α7 tail binding site, besides a previously characterized Rpt5 binding site. The need for these two binding sites, which are on different proteasome subcomplexes, explains the specificity of Ecm29 for proteasome holoenzymes. We propose that alterations in the relative position of these two sites in different conformations of the proteasome provides Ecm29 the ability to preferentially bind specific proteasome conformations. PMID:27302526

  6. Molecular cloning of the mouse proteasome subunits MC14 and MECL-1: reciprocally regulated tissue expression of interferon-gamma-modulated proteasome subunits.

    PubMed

    Stohwasser, R; Standera, S; Peters, I; Kloetzel, P M; Groettrup, M

    1997-05-01

    The primary structures of the interferon-gamma-inducible mouse 20S proteasome subunit MECL-1 and its alternate homolog MC14 were determined. Northern analysis of mouse tissues revealed that MECL-1 mRNA predominantly occurred in thymus, lymph nodes, and spleen, whereas small amounts were detected in non-lymphoid tissues such as kidney, muscle, and testis. Unexpectedly, probing RNA blots with MC14 showed that tissues with high MECL-1 expression contained little MC14 and vice versa. A very similar reciprocal tissue expression was subsequently found for the homologous subunit pairs LMP2 and delta as well as LMP7 and MB1. The subunit protein composition of 20S proteasomes purified from liver, thymus, and lung reflected RNA expression. The impact of a regulated reciprocal tissue expression is discussed with respect to thymic selection and the induction of tolerance in potentially autoreactive T cells. PMID:9174609

  7. Identification of proteasome subunit beta type 2 associated with deltamethrin detoxification in Drosophila Kc cells by cDNA microarray analysis and bioassay analyses.

    PubMed

    Hu, Junli; Jiao, Dongxu; Xu, Qin; Ying, Xiaoli; Liu, Wei; Chi, Qingping; Ye, Yuting; Li, Xueyu; Cheng, Luogen

    2016-05-10

    Insecticide deltamethrin resistance has presented a difficult obstacle for pest control and the resistance development is complex and associated with many genes. To better understand the possible molecular mechanisms involved in DM stress, in this study, cDNA microarray analysis was employed. 448 differentially expressed genes with at least a 2-fold expression difference were identified in Drosophila cells after DM exposure. Moreover, some genes were confirmed with qPCR, which yielded results consistent with the microarray analysis. Three members of the ubiquitin-proteasome system were significantly elevated in DM-stressed cells, suggesting that the ubiquitin-proteasome pathway may play an important role in DM detoxification. The proteasome beta2 subunit (Prosbeta2) is a member of 20S proteasome subunit family, which forms the proteolytic core of 26S proteasome. Whether Prosbeta2 participates in DM detoxification requires further study. RNAi and heterologous expression were conducted to investigate the contribution of Prosbeta2 in DM detoxification. The results revealed Prosbeta2 knockdown significantly reduce the level of DM detoxification in RNAi-treated cells after 48 h. Overexpression of Prosbeta2 increased cellular viability. These detoxification results represent the first evidence that Prosbeta2 plays a role in the detoxification of DM, which may provide new idea and target for studying the molecular mechanisms of insect resistance. PMID:26850132

  8. Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein.

    PubMed Central

    Hampton, R Y; Gardner, R G; Rine, J

    1996-01-01

    3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), a key enzyme of sterol synthesis, is an integral membrane protein of the endoplasmic reticulum (ER). In both humans and yeast, HMG-R is degraded at or in the ER. The degradation of HMG-R is regulated as part of feedback control of the mevalonate pathway. Neither the mechanism of degradation nor the nature of the signals that couple the degradation of HMG-R to the mevalonate pathway is known. We have launched a genetic analysis of the degradation of HMG-R in Saccharomyces cerevisiae using a selection for mutants that are deficient in the degradation of Hmg2p, an HMG-R isozyme. The underlying genes are called HRD (pronounced "herd"), for HMG-CoA reductase degradation. So far we have discovered mutants in three genes: HRD1, HRD2, and HRD3. The sequence of the HRD2 gene is homologous to the p97 activator of the 26S proteasome. This p97 protein, also called TRAP-2, has been proposed to be a component of the mature 26S proteasome. The hrd2-1 mutant had numerous pleiotropic phenotypes expected for cells with a compromised proteasome, and these phenotypes were complemented by the human TRAP-2/p97 coding region. In contrast, HRD1 and HRD3 genes encoded previously unknown proteins predicted to be membrane bound. The Hrd3p protein was homologous to the Caenorhabditis elegans sel-1 protein, a negative regulator of at least two different membrane proteins, and contained an HRD3 motif shared with several other proteins. Hrd1p had no full-length homologues, but contained an H2 ring finger motif. These data suggested a model of ER protein degradation in which the Hrd1p and Hrd3p proteins conspire to deliver HMG-R to the 26S proteasome. Moreover, our results lend in vivo support to the proposed role of the p97/TRAP-2/Hrd2p protein as a functionally important component of the 26S proteasome. Because the HRD genes were required for the degradation of both regulated and unregulated substrates of ER degradation, the HRD genes are the

  9. A third interferon-gamma-induced subunit exchange in the 20S proteasome.

    PubMed

    Groettrup, M; Kraft, R; Kostka, S; Standera, S; Stohwasser, R; Kloetzel, P M

    1996-04-01

    The 20S proteasome is a protease complex of functional importance for antigen processing. Two of the 14 proteasome subunits, delta and MB1, can be replaced by the major histocompatibility complex (MHC)-encoded and interferon-gamma (IFN-gamma)-inducible subunits LMP2 and LMP7, respectively. LMP2 and LMP7 alter the cleavage site specificity of the 20S proteasome and are required for the efficient generation of T cell epitopes from a number of viral proteins and for optimal MHC class I cell surface expression. We compared the 20S proteasome subunit pattern from IFN-gamma-induced and non-induced mouse fibroblasts on two-dimensional gels and identified a third subunit exchange by microsequencing: the non-MHC-encoded subunit MECL-1 is induced by IFN-gamma and replaces a sofar barely characterized beta subunit designated 'MC14'. In analogy to LMP2 and LMP7, MECL-1 may be functional in MHC class I-restricted antigen presentation. PMID:8625980

  10. [Cloning, expression and characterization of a gene encoding alpha2 subunit of the proteasome in Schistosoma japonicum].

    PubMed

    Hong, Yang; Han, Hongxiao; Peng, Jinbiao; Li, Ye; Shi, Yaojun; Fu, Zhiqiang; Liu, Jinming; Li, Xiangrui; Lin, Jiaojiao

    2010-04-01

    The 26S proteasome is a proteolytic complex responsible for the degradation of the vast majority of eukaryotic proteins. Regulated proteolysis by the proteasome is thought to influence cell cycle progression, transcriptional control, and other critical cellular processes. A novel Schistosoma japonicum gene (GenBank Accession No. AY813725) proteasome alpha2 subunit (SjPSMA2) was cloned. Sequence analysis revealed that the ORF of SjPSMA2 gene contains 708 nucleotides encoding 235 amino acids, and the molecular weight was estimated to be 25.84 kDa. Real-time PCR analysis showed that this gene expressed in 7 d, 13 d, 18 d, 23 d, 32 d and 42 d schistosoma. The mRNA level of SjPSMA2 was lower in 7 d and 23 d schistosomulum than that in other stages. The SjPSMA2 cDNA fragment was subcloned into an expression vector pET28a(+) and transformed into E. coli BL21 (DE3) cells. After induction with IPTCQ the 30 kDa fusion protein was produced as included bodies. Western-blotting revealed that the fusion protein could be recognized by the rabbit serum anti-Schistosoma japonicum adult worm antigen preparation, and the protein in native could be detected. After immunization of BALB/c mice with the fusion protein, the reduction rates of worm counts and liver egg counts were 12.33% and 35.23%. ELISA results revealed that the vaccinated group showed a significant increase in the level of IgG antibody. This study provided an important basis for investigating the regulation mechanism of the proteasome during the development of Schistosoma japonicum. PMID:20575440

  11. Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production.

    PubMed

    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 I; Rother, Kristina; Hildebrand, Peter W; Brogan, Paul; Krüger, Elke; Aksentijevich, Ivona; Goldbach-Mansky, Raphaela

    2015-11-01

    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

  12. 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

  13. Novel internally quenched substrate of the trypsin-like subunit of 20S eukaryotic proteasome.

    PubMed

    Gruba, Natalia; Wysocka, Magdalena; Brzezińska, Magdalena; Debowski, Dawid; Rolka, Krzysztof; Martin, Nathaniel I; Lesner, Adam

    2016-09-01

    This article describes the synthesis, using combinatorial chemistry, of internally quenched substrates of the trypsin-like subunit of human 20S proteasome. Such substrates were optimized in both the nonprime and prime regions of the peptide chain. Two were selected as the most susceptible for proteasomal proteolysis with excellent kinetic parameters: (i) ABZ-Val-Val-Ser-Arg-Ser-Leu-Gly-Tyr(3-NO2)-NH2 (kcat/KM = 934,000 M(-1) s(-1)) and (ii) ABZ-Val-Val-Ser-GNF-Ala-Met-Gly-Tyr(3-NO2)-NH2 (kcat/KM = 1,980,000 M(-1) s(-1)). Both compounds were efficiently hydrolyzed by the 20S proteasome at picomolar concentrations, demonstrating significant selectivity over other proteasome entities. PMID:26314791

  14. Degradation of the encephalomyocarditis virus and hepatitis A virus 3C proteases by the ubiquitin/26S proteasome system in vivo.

    PubMed

    Schlax, Peter E; Zhang, Jin; Lewis, Elizabeth; Planchart, Antonio; Lawson, T Glen

    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. PMID:17150238

  15. 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.

  16. The Arabidopsis F-Box Protein CORONATINE INSENSITIVE1 Is Stabilized by SCFCOI1 and Degraded via the 26S Proteasome Pathway[C][W

    PubMed Central

    Yan, Jianbin; Li, Haiou; Li, Shuhua; Yao, Ruifeng; Deng, Haiteng; Xie, Qi; Xie, Daoxin

    2013-01-01

    Jasmonate regulates critical aspects of plant development and defense. The F-box protein CORONATINE INSENSITIVE1 (COI1) functions as a jasmonate receptor and forms Skp1/Cullin1/F-box protein COI1 (SCFCOI1) complexes with Arabidopsis thaliana Cullin1 and Arabidopsis Skp1-like1 (ASK1) to recruit its substrate jasmonate ZIM-domain proteins for ubiquitination and degradation. Here, we reveal a mechanism regulating COI1 protein levels in Arabidopsis. Genetic and biochemical analysis and in vitro degradation assays demonstrated that the COI1 protein was initially stabilized by interacting with ASK1 and further secured by assembly into SCFCOI1 complexes, suggesting a function for SCFCOI1 in the stabilization of COI1 in Arabidopsis. Furthermore, we show that dissociated COI1 is degraded through the 26S proteasome pathway, and we identified the 297th Lys residue as an active ubiquitination site in COI1. Our data suggest that the COI1 protein is strictly regulated by a dynamic balance of SCFCOI1-mediated stabilization and 26S proteasome–mediated degradation and thus maintained at a protein level essential for proper biological functions in Arabidopsis development and defense responses. PMID:23386265

  17. Clinical activity of carfilzomib correlates with inhibition of multiple proteasome subunits: application of a novel pharmacodynamic assay.

    PubMed

    Lee, Susan J; Levitsky, Konstantin; Parlati, Francesco; Bennett, Mark K; Arastu-Kapur, Shirin; Kellerman, Lois; Woo, Tina F; Wong, Alvin F; Papadopoulos, Kyriakos P; Niesvizky, Ruben; Badros, Ashraf Z; Vij, Ravi; Jagannath, Sundar; Siegel, David; Wang, Michael; Ahmann, Gregory J; Kirk, Christopher J

    2016-06-01

    While proteasome inhibition is a validated therapeutic approach for multiple myeloma (MM), inhibition of individual constitutive proteasome (c20S) and immunoproteasome (i20S) subunits has not been fully explored owing to a lack of effective tools. We utilized the novel proteasome constitutive/immunoproteasome subunit enzyme-linked immunosorbent (ProCISE) assay to quantify proteasome subunit occupancy in samples from five phase I/II and II trials before and after treatment with the proteasome inhibitor carfilzomib. Following the first carfilzomib dose (15-56 mg/m(2) ), dose-dependent inhibition of c20S and i20S chymotrypsin-like active sites was observed [whole blood: ≥67%; peripheral blood mononuclear cells (PBMCs): ≥75%]. A similar inhibition profile was observed in bone marrow-derived CD138(+) tumour cells. Carfilzomib-induced proteasome inhibition was durable, with minimal recovery in PBMCs after 24 h but near-complete recovery between cycles. Importantly, the ProCISE assay can be used to quantify occupancy of individual c20S and i20S subunits. We observed a relationship between MM patient response (n = 29), carfilzomib dose and occupancy of multiple i20S subunits, where greater occupancy was associated with an increased likelihood of achieving a clinical response at higher doses. ProCISE represents a new tool for measuring proteasome inhibitor activity in clinical trials and relating drug action to patient outcomes. PMID:27071340

  18. Mutational analysis of subunit i beta2 (MECL-1) demonstrates conservation of cleavage specificity between yeast and mammalian proteasomes.

    PubMed

    Salzmann, U; Kral, S; Braun, B; Standera, S; Schmidt, M; Kloetzel, P M; Sijts, A

    1999-07-01

    Proteasomes are the major protein-degrading complexes in the cytosol and regulate many cellular processes. To examine the functional importance of the MC14/MECL-1 proteasome active site subunits, cell lines expressing a catalytically inactive form of MECL-1 were established. Whereas mutant MECL-1 was readily incorporated into cytosolic proteasomes, replacing the constitutive MC14 subunit, removal of the prosequence was incomplete indicating that its processing required autocatalytic cleavage. Functional analyses showed that the absence of the MC14/MECL-1 active sites abrogated proteasomal trypsin-like activity, but did not affect other catalytic activities. Our data demonstrate a conservation of cleavage specificity between mammalian and yeast proteasomes. PMID:10413086

  19. The subunits MECL-1 and LMP2 are mutually required for incorporation into the 20S proteasome

    PubMed Central

    Groettrup, Marcus; Standera, Sybille; Stohwasser, Ralf; Kloetzel, Peter M.

    1997-01-01

    Processing of antigens for presentation by major histocompatibility complex (MHC) class I molecules requires the activity of the proteasome. The 20S proteasome complex is composed of 14 different subunits, 2 of which can be substituted by the interferon γ (IFN-γ)-inducible and MHC-encoded subunits LMP2 and LMP7 (low molecular mass poylpeptides 2 and 7). A third subunit, MECL-1, is inducible by IFN-γ but is encoded outside the MHC. Here we show by cotransfection experiments that the incorporation of MECL-1 into the 20S proteasome is directly dependent on the expression of LMP2 but independent of LMP7. Conversely, the uptake of LMP2 is strongly enhanced by MECL-1 expression. The expression of MECL-1 caused a replacement of the homologous subunit Z in the 20S proteasome complex. LMP2 is required for MECL-1 incorporation at the level of proteasome precursor formation that guarantees the concerted incorporation of two IFN-γ-inducible proteasome subunits encoded inside and outside the MHC. The obligatory coincorporation of MECL-1 and LMP2 is an important parameter for the interpretation of results obtained with LMP2-deficient cell lines and mice as well as for the design of experiments addressing the function of MECL-1 in antigen presentation. PMID:9256419

  20. Plasminogen Activator Inhibitor Type 1 Interacts with α3 Subunit of Proteasome and Modulates Its Activity*

    PubMed Central

    Boncela, Joanna; Przygodzka, Patrycja; Papiewska-Pajak, Izabela; Wyroba, Elzbieta; Osinska, Magdalena; Cierniewski, Czeslaw S.

    2011-01-01

    Plasminogen activator inhibitor type-1 (PAI-1), a multifunctional protein, is an important physiological regulator of fibrinolysis, extracellular matrix homeostasis, and cell motility. Recent observations show that PAI-1 may also be implicated in maintaining integrity of cells, especially with respect to cellular proliferation or apoptosis. In the present study we provide evidence that PAI-1 interacts with proteasome and affects its activity. First, by using the yeast two-hybrid system, we found that the α3 subunit of proteasome directly interacts with PAI-1. Then, to ensure that the PAI-1-proteasome complex is formed in vivo, both proteins were coimmunoprecipitated from endothelial cells and identified with specific antibodies. The specificity of this interaction was evidenced after transfection of HeLa cells with pCMV-PAI-1 and coimmunoprecipitation of both proteins with anti-PAI-1 antibodies. Subsequently, cellular distribution of the PAI-1-proteasome complexes was established by immunogold staining and electron microscopy analyses. Both proteins appeared in a diffuse cytosolic pattern but also could be found in a dense perinuclear and nuclear location. Furthermore, PAI-1 induced formation of aggresomes freely located in endothelial cytoplasm. Increased PAI-1 expression abrogated degradation of degron analyzed after cotransfection of HeLa cells with pCMV-PAI-1 and pd2EGFP-N1 and prevented degradation of p53 as well as IκBα, as evidenced both by confocal microscopy and Western immunoblotting. PMID:21135093

  1. Natural alleles of a proteasome α2 subunit gene contribute to thermotolerance and adaptation of African rice.

    PubMed

    Li, Xin-Min; Chao, Dai-Yin; Wu, Yuan; Huang, Xuehui; Chen, Ke; Cui, Long-Gang; Su, Lei; Ye, Wang-Wei; Chen, Hao; Chen, Hua-Chang; Dong, Nai-Qian; Guo, Tao; Shi, Min; Feng, Qi; Zhang, Peng; Han, Bin; Shan, Jun-Xiang; Gao, Ji-Ping; Lin, Hong-Xuan

    2015-07-01

    Global warming threatens many aspects of human life, for example, by reducing crop yields. Breeding heat-tolerant crops using genes conferring thermotolerance is a fundamental way to help deal with this challenge. Here we identify a major quantitative trait locus (QTL) for thermotolerance in African rice (Oryza glaberrima), Thermo-tolerance 1 (TT1), which encodes an α2 subunit of the 26S proteasome involved in the degradation of ubiquitinated proteins. Ubiquitylome analysis indicated that OgTT1 protects cells from heat stress through more efficient elimination of cytotoxic denatured proteins and more effective maintenance of heat-response processes than achieved with OsTT1. Variation in TT1 has been selected for on the basis of climatic temperature and has had an important role in local adaptation during rice evolution. In addition, we found that overexpression of OgTT1 was associated with markedly enhanced thermotolerance in rice, Arabidopsis and Festuca elata. This discovery may lead to an increase in crop security in the face of the ongoing threat of global warming. PMID:25985140

  2. 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

  3. Spatial arrangement and functional role of α subunits of proteasome activator PA28 in hetero-oligomeric form

    SciTech Connect

    Sugiyama, Masaaki; Sahashi, Hiroki; Kurimoto, Eiji; Takata, Shin-ichi; Yagi, Hirokazu; Kanai, Keita; Sakata, Eri; Minami, Yasufumi; Tanaka, Keiji; Kato, Koichi

    2013-03-01

    Highlights: ► Homologous α and β subunits are alternatively arranged in the PA28 heptameric ring. ► The flexible loops of the three α subunits surround the site of substrate entry. ► The loops serve as gatekeepers that selectively hinder passage of longer peptides. - Abstract: A major form of proteasome activator PA28 is a heteroheptamer composed of interferon-γ-inducible α and β subunits, which share approximately 50% amino acid identity and possess distinct insert loops. This activator forms a complex with the 20S proteasome and thereby stimulates proteasomal degradation of peptides in an ATP-independent manner, giving rise to smaller antigenic peptides presented by major histocompatibility complex class I molecules. In this study, we performed biophysical and biochemical characterization of the structure and function of the PA28 hetero-oligomer. Deuteration-assisted small-angle neutron scattering demonstrated three α and four β subunits are alternately arranged in the heptameric ring. In this arrangement, PA28 loops surround the central pore of the heptameric ring (site for peptide entry). Activating the 20S proteasome with a PA28 mutant that lacked the α subunit loops cleaved model substrates longer than a nonapeptide with better efficiency when compared to wild-type PA28. Based on these data, we hypothesize that the flexible PA28 loops act as gatekeepers, which function to select the length of peptide substrates to be transported between the proteolytic chamber and the extra-proteasomal medium.

  4. alpha5 subunit in Trypanosoma brucei proteasome can self-assemble to form a cylinder of four stacked heptamer rings.

    PubMed Central

    Yao, Y; Toth, C R; Huang, L; Wong, M L; Dias, P; Burlingame, A L; Coffino, P; Wang, C C

    1999-01-01

    The proteasomes have a central role in catalysing protein degradation among both prokaryotes and eukaryotes. The 20 S proteasome constitutes their catalytic core. In studying the structure of Trypanosoma brucei 20 S proteasomes, we isolated by two-dimensional (2D) gel electrophoresis a 27 kDa subunit protein with an estimated pI of 4.7 and subjected it to mass spectrometric analysis. A tryptic peptide sequence from the protein was found identical with that of the rat alpha5 subunit. With the use of antiserum against T. brucei 20 S proteasomes to screen a T. b. rhodesiense lambda expression cDNA library, we obtained a cDNA clone encoding a full-length protein of 246 amino acid residues with a calculated molecular mass of 27174 Da and a pI of 4.71. It bears 50. 0% and 46.3% sequence identity with rat and yeast proteasome subunit alpha5 respectively, and matches all the peptide sequences derived from MS of the 2D gel-purified protein. The protein is thus designated the alpha5 subunit of T. brucei 20 S proteasome (TbPSA5). The recombinant protein, expressed in plasmid-transformed Escherichia coli, was found in a 27 kDa monomer form as well as polymerized forms with estimated molecular masses ranging from 190 to 800 kDa. Under the electron microscope, the most highly polymerized forms bear the appearance of cylinders of four-stacked heptamer rings with an estimated outer diameter of 14.5 nm and a length of 18 nm, which were immunoprecipitable by anti-(T. brucei 20 S proteasome) antiserum. In view of the documented self-assembly of the archaeon proteasome alpha subunit into double heptamer rings and the spontaneous assembly of the two alpha subunits from the 20 S proteasome of Rhodococcus erythropolis, the self-assembly of the T. brucei alpha subunit might reflect a common feature of proteasome biogenesis shared by prokaryotes and primitive eukaryotes such as the trypanosomes but apparently lost among the higher forms of eukaryote such as the yeast and the mammals. PMID

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

    PubMed

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

    2016-06-01

    Deregulation of the ubiquitin-proteasome pathway has been frequently observed in a number of malignancies. Using quantitative Western blotting of normal and matched tumour tissue, we here identified a significant increase in the 19S proteasome subunit Rpt4 in response to chemoradiation in locally advanced rectal cancer patients with unfavourable outcome. We therefore explored the potential of Rpt4 reduction as a therapeutic strategy in colorectal cancer (CRC). Utilizing siRNA to down regulate Rpt4 expression, we show that silencing of Rpt4 reduced proteasomal activity and induced endoplasmic reticulum stress. Gene silencing of Rpt4 also inhibited cell proliferation, reduced clonogenic survival and induced apoptosis in HCT-116 colon cancer cells. We next developed a cell penetrating peptide-based nanoparticle delivery system to achieve in vivo gene silencing of Rpt4. Administration of Rpt4 siRNA nanoparticles reduced tumour growth and improved survival in a HCT-116 colon cancer xenograft tumour model in vivo. Collectively, our data suggest that inhibition of Rpt4 represents a novel strategy for the treatment of CRC. PMID:26997367

  6. Inhibition on Proteasome β1 Subunit Might Contribute to the Anti-Cancer Effects of Fangchinoline in Human Prostate Cancer Cells

    PubMed Central

    Sun, Peng; Feng, Li-Xing; Liu, Miao; Hu, Li-Hong; Wu, Wan-Ying; Jiang, Bao-Hong; Yang, Min; Qu, Xiao-Bo; Guo, De-An; Liu, Xuan

    2015-01-01

    Fangchinoline is a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae S. Moore. Fangchinoline and its structure analogue, tetrandrine, exhibited direct binding affinity with recombinant human proteasome β1 subunit and also inhibited its activity in vitro. In cultured prostate PC-3 cells and LnCap cells, fangchinoline could dose-dependently inhibit cell proliferation and caspase-like activity of cellular proteasome which was mediated by proteasome β1 subunit. The inhibitive effect of fangchinoline on caspase-like activity of proteasome was also observed in purified human erythrocyte 20S proteasome. In PC-3 cells, fangchinoline induced cell cycle arrest at G0/G1 phase and apoptosis. Treatment of PC-3 tumor-bearing nude mice with fangchinoline inhibited tumor growth, induced apoptosis and also caused decrease in proteasome activities in tumor xenografts. Dose-dependent and time-dependent accumulation of ubiquitinated proteins and important proteasome substrates such as p27, Bax and IκB-α were observed in fangchinoline-treated cells. Over-expression of proteasome β1 subunit by plasmid transfection increased sensitivity of cells to the cytotoxicity of fangchinoline while knockdown of proteasome β1 subunit ameliorated cytotoxicity of fangchinoline in PC-3 cells. Results of the present study suggested that proteasome inhibition was involved in the anti-cancer effects of fangchinoline. Fangchinoline and its structure analogues might be new natural proteasome inhibitors targeting β1 subunit. PMID:26512898

  7. Inhibition on Proteasome β1 Subunit Might Contribute to the Anti-Cancer Effects of Fangchinoline in Human Prostate Cancer Cells.

    PubMed

    Li, Dong; Lu, Yu; Sun, Peng; Feng, Li-Xing; Liu, Miao; Hu, Li-Hong; Wu, Wan-Ying; Jiang, Bao-Hong; Yang, Min; Qu, Xiao-Bo; Guo, De-An; Liu, Xuan

    2015-01-01

    Fangchinoline is a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae S. Moore. Fangchinoline and its structure analogue, tetrandrine, exhibited direct binding affinity with recombinant human proteasome β1 subunit and also inhibited its activity in vitro. In cultured prostate PC-3 cells and LnCap cells, fangchinoline could dose-dependently inhibit cell proliferation and caspase-like activity of cellular proteasome which was mediated by proteasome β1 subunit. The inhibitive effect of fangchinoline on caspase-like activity of proteasome was also observed in purified human erythrocyte 20S proteasome. In PC-3 cells, fangchinoline induced cell cycle arrest at G0/G1 phase and apoptosis. Treatment of PC-3 tumor-bearing nude mice with fangchinoline inhibited tumor growth, induced apoptosis and also caused decrease in proteasome activities in tumor xenografts. Dose-dependent and time-dependent accumulation of ubiquitinated proteins and important proteasome substrates such as p27, Bax and IκB-α were observed in fangchinoline-treated cells. Over-expression of proteasome β1 subunit by plasmid transfection increased sensitivity of cells to the cytotoxicity of fangchinoline while knockdown of proteasome β1 subunit ameliorated cytotoxicity of fangchinoline in PC-3 cells. Results of the present study suggested that proteasome inhibition was involved in the anti-cancer effects of fangchinoline. Fangchinoline and its structure analogues might be new natural proteasome inhibitors targeting β1 subunit. PMID:26512898

  8. Structure of Rpn10 and Its Interactions with Polyubiquitin Chains and the Proteasome Subunit Rpn12*

    PubMed Central

    Riedinger, Christiane; Boehringer, Jonas; Trempe, Jean-Francois; Lowe, Edward D.; Brown, Nicholas R.; Gehring, Kalle; Noble, Martin E. M.; Gordon, Colin; Endicott, Jane A.

    2010-01-01

    Schizosaccharomyces pombe Rpn10 (SpRpn10) is a proteasomal ubiquitin (Ub) receptor located within the 19 S regulatory particle where it binds to subunits of both the base and lid subparticles. We have solved the structure of full-length SpRpn10 by determining the crystal structure of the von Willebrand factor type A domain and characterizing the full-length protein by NMR. We demonstrate that the single Ub-interacting motif (UIM) of SpRpn10 forms a 1:1 complex with Lys48-linked diUb, which it binds selectively over monoUb and Lys63-linked diUb. We further show that the SpRpn10 UIM binds to SpRpn12, a subunit of the lid subparticle, with an affinity comparable with Lys48-linked diUb. This is the first observation of a UIM binding other than a Ub fold and suggests that SpRpn12 could modulate the activity of SpRpn10 as a proteasomal Ub receptor. PMID:20739285

  9. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene.

    PubMed Central

    Kurtzman, C P; Robnett, C J

    1997-01-01

    Clinically important species of Candida and related organisms were compared for extent of nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region is sufficiently variable to allow reliable separation of all known clinically significant yeast species. Of the 204 described species examined, 21 appeared to be synonyms of previously described organisms. Phylogenetic relationships among the species are presented. PMID:9114410

  10. Proteasome β5i Subunit Deficiency Affects Opsonin Synthesis and Aggravates Pneumococcal Pneumonia

    PubMed Central

    Kirschner, Felicia; Reppe, Katrin; Andresen, Nadine; Witzenrath, Martin; Ebstein, Frédéric; Kloetzel, Peter-Michael

    2016-01-01

    Immunoproteasomes, harboring the active site subunits β5i/LMP7, β1i/LMP2, and β2i/MECL1 exert protective, regulatory or modulating functions during infection-induced immune responses. Immunoproteasomes are constitutively expressed in hematopoietic derived cells, constituting the first line of defense against invading pathogens. To clarify the impact of immunoproteasomes on the innate immune response against Streptococcus pneumoniae, we characterized the progression of disease and analyzed the systemic immune response in β5i/LMP7-/- mice. Our data show that β5i/LMP7 deficiency, which affected the subunit composition of proteasomes in murine macrophages and liver, was accompanied by reduced transcription of genes encoding immune modulating molecules such as pentraxins, ficolins, and collectins. The diminished opsonin expression suggested an impaired humoral immune response against invading pneumococci resulting in an aggravated systemic dissemination of S. pneumoniae in β5i/LMP7-/- mice. The impaired bacterial elimination in β5i/LMP7-/- mice was accompanied by an aggravated course of pneumonia with early mortality as a consequence of critical illness during the late phase of disease. In summary our results highlight an unsuspected role for immuno-subunits in modulating the innate immune response to extracellular bacterial infections. PMID:27100179

  11. Proteasome β5i Subunit Deficiency Affects Opsonin Synthesis and Aggravates Pneumococcal Pneumonia.

    PubMed

    Kirschner, Felicia; Reppe, Katrin; Andresen, Nadine; Witzenrath, Martin; Ebstein, Frédéric; Kloetzel, Peter-Michael

    2016-01-01

    Immunoproteasomes, harboring the active site subunits β5i/LMP7, β1i/LMP2, and β2i/MECL1 exert protective, regulatory or modulating functions during infection-induced immune responses. Immunoproteasomes are constitutively expressed in hematopoietic derived cells, constituting the first line of defense against invading pathogens. To clarify the impact of immunoproteasomes on the innate immune response against Streptococcus pneumoniae, we characterized the progression of disease and analyzed the systemic immune response in β5i/LMP7-/- mice. Our data show that β5i/LMP7 deficiency, which affected the subunit composition of proteasomes in murine macrophages and liver, was accompanied by reduced transcription of genes encoding immune modulating molecules such as pentraxins, ficolins, and collectins. The diminished opsonin expression suggested an impaired humoral immune response against invading pneumococci resulting in an aggravated systemic dissemination of S. pneumoniae in β5i/LMP7-/- mice. The impaired bacterial elimination in β5i/LMP7-/- mice was accompanied by an aggravated course of pneumonia with early mortality as a consequence of critical illness during the late phase of disease. In summary our results highlight an unsuspected role for immuno-subunits in modulating the innate immune response to extracellular bacterial infections. PMID:27100179

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

    PubMed Central

    Shi, Zhi; Li, Zenggang; Li, Zijian; Cheng, Kejun; Du, Yuhong; Fu, Haian; Khuri, Fadlo R.

    2014-01-01

    The cyclin-dependent kinase inhibitor 1A (CDKN1A), p21/Cip1, is a vital cell cycle regulator, dysregulation of which has been associated with a large number of human malignancies. One critical mechanism that controls p21 function is through its degradation, which allows the activation of its associated cell cycle promoting kinases, CDK2 and CDK4. Thus, delineating how p21 is stabilized and degraded will enhance our understanding of cell growth control and offer a basis for potential therapeutic interventions. Here, we report a novel regulatory mechanism that controls the dynamic status of p21 through its interaction with Cdk5 and Abl enzyme substrate 1 (Cables1). Cables1 has a proposed role as a tumor suppressor. We found that upregulation of Cables1 protein was correlated with increased half-life of p21 protein, which was attributed to Cables1/p21 complex formation and supported by their co-localization in the nucleus. Mechanistically, Cables1 interferes with the proteasome (Prosome, Macropain) subunit alpha type 3 (PSMA3) binding to p21 and protects p21 from PSMA3-mediated proteasomal degradation. Moreover, silencing of p21 partially reverses the ability of Cables1 to induce cell death and inhibit cell proliferation. In further support of a potential pathophysiological role of Cables1, the expression level of Cables1 is tightly associated with p21 in both cancer cell lines and human lung cancer patient tumor samples. Together, these results suggest Cables1 as a novel p21 regulator through maintaining p21 stability, and support the model that the tumor suppressive function of Cables1 occurs at least in part through enhancing the tumor suppressive activity of p21. PMID:24975575

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

    PubMed

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

    2016-07-15

    The ubiquitin/proteasome system is the major protein degradation pathway in eukaryotes with several key catalytic cores. Targeting the β5 subunit with small-molecule inhibitors is an established therapeutic strategy for hematologic cancers. Herein, we report a mouse-trap-like conformational change that influences molecular recognition depending on the substitution pattern of a bound ligand. Variation of the size of P1 residues from the highly β5-selective proteasome inhibitor BSc2118 allows for discrimination between inhibitory strength and substrate conversion. We found that increasing molecular size strengthens inhibition, whereas decreasing P1 size accelerates substrate conversion. Evaluation of substrate hydrolysis after silencing of β5 activity reveals significant residual activity for large residues exclusively. Thus, classification of the β5 subunit as chymotrypsin-like and the use of the standard tyrosine-containing substrate should be reconsidered. PMID:27111844

  14. The porcine gene TBP10 encodes a protein homologous to the human tat-binding protein/26S protease subunit family.

    PubMed

    Leeb, T; Rettenberger, G; Breech, J; Hameister, H; Brenig, B

    1996-03-01

    We have cloned a porcine gene, designated TBP1O, that belongs to the Tat-binding protein/26S protease subunit family. The genomic structure of the porcine TBP1O gene was analyzed after isolation of three overlapping genomic phage lambda clones. The TBP10 gene harbors 12 exons spanning 4.5 kb of chromosomal DNA. The TBP1O gene was assigned to Chromosome (Chr) 12 by fluorescence in situ hybridization (FISH) on metaphase chromosomes. The chromosomal location was confirmed by PCR analysis of a porcine-rodent hybrid cell panel. The TBP1O protein is encoded by a 1221 nucleotide cDNA and has a molecular mass of 45.6 kDa. The predicted amino acid sequence has highest similarity to the human and bovine p45 subunit of the 26S protease and the human transcription factor TRIP1. Further similarities were detected to the slime mold protein DdTBP1O and the Schizosaccharomyces pombe and Saccharomyces cerevisiae protein SUG1. Like DdTBP1O and other members of the protein family, the porcine TBP1O harbors a leucine zipper motif in the N-terminal region and a domain characteristics of ATP-dependent proteases in the C-terminal region. PMID:8833236

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

    PubMed

    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

  16. 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

  17. Localization of the proteasomal ubiquitin receptors Rpn10 and Rpn13 by electron cryomicroscopy

    PubMed Central

    Sakata, Eri; Bohn, Stefan; Mihalache, Oana; Kiss, Petra; Beck, Florian; Nagy, Istvan; Nickell, Stephan; Tanaka, Keiji; Saeki, Yasushi; Förster, Friedrich; Baumeister, Wolfgang

    2012-01-01

    Two canonical subunits of the 26S proteasome, Rpn10 and Rpn13, function as ubiquitin (Ub) receptors. The mutual arrangement of these subunits—and all other non-ATPase subunits—in the regulatory particle is unknown. Using electron cryomicroscopy, we calculated difference maps between wild-type 26S proteasome from Saccharomyces cerevisiae and deletion mutants (rpn10Δ, rpn13Δ, and rpn10Δrpn13Δ). These maps allowed us to localize the two Ub receptors unambiguously. Rpn10 and Rpn13 mapped to the apical part of the 26S proteasome, above the N-terminal coiled coils of the AAA-ATPase heterodimers Rpt4/Rpt5 and Rpt1/Rpt2, respectively. On the basis of the mutual positions of Rpn10 and Rpn13, we propose a model for polyubiquitin binding to the 26S proteasome. PMID:22215586

  18. The over-expression of the β2 catalytic subunit of the proteasome decreases homologous recombination and impairs DNA double-strand break repair in human cells.

    PubMed

    Collavoli, Anita; Comelli, Laura; Cervelli, Tiziana; Galli, Alvaro

    2011-01-01

    By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p. PMID:21660142

  19. The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

    PubMed Central

    Collavoli, Anita; Comelli, Laura; Cervelli, Tiziana; Galli, Alvaro

    2011-01-01

    By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p. PMID:21660142

  20. The 19S proteasome subunit Rpt3 regulates distribution of CENP-A by associating with centromeric chromatin.

    PubMed

    Kitagawa, Teppei; Ishii, Kojiro; Takeda, Kojiro; Matsumoto, Tomohiro

    2014-01-01

    CENP-A, a variant of histone H3, is incorporated into centromeric chromatin and plays a role during kinetochore establishment. In fission yeast, the localization of CENP-A is limited to a region spanning 10-20 kb of the core domain of the centromere. Here, we report a mutant (rpt3-1) in which this region is expanded to 40-70 kb. Likely due to abnormal distribution of CENP-A, this mutant exhibits chromosome instability and enhanced gene silencing. Interestingly, the rpt3(+) gene encodes a subunit of the 19S proteasome, which localizes to the nuclear membrane. Although Rpt3 associates with centromeric chromatin, the mutant protein has lost this localization. A loss of the cut8(+) gene encoding an anchor of the proteasome to the nuclear membrane causes similar phenotypes as observed in the rpt3-1 mutant. Thus, we propose that the proteasome (or its subcomplex) associates with centromeric chromatin and regulates distribution of CENP-A. PMID:24710126

  1. 1.15 Å resolution structure of the proteasome-assembly chaperone Nas2 PDZ domain

    SciTech Connect

    Singh, Chingakham R.; Lovell, Scott; Mehzabeen, Nurjahan; Chowdhury, Wasimul Q.; Geanes, Eric S.; Battaile, Kevin P.; Roelofs, Jeroen

    2014-03-25

    The proteasome-assembly chaperone Nas2 binds to the proteasome subunit Rpt5 using its PDZ domain. The structure of the Nas2 PDZ domain has been determined. The 26S proteasome is a 2.5 MDa protease dedicated to the degradation of ubiquitinated proteins in eukaryotes. The assembly of this complex containing 66 polypeptides is assisted by at least nine proteasome-specific chaperones. One of these, Nas2, binds to the proteasomal AAA-ATPase subunit Rpt5. The PDZ domain of Nas2 binds to the C-terminal tail of Rpt5; however, it does not require the C-terminus of Rpt5 for binding. Here, the 1.15 Å resolution structure of the PDZ domain of Nas2 is reported. This structure will provide a basis for further insights regarding the structure and function of Nas2 in proteasome assembly.

  2. Growth and developmental functions of a human immunodeficiency virus Tat-binding protein/26S protease subunit homolog from Dictyostelium discoideum.

    PubMed Central

    Cao, J G; Firtel, R A

    1995-01-01

    We have characterized a newly identified gene from Dictyostelium discoideum, DdTBP alpha, that encodes a member of the family of eukaryotic proteins. These proteins contain a conserved ATPase domain, include subunits of the 26S protease subunit, and are homologous to the mammalian human immunodeficiency virus Tat-binding protein TBP1. While information indicates that some family members are involved in the regulation of transcription in mammalian and yeast cells during growth, these proteins are also involved in other cellular functions, and nothing is known about their possible function in multicellular development. The Dictyostelium DdTBP alpha gene is developmentally regulated, with its expression at the highest levels occurring during growth and early development. The gene is present in two copies in the genome. Disruption of one copy by homologous recombination leads to aberrant morphogenesis, which lasts from the formation of the first finger until the onset of culmination. The gene appears to be essential for growth since we were unable to obtain a complete null phenotype and since expression of an inducible antisense construct in the partial null background resulted in cell death. Expression of the antisense construct during development accentuated the partial null phenotype and also resulted in very abnormal fruiting bodies. Overexpression of DdTBP alpha from its own promoter leads to very large multinucleated vegetative cells when the cells are grown in suspension culture. When the cells are plated onto petri dishes in growth medium, they rapidly split into multiple cells containing one to two nuclei, in a manner similar to that of wild-type cells. Overexpressing cells are significantly delayed in forming a multicellular aggregate, but development proceeds normally once the first finger stage is reached. The results indicate that DdTBP alpha plays an important role in regulating both growth and morphogenesis in D. discoideum. PMID:7862164

  3. 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

  4. IDENTIFICATION OF PROTEASOME ALPHA6 SUBUNIT ASSOCIATED WITH DELTAMETHRIN RESISTANCE IN Drosophila melanogaster Kc CELLS.

    PubMed

    Hu, Junli; Xu, Qin; Chi, Qingping; Liu, Wei; Li, Fengliang; Cheng, Luogen

    2016-02-01

    Differential expression of the proteasome alpha6 (prosalpha6) was previously reported between Plutella xylostella strains that are resistant or susceptible to the pesticide deltamethrin (DM). This finding indicated that the prosalpha6 may be involved in DM resistance. In this article, qPCR analysis revealed that the prosalpha6 was also significantly upregulated in Drosophila Kc cells treated with DM. To better understand the contribution of prosalpha6 in DM resistance, RNA interference, heterologous expression, and a proteasome inhibitor (MG-132) were used. MG-132 was used to suppress proteasomal activity, and the dsRNA was designed to block the function of prosalpha6. The results indicated that both MG-132 and prosalpha6 knockdown decreased the cellular viability following DM treatment. Prosalpha6 was cloned and transfected into Drosophila Kc cells. The result showed that overexpression of prosalpha6 in Drosophila Kc cells conferred some protection against DM. Taken together, our results indicate that prosalpha6 is involved in Drosophila cells DM resistance. PMID:26764169

  5. Structural disorder and its role in proteasomal degradation.

    PubMed

    Aufderheide, Antje; Unverdorben, Pia; Baumeister, Wolfgang; Förster, Friedrich

    2015-09-14

    The ubiquitin proteasome system is responsible for the controlled degradation of a vast number of intracellular proteins. It targets misfolded or otherwise aberrant proteins as well as proteins no longer needed at a given point in time. The 26S proteasome is a large macromolecular machine comprising 33 distinct subunits as well as a number of transiently associating cofactors. Being essentially a non-specific protease, specificity is conferred by the ubiquitin system, which selects and marks substrates for degradation. Here, we review our current understanding of the structure and function of the 26S proteasome; in doing so we highlight the role of disordered protein regions. Disordered segments in substrates promote their degradation, whereas low complexity regions prevent their proteolysis. In the 26S proteasome itself a main role of disordered segments seems to be rendering the ubiquitin receptors mobile, possibly supporting recruitment of polyubiquitylated substrates. Thus, these structural features of substrates as well as of the 26S proteasome itself likely play important roles at different stages of the protein degradation process. PMID:26226424

  6. cDNA cloning, characterization, and developmental expression of the 20S proteasome alpha5 subunit in the Mediterranean fruit fly Ceratitis capitata.

    PubMed

    Verras, Meletios; Gourzi, Polyxeni; Kalosaka, Katerina; Zacharopoulou, Antigone; Mintzas, Anastassios C

    2008-03-01

    In the present study, we report the cDNA cloning, characterization, and developmental expression of the 20S proteasome alpha5 subunit from the Mediterranean fruit fly Ceratitis capitata (medfly). Using an RT-PCR fragment that corresponds to the amino-terminal region of the Drosophila melanogaster 20S proteasome alpha5 subunit, we isolated a 987-bp cDNA that encodes the complete coding region of the medfly ortholog, which was named CcPSMA5. CcPSMA5 consists of 241 amino acids and has a predicted molecular weight of 26.4 kDa and pI 4.75. Comparison of the CcPSMA5 amino acid sequence with the sequences of all known 20S proteasome alpha5 subunits from different organisms indicated that the medfly 20S proteasome alpha5 subunit has the strongest homology to that of Drosophila. In situ hybridization showed that the CcPSMA5 gene is mapped in the region 44B of chromosome 4. Northern blot hybridization analysis showed that the CcPSMA5 mRNA has a size of approximately 1.2 kb. High levels of the CcPSMA5 mRNA were detected in freshly laid eggs, indicating that they were maternally deposited. The mRNA expression pattern during medfly development suggests that the CcPSMA5 gene is upregulated before mid-embryogenesis and at the onset of metamorphosis. PMID:18163525

  7. Protein Interaction between Ameloblastin and Proteasome Subunit α Type 3 Can Facilitate Redistribution of Ameloblastin Domains within Forming Enamel.

    PubMed

    Geng, Shuhui; White, Shane N; Paine, Michael L; Snead, Malcolm L

    2015-08-21

    Enamel is a bioceramic tissue composed of thousands of hydroxyapatite crystallites aligned in parallel within boundaries fabricated by a single ameloblast cell. Enamel is the hardest tissue in the vertebrate body; however, it starts development as a self-organizing assembly of matrix proteins that control crystallite habit. Here, we examine ameloblastin, a protein that is initially distributed uniformly across the cell boundary but redistributes to the lateral margins of the extracellular matrix following secretion thus producing cell-defined boundaries within the matrix and the mineral phase. The yeast two-hybrid assay identified that proteasome subunit α type 3 (Psma3) interacts with ameloblastin. Confocal microscopy confirmed Psma3 co-distribution with ameloblastin at the ameloblast secretory end piece. Co-immunoprecipitation assay of mouse ameloblast cell lysates with either ameloblastin or Psma3 antibody identified each reciprocal protein partner. Protein engineering demonstrated that only the ameloblastin C terminus interacts with Psma3. We show that 20S proteasome digestion of ameloblastin in vitro generates an N-terminal cleavage fragment consistent with the in vivo pattern of ameloblastin distribution. These findings suggest a novel pathway participating in control of protein distribution within the extracellular space that serves to regulate the protein-mineral interactions essential to biomineralization. PMID:26070558

  8. Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S β5-subunit

    PubMed Central

    Blackburn, Christopher; Gigstad, Kenneth M.; Hales, Paul; Garcia, Khristofer; Jones, Matthew; Bruzzese, Frank J.; Barrett, Cynthia; Liu, Jane X.; Soucy, Teresa A.; Sappal, Darshan S.; Bump, Nancy; Olhava, Edward J.; Fleming, Paul; Dick, Lawrence R.; Tsu, Christopher; Sintchak, Michael D.; Blank, Jonathan L.

    2010-01-01

    The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome used on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin–proteasome system in cells. We show that these compounds are entirely selective for the β5 (chymotrypsin-like) site over the β1 (caspase-like) and β2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC50 values for the human 20S β5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin–luciferase reporter, activation of NFκB (nuclear factor κB) in response to TNF-α (tumour necrosis factor-α) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the β5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the β5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells. PMID:20632995

  9. Characterization of a new series of non-covalent proteasome inhibitors with exquisite potency and selectivity for the 20S [beta]5-subunit

    SciTech Connect

    Blackburn, Christopher; Gigstad, Kenneth M.; Hales, Paul; Garcia, Khristofer; Jones, Matthew; Bruzzese, Frank J.; Barrett, Cynthia; Liu, Jane X.; Soucy, Teresa A.; Sappal, Darshan S.; Bump, Nancy; Olhava, Edward J.; Fleming, Paul; Dick, Lawrence R.; Tsu, Christopher; Sintchak, Michael D.; Blank, Jonathan L.

    2012-04-30

    The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome used on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin-proteasome system in cells. We show that these compounds are entirely selective for the {beta}5 (chymotrypsin-like) site over the {beta}1 (caspase-like) and {beta}2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC{sub 50} values for the human 20S {beta}5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin-luciferase reporter, activation of NF{Kappa}B (nuclear factor {Kappa}B) in response to TNF-{alpha} (tumor necrosis factor-{alpha}) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the {beta}5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the {beta}5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.

  10. Association Analysis of Proteasome Subunits and Transporter Associated with Antigen Processing on Chinese Patients with Parkinson's Disease

    PubMed Central

    Mo, Ming-Shu; Huang, Wei; Sun, Cong-Cong; Zhang, Li-Min; Cen, Luan; Xiao, You-Sheng; Li, Guo-Fei; Yang, Xin-Ling; Qu, Shao-Gang; Xu, Ping-Yi

    2016-01-01

    Background: Proteasome subunits (PSMB) and transporter associated with antigen processing (TAP) loci are located in the human leukocyte antigen (HLA) Class II region play important roles in immune response and protein degradation in neurodegenerative diseases. This study aimed to explore the association between single nucleotide polymorphisms (SNPs) of PSMB and TAP and Parkinson's disease (PD). Methods: A case–control study was conducted by genotyping SNPs in PSMB8, PSMB9, TAP1, and TAP2 genes in the Chinese population. Subjects included 542 sporadic patients with PD and 674 healthy controls. Nine identified SNPs in PSMB8, PSMB9, TAP1, and TAP2 were genotyped through SNaPshot testing. Results: The stratified analysis of rs17587 was specially performed on gender. Data revealed that female patients carry a higher frequency of rs17587-G/G versus (A/A + G/A) compared with controls. But there was no significant difference with respect to the genotypic frequencies of the SNPs in PSMB8, TAP1, and TAP2 loci in PD patients. Conclusion: Chinese females carrying the rs17587-G/G genotype in PSMB9 may increase a higher risk for PD, but no linkage was found between other SNPs in HLA Class II region and PD. PMID:27098790

  11. Genetics of proteasome diseases.

    PubMed

    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

  12. Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome

    PubMed Central

    Tsvetkov, Peter; Mendillo, Marc L; Zhao, Jinghui; Carette, Jan E; Merrill, Parker H; Cikes, Domagoj; Varadarajan, Malini; van Diemen, Ferdy R; Penninger, Josef M; Goldberg, Alfred L; Brummelkamp, Thijn R; Santagata, Sandro; Lindquist, Susan

    2015-01-01

    Proteasomes are central regulators of protein homeostasis in eukaryotes. Proteasome function is vulnerable to environmental insults, cellular protein imbalance and targeted pharmaceuticals. Yet, mechanisms that cells deploy to counteract inhibition of this central regulator are little understood. To find such mechanisms, we reduced flux through the proteasome to the point of toxicity with specific inhibitors and performed genome-wide screens for mutations that allowed cells to survive. Counter to expectation, reducing expression of individual subunits of the proteasome's 19S regulatory complex increased survival. Strong 19S reduction was cytotoxic but modest reduction protected cells from inhibitors. Protection was accompanied by an increased ratio of 20S to 26S proteasomes, preservation of protein degradation capacity and reduced proteotoxic stress. While compromise of 19S function can have a fitness cost under basal conditions, it provided a powerful survival advantage when proteasome function was impaired. This means of rebalancing proteostasis is conserved from yeast to humans. DOI: http://dx.doi.org/10.7554/eLife.08467.001 PMID:26327695

  13. Evolution of Proteasome Regulators in Eukaryotes

    PubMed Central

    Fort, Philippe; Kajava, Andrey V.; Delsuc, Fredéric; Coux, Olivier

    2015-01-01

    All living organisms require protein degradation to terminate biological processes and remove damaged proteins. One such machine is the 20S proteasome, a specialized barrel-shaped and compartmentalized multicatalytic protease. The activity of the 20S proteasome generally requires the binding of regulators/proteasome activators (PAs), which control the entrance of substrates. These include the PA700 (19S complex), which assembles with the 20S and forms the 26S proteasome and allows the efficient degradation of proteins usually labeled by ubiquitin tags, PA200 and PA28, which are involved in proteolysis through ubiquitin-independent mechanisms and PI31, which was initially identified as a 20S inhibitor in vitro. Unlike 20S proteasome, shown to be present in all Eukaryotes and Archaea, the evolutionary history of PAs remained fragmentary. Here, we made a comprehensive survey and phylogenetic analyses of the four types of regulators in 17 clades covering most of the eukaryotic supergroups. We found remarkable conservation of each PA700 subunit in all eukaryotes, indicating that the current complex PA700 structure was already set up in the last eukaryotic common ancestor (LECA). Also present in LECA, PA200, PA28, and PI31 showed a more contrasted evolutionary picture, because many lineages have subsequently lost one or two of them. The paramount conservation of PA700 composition in all eukaryotes and the dynamic evolution of PA200, PA28, and PI31 are discussed in the light of current knowledge on their physiological roles. PMID:25943340

  14. Site-specific Proteasome Phosphorylation Controls Cell Proliferation and Tumorigenesis

    PubMed Central

    Guo, Xing; Wang, Xiaorong; Wang, Zhiping; Banerjee, Sourav; Yang, Jing; Huang, Lan; Dixon, Jack E.

    2015-01-01

    Despite the fundamental importance of proteasomal degradation in cells, little is known about whether and how the 26S proteasome itself is regulated in coordination with various physiological processes. Here we show that the proteasome is dynamically phosphorylated during cell cycle at Thr25 of the 19S subunit Rpt3. CRISPR/Cas9-mediated genome editing, RNA interference and biochemical studies demonstrate that blocking Rpt3-Thr25 phosphorylation markedly impairs proteasome activity and impedes cell proliferation. Through a kinome-wide screen, we have identified dual-specificity tyrosine-regulated kinase 2 (DYRK2) as the primary kinase that phosphorylates Rpt3-Thr25, leading to enhanced substrate translocation and degradation. Importantly, loss of the single phosphorylation of Rpt3-Thr25 or knockout of DYRK2 significantly inhibits tumor formation by proteasome-addicted human breast cancer cells in mice. These findings define an important mechanism for proteasome regulation and demonstrate the biological significance of proteasome phosphorylation in regulating cell proliferation and tumorigenesis. PMID:26655835

  15. Overexpression of Dsk2/dUbqln results in severe developmental defects and lethality in Drosophila melanogaster that can be rescued by overexpression of the p54/Rpn10/S5a proteasomal subunit.

    PubMed

    Lipinszki, Zoltán; Pál, Margit; Nagy, Olga; Deák, Péter; Hunyadi-Gulyas, Eva; Udvardy, Andor

    2011-12-01

    Polyubiquitin receptors execute the targeting of polyubiquitylated proteins to the 26S proteasome. In vitro studies indicate that disturbance of the physiological balance among different receptor proteins impairs the proteasomal degradation of polyubiquitylated proteins. To study the physiological consequences of shifting the in vivo equilibrium between the p54/Rpn10 proteasomal and the Dsk2/dUbqln extraproteasomal polyubiquitin receptors, transgenic Drosophila lines were constructed in which the overexpression or RNA interference-mediated silencing of these receptors can be induced. Flies overexpressing Flag-p54 were viable and fertile, without any detectable morphological abnormalities, although detectable accumulation of polyubiquitylated proteins demonstrated a certain level of proteolytic disturbance. Flag-p54 was assembled into the 26S proteasome and could fully complement the lethal phenotype of a p54 null mutant Drosophila line. The overexpression of Dsk2 caused severe morphological abnormalities in the late pupal stages, leading to pharate adult lethality, accompanied by a huge accumulation of highly polyubiquitylated proteins. The lethal phenotype of Dsk2 overexpression could be rescued in a double transgenic line coexpressing Flag-Dsk2 and Flag-p54. Although the double transgenic line was viable and fertile, it did not restore the proteolytic defects; the accumulation of the highly polyubiquitylated proteins was even more severe in the double transgenic line. Significant differences were found in the Dsk2-26S proteasome interaction in Drosophila melanogaster as compared with Saccharomyces cerevisiae. In yeast, Dsk2 can interact only with ΔRpn10 proteasomes and not with the wild-type one. In Drosophila, Dsk2 does not interact with Δp54 proteasomes, but the interaction can be fully restored by complementing the Δp54 deletion with Flag-p54. PMID:21973017

  16. Proteasome activation is a mechanism for pyrazolone small molecules displaying therapeutic potential in amyotrophic lateral sclerosis.

    PubMed

    Trippier, Paul C; Zhao, Kevin Tianmeng; Fox, Susan G; Schiefer, Isaac T; Benmohamed, Radhia; Moran, Jason; Kirsch, Donald R; Morimoto, Richard I; Silverman, Richard B

    2014-09-17

    Amyotrophic lateral sclerosis (ALS) is a progressive and ultimately fatal neurodegenerative disease. Pyrazolone containing small molecules have shown significant disease attenuating efficacy in cellular and murine models of ALS. Pyrazolone based affinity probes were synthesized to identify high affinity binding partners and ascertain a potential biological mode of action. Probes were confirmed to be neuroprotective in PC12-SOD1(G93A) cells. PC12-SOD1(G93A) cell lysates were used for protein pull-down, affinity purification, and subsequent proteomic analysis using LC-MS/MS. Proteomics identified the 26S proteasome regulatory subunit 4 (PSMC1), 26S proteasome regulatory subunit 6B (PSMC4), and T-complex protein 1 (TCP-1) as putative protein targets. Coincubation with appropriate competitors confirmed the authenticity of the proteomics results. Activation of the proteasome by pyrazolones was demonstrated in the absence of exogenous proteasome inhibitor and by restoration of cellular protein degradation of a fluorogenic proteasome substrate in PC12-SOD1(G93A) cells. Importantly, supplementary studies indicated that these molecules do not induce a heat shock response. We propose that pyrazolones represent a rare class of molecules that enhance proteasomal activation in the absence of a heat shock response and may have therapeutic potential in ALS. PMID:25001311

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

    PubMed Central

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

    2013-01-01

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

  18. Linkage of TATA-binding protein and proteasome subunit C5 genes in mice and humans reveals synteny conserved between mammals and invertebrates.

    PubMed

    Trachtulec, Z; Hamvas, R M; Forejt, J; Lehrach, H R; Vincek, V; Klein, J

    1997-08-15

    The TATA-binding protein (TBP) is a factor required for the transcription of all classes of eukaryotic genes. Here, we demonstrate that in the mouse the TBP-encoding gene (Tbp) resides next to the proteasomal subunit C5-encoding gene (Psmb1). The genes are located on mouse chromosome 17 in the t complex within the Hybrid sterility 1 (Hst1) region. We demonstrate that the homologous human genes (TBP AND PSMB1) are tightly linked on the long arm of chromosome 6, in a region syntenic with the proximal part of mouse chromosome 17. The mouse Tbp and Psmb1 and the human TBP and PSMB1 genes are transcribed in the opposite orientation. The TATA-binding protein and proteasomal subunit C5 genes are also linked on chromosome III of Caenorhabditis elegans, and together they are linked to other genes whose homologs map to human chromosome 6 and mouse chromosome 17. In the Drosophila genome, the housekeeping TATA-binding protein gene maps close to two other genes with homologs in the mammalian major histocompatibility complex. There thus exists conserved synteny of unrelated genes between mammals and invertebrates. PMID:9286694

  19. cAMP stimulates the ubiquitin/proteasome pathway in rat spinal cord neurons.

    PubMed

    Myeku, Natura; Wang, Hu; Figueiredo-Pereira, Maria E

    2012-10-11

    Proteasome impairment and accumulation of ubiquitinated proteins are implicated in neurodegeneration associated with different forms of spinal cord injury. We show herein that elevating cAMP in rat spinal cord neurons increases 26S proteasome activity in a protein kinase A-dependent manner. Treating spinal cord neurons with dibutyryl-cAMP (db-cAMP) also raised the levels of various components of the UPP including proteasome subunits Rpt6 and β5, polyubiquitin shuttling factor p62/sequestosome1, E3 ligase CHIP, AAA-ATPase p97 and the ubiquitin gene ubB. Finally, db-cAMP reduced the accumulation of ubiquitinated proteins, proteasome inhibition, and neurotoxicity triggered by the endogenous product of inflammation prostaglandin J2. We propose that optimizing the effects of cAMP/PKA-signaling on the UPP could offer an effective therapeutic approach to prevent UPP-related proteotoxicity in spinal cord neurons. PMID:22982149

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

    PubMed

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

    2008-06-01

    In amyotrophic lateral sclerosis caused by mutations in Cu/Zn-superoxide dismutase (SOD1), altered solubility and aggregation of the mutant protein implicates failure of pathways for detecting and catabolizing misfolded proteins. Our previous studies demonstrated early reduction of proteasome-mediated proteolytic activity in lumbar spinal cord of SOD1(G93A) transgenic mice, tissue particularly vulnerable to disease. The purpose of this study was to identify any underlying abnormalities in proteasomal structure. In lumbar spinal cord of pre-symptomatic mice [postnatal day 45 (P45) and P75], normal levels of structural 20S alpha subunits were incorporated into 20S/26S proteasomes; however, proteasomal complexes separated by native gel electrophoresis showed decreased immunoreactivity with antibodies to beta3, a structural subunit of the 20S proteasome core, and beta5, the subunit with chymotrypsin-like activity. This occurred prior to increase in beta5i immunoproteasomal subunit. mRNA levels were maintained and no association of mutant SOD1 with proteasomes was identified, implicating post-transcriptional mechanisms. mRNAs also were maintained in laser captured motor neurons at a later stage of disease (P100) in which multiple 20S proteins are reduced relative to the surrounding neuropil. Increase in detergent-insoluble, ubiquitinated proteins at P75 provided further evidence of stress on mechanisms of protein quality control in multiple cell types prior to significant motor neuron death. PMID:18315558

  1. Biochemical analysis of proteasomes from mouse microglia: induction of immunoproteasomes by interferon-gamma and lipopolysaccharide.

    PubMed

    Stohwasser, R; Giesebrecht, J; Kraft, R; Müller, E C; Häusler, K G; Kettenmann, H; Hanisch, U K; Kloetzel, P M

    2000-02-15

    The 20S proteasome is a multicatalytic threonine protease and serves to process peptides that are subsequently presented as antigenic epitopes by MHC class I molecules. In the brain, microglial cells are the major antigen presenting cells and they respond sensitive to pathologic events. We used cultured mouse microglia and a microglial cell line, the BV-2 line, as a model to study the correlation between microglial activation parameters and structural plasticity of the 20S/26S proteasome. Lipopolysaccharide (LPS)- or interferon-gamma (IFN-gamma)-stimulated microglia or BV-2 cells exhibit properties of activated microglia such as high levels of TNFalpha and IL-6 release. In response to IFN-gamma or LPS, three constitutive beta subunits (beta1/Delta, beta2/MC14, beta5/MB1) were replaced by the immunoproteasome subunits ibeta1/LMP2, ibeta2/MECL-1, and ibeta5/LMP7, indicating that activated microglia adapts its proteasomal subunit composition to the requirements of an optimized MHC class I epitope processing. Induction of immunoproteasomes in BV-2 cells was solely provoked by IFN-gamma, but not by LPS. Moreover, LPS (but not IFN-gamma) triggered the expression of a novel protein of approximately 50 kD as part of the proteasome activator PA700, that is the substrate-recognizing and unfolding unit of the 26S proteasome. These results indicate that both the 20S core protease as well as the proteasome activator PA700 are targets of modulatory subunit replacements or transient association of regulatory components in the course of microglial activation. PMID:10652445

  2. The effects of anti-DNA topoisomerase II drugs, etoposide and ellipticine, are modified in root meristem cells of Allium cepa by MG132, an inhibitor of 26S proteasomes.

    PubMed

    Żabka, Aneta; Winnicki, Konrad; Polit, Justyna Teresa; Maszewski, Janusz

    2015-11-01

    DNA topoisomerase II (Topo II), a highly specialized nuclear enzyme, resolves various entanglement problems concerning DNA that arise during chromatin remodeling, transcription, S-phase replication, meiotic recombination, chromosome condensation and segregation during mitosis. The genotoxic effects of two Topo II inhibitors known as potent anti-cancer drugs, etoposide (ETO) and ellipticine (EPC), were assayed in root apical meristem cells of Allium cepa. Despite various types of molecular interactions between these drugs and DNA-Topo II complexes at the chromatin level, which have a profound negative impact on the genome integrity (production of double-strand breaks, chromosomal bridges and constrictions, lagging fragments of chromosomes and their uneven segregation to daughter cell nuclei), most of the elicited changes were apparently similar, regarding both their intensity and time characteristics. No essential changes between ETO- and EPC-treated onion roots were noticed in the frequency of G1-, S-, G2-and M-phase cells, nuclear morphology, chromosome structures, tubulin-microtubule systems, extended distribution of mitosis-specific phosphorylation sites of histone H3, and the induction of apoptosis-like programmed cell death (AL-PCD). However, the important difference between the effects induced by the ETO and EPC concerns their catalytic activities in the presence of MG132 (proteasome inhibitor engaged in Topo II-mediated formation of cleavage complexes) and relates to the time-variable changes in chromosomal aberrations and AL-PCD rates. This result implies that proteasome-dependent mechanisms may contribute to the course of physiological effects generated by DNA lesions under conditions that affect the ability of plant cells to resolve topological problems that associated with the nuclear metabolic activities. PMID:26233708

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

    PubMed

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

    2016-08-01

    The 20S catalytic core of the human 26S proteasome can be secreted from cells, and high levels of extracellular 20S proteasome have been linked to many types of cancers and autoimmune diseases. Several diagnostic approaches have been developed that detect 20S proteasome activity in plasma, but these suffer from problems with efficiency and sensitivity. In this report, we describe the optimization and synthesis of an internally quenched fluorescent substrate of the 20S proteasome, and investigate its use as a potential diagnostic test in bladder cancer. This peptide, 2-aminobenzoic acid (ABZ)-Val-Val-Ser-Tyr-Ala-Met-Gly-Tyr(3-NO2 )-NH2 , is cleaved by the chymotrypsin 20S proteasome subunit and displays an excellent specificity constant value (9.7 × 10(5)  m(-1) ·s(-1) ) and a high kcat (8 s(-1) ). Using this peptide, we identified chymotrypsin-like proteasome activity in the majority of urine samples obtained from patients with bladder cancer, whereas the proteasome activity in urine samples from healthy volunteers was below the detection limit (0.5 pm). These findings were confirmed by an inhibitory study and immunochemistry methods. PMID:27326540

  4. Molecular Architecture and Assembly of the Eukaryotic Proteasome

    PubMed Central

    Tomko, Robert J.; Hochstrasser, Mark

    2013-01-01

    The eukaryotic ubiquitin-proteasome system is responsible for most cellular quality-control and regulatory protein degradation. Its substrates, which are usually modified by polymers of ubiquitin, are ultimately degraded by the 26S proteasome. This 2.6 MDa protein complex is separated into a barrel-shaped proteolytic 20S core particle (CP) of 28 subunits capped on one or both ends by a 19S regulatory particle (RP) comprising at least 19 subunits. The RP coordinates substrate recognition, removal of substrate polyubiquitin chains, and substrate unfolding and translocation into the CP for degradation. While many atomic structures of the CP have been determined, the RP has resisted high-resolution analysis. Recently, however, a combination of cryo-electron microscopy (cryo-EM), biochemical analysis, and crystal structure determination of several RP subunits has yielded a near-atomic resolution view of much of the complex. Major new insights into chaperone-assisted proteasome assembly have also recently been made. Here we review these novel findings. PMID:23495936

  5. The proteasome: a macromolecular assembly designed for controlled proteolysis.

    PubMed Central

    Zwickl, P; Voges, D; Baumeister, W

    1999-01-01

    In eukaryotic cells, the vast majority of proteins in the cytosol and nucleus are degraded via the proteasome-ubiquitin pathway. The 26S proteasome is a huge protein degradation machine of 2.5 MDa, built of approximately 35 different subunits. It contains a proteolytic core complex, the 20S proteasome and one or two 19S regulatory complexes which associate with the termini of the barrel-shaped 20S core. The 19S regulatory complex serves to recognize ubiquitylated target proteins and is implicated to have a role in their unfolding and translocation into the interior of the 20S complex where they are degraded into oligopeptides. While much progress has been made in recent years in elucidating the structure, assembly and enzymatic mechanism of the 20S complex, our knowledge of the functional organization of the 19S regulator is rather limited. Most of its subunits have been identified, but specific functions can be assigned to only a few of them. PMID:10582236

  6. Structural Biology of the Proteasome

    PubMed Central

    Kish-Trier, Erik

    2016-01-01

    The proteasome refers to a collection of complexes centered on the 20S proteasome core particle, a complex of 28 subunits that houses proteolytic sites in its hollow interior. Proteasomes are found in eukaryotes, archaea, and some eubacteria, and their activity is critical for many cellular pathways. Important advances include inhibitor binding studies and the structure of the immunoproteasome, whose specificity is altered by incorporation of inducible catalytic subunits. The inherent repression of the 20S CP is relieved by the ATP-independent activators, 11S and Blm10/PA200, whose structures reveal principles of proteasome mechanism. The structure of the ATP-dependent 19S regulatory particle, which mediates degradation of polyubiquitylated proteins, is being revealed by a combination of crystal or NMR structures of individual subunits and electron microscopy reconstruction of the intact complex. Other recent structural advances inform about mechanisms of assembly and the role of conformational changes in the functional cycle. PMID:23414347

  7. Docking of the Proteasomal ATPases’ C-termini in the 20S Proteasomes alpha Ring Opens the Gate for Substrate Entry

    PubMed Central

    Smith, David M.; Chang, Shih-Chung; Park, Soyeon; Finley, Daniel; Cheng, Yifan; Goldberg, Alfred

    2007-01-01

    Summary The 20S proteasome functions in protein degradation in eukaryotes together with the 19S ATPases or in archaea with the homologous PAN ATPase complex. PAN and the 19S ATPases contain a conserved C-terminal hydrophobic-tyrosine-X motif (HbYX). We show that these residues are essential for PAN to associate with the 20S and open its gated-channel for substrate entry. Upon ATP binding, these C-terminal residues bind to pockets between the 20S’s α-subunits. Furthermore, seven-residue peptides from PAN’s C-terminus that contain the HbYX motif also bind to these sites and induce gate-opening in both archaeal and mammalian 20S proteasomes. Gate-opening could be induced by short C-terminal peptides from the 19S ATPase subunits, Rpt2 and Rpt5, but not by ones from PA28/26, which lack the HbYX motif and cause gate-opening by distinct mechanism. The C-terminal YX residues in the 19S ATPases were also shown to be critical for gating and stability of mammalian and yeast 26S proteasomes. Thus, the C-termini of the proteasomal ATPases function like a “key-in-a-lock” to induce gate-opening and allow substrate entry. PMID:17803938

  8. Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11

    PubMed Central

    Pathare, Ganesh Ramnath; Nagy, István; Śledź, Paweł; Anderson, Daniel J.; Zhou, Han-Jie; Pardon, Els; Steyaert, Jan; Förster, Friedrich; Bracher, Andreas; Baumeister, Wolfgang

    2014-01-01

    The ATP-dependent degradation of polyubiquitylated proteins by the 26S proteasome is essential for the maintenance of proteome stability and the regulation of a plethora of cellular processes. Degradation of substrates is preceded by the removal of polyubiquitin moieties through the isopeptidase activity of the subunit Rpn11. Here we describe three crystal structures of the heterodimer of the Mpr1–Pad1–N-terminal domains of Rpn8 and Rpn11, crystallized as a fusion protein in complex with a nanobody. This fusion protein exhibits modest deubiquitylation activity toward a model substrate. Full activation requires incorporation of Rpn11 into the 26S proteasome and is dependent on ATP hydrolysis, suggesting that substrate processing and polyubiquitin removal are coupled. Based on our structures, we propose that premature activation is prevented by the combined effects of low intrinsic ubiquitin affinity, an insertion segment acting as a physical barrier across the substrate access channel, and a conformationally unstable catalytic loop in Rpn11. The docking of the structure into the proteasome EM density revealed contacts of Rpn11 with ATPase subunits, which likely stabilize the active conformation and boost the affinity for the proximal ubiquitin moiety. The narrow space around the Rpn11 active site at the entrance to the ATPase ring pore is likely to prevent erroneous deubiquitylation of folded proteins. PMID:24516147

  9. Bacterial Proteasomes

    PubMed Central

    Jastrab, Jordan B.; Darwin, K. Heran

    2015-01-01

    Interest in bacterial proteasomes was sparked by the discovery that proteasomal degradation is required for the pathogenesis of Mycobacterium tuberculosis, one of the world's deadliest pathogens. Although bacterial proteasomes are structurally similar to their eukaryotic and archaeal homologs, there are key differences in their mechanisms of assembly, activation, and substrate targeting for degradation. In this article, we compare and contrast bacterial proteasomes with their archaeal and eukaryotic counterparts, and we discuss recent advances in our understanding of how bacterial proteasomes function to influence microbial physiology. PMID:26488274

  10. 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. PMID:27137187

  11. Altered Composition of Liver Proteasome Assemblies Contributes to Enhanced Proteasome Activity in the Exceptionally Long-Lived Naked Mole-Rat

    PubMed Central

    Rodriguez, Karl A.; Edrey, Yael H.; Osmulski, Pawel; Gaczynska, Maria; Buffenstein, Rochelle

    2012-01-01

    The longest-lived rodent, the naked mole-rat (Bathyergidae; Heterocephalus glaber), maintains robust health for at least 75% of its 32 year lifespan, suggesting that the decline in genomic integrity or protein homeostasis routinely observed during aging, is either attenuated or delayed in this extraordinarily long-lived species. The ubiquitin proteasome system (UPS) plays an integral role in protein homeostasis by degrading oxidatively-damaged and misfolded proteins. In this study, we examined proteasome activity in naked mole-rats and mice in whole liver lysates as well as three subcellular fractions to probe the mechanisms behind the apparently enhanced effectiveness of UPS. We found that when compared with mouse samples, naked mole-rats had significantly higher chymotrypsin-like (ChT-L) activity and a two-fold increase in trypsin-like (T-L) in both whole lysates as well as cytosolic fractions. Native gel electrophoresis of the whole tissue lysates showed that the 20S proteasome was more active in the longer-lived species and that 26S proteasome was both more active and more populous. Western blot analyses revealed that both 19S subunits and immunoproteasome catalytic subunits are present in greater amounts in the naked mole-rat suggesting that the observed higher specific activity may be due to the greater proportion of immunoproteasomes in livers of healthy young adults. It thus appears that proteasomes in this species are primed for the efficient removal of stress-damaged proteins. Further characterization of the naked mole-rat proteasome and its regulation could lead to important insights on how the cells in these animals handle increased stress and protein damage to maintain a longer health in their tissues and ultimately a longer life. PMID:22567116

  12. Proteasome inhibitors.

    PubMed

    Teicher, Beverly A; Tomaszewski, Joseph E

    2015-07-01

    Proteasome inhibitors have a 20 year history in cancer therapy. The first proteasome inhibitor, bortezomib (Velcade, PS-341), a break-through multiple myeloma treatment, moved rapidly through development from bench in 1994 to first approval in 2003. Bortezomib is a reversible boronic acid inhibitor of the chymotrypsin-like activity of the proteasome. Next generation proteasome inhibitors include carfilzomib and oprozomib which are irreversible epoxyketone proteasome inhibitors; and ixazomib and delanzomib which are reversible boronic acid proteasome inhibitors. Two proteasome inhibitors, bortezomib and carfilzomib are FDA approved drugs and ixazomib and oprozomib are in late stage clinical trials. All of the agents are potent cytotoxics. The disease focus for all the proteasome inhibitors is multiple myeloma. This focus arose from clinical observations made in bortezomib early clinical trials. Later preclinical studies confirmed that multiple myeloma cells were indeed more sensitive to proteasome inhibitors than other tumor cell types. The discovery and development of the proteasome inhibitor class of anticancer agents has progressed through a classic route of serendipity and scientific investigation. These agents are continuing to have a major impact in their treatment of hematologic malignancies and are beginning to be explored as potential treatment agent for non-cancer indications. PMID:25935605

  13. Regulated protein turnover: snapshots of the proteasome in action

    PubMed Central

    Bhattacharyya, Sucharita; Yu, Houqing; Mim, Carsten; Matouschek, Andreas

    2015-01-01

    The ubiquitin-proteasome system (UPS) is the main ATP-dependent protein degradation pathway in the cytosol and nucleus of eukaryotic cells. At its centre is the 26S proteasome, which degrades regulatory proteins and mis-folded or damaged proteins. In a major breakthrough, several groups have determined high-resolution structures of the entire 26S proteasome particle in different nucleotide conditions and with and without substrate using cryo-electron microscopy combined with other techniques. These structures bring some surprising insights into the functional mechanism of the proteasome and will provide invaluable guidance for genetic and biochemical studies of this key regulatory system. PMID:24452470

  14. Direct cellular delivery of human proteasomes to delay tau aggregation.

    PubMed

    Han, Dong Hoon; Na, Hee-Kyung; Choi, Won Hoon; Lee, Jung Hoon; Kim, Yun Kyung; Won, Cheolhee; Lee, Seung-Han; Kim, Kwang Pyo; Kuret, Jeff; Min, Dal-Hee; Lee, Min Jae

    2014-01-01

    The 26S proteasome is the primary machinery that degrades ubiquitin (Ub)-conjugated proteins, including many proteotoxic proteins implicated in neurodegeneraton. It has been suggested that the elevation of proteasomal activity is tolerable to cells and may be beneficial to prevent the accumulation of protein aggregates. Here we show that purified proteasomes can be directly transported into cells through mesoporous silica nanoparticle-mediated endocytosis. Proteasomes that are loaded onto nanoparticles through non-covalent interactions between polyhistidine tags and nickel ions fully retain their proteolytic activity. Cells treated with exogenous proteasomes are more efficient in degrading overexpressed human tau than endogenous proteasomal substrates, resulting in decreased levels of tau aggregates. Moreover, exogenous proteasome delivery significantly promotes cell survival against proteotoxic stress caused by tau and reactive oxygen species. These data demonstrate that increasing cellular proteasome activity through the direct delivery of purified proteasomes may be an effective strategy for reducing cellular levels of proteotoxic proteins. PMID:25476420

  15. 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. PMID:27053109

  16. [Proteasome inhibitor].

    PubMed

    Yagi, Hideo

    2014-06-01

    The ubiquitin-proteasome system plays an essential role in degradation of eukaryotic intracellular protein, including cell cycle regulation, cell growth and proliferation, and survival. Cancer cells generally have higher level of proteasome activity compared with normal cells, suggesting proteasome inhibition could be therapeutic target in oncology. Bortezomib, the first proteasome inhibitor introduced into the clinic, is approved for the treatment of patients with multiple myeloma (MM). Although it was approved as single agent in the relapsed setting, bortezomib is now predominantly used in combination with conventional and novel targeted agents because bortezomib has demonstrated additive and synergistic activity in preclinical studies. Recently, several second-generation proteasome inhibitors, such as carfilzomib and MLN9708, have been developed and entered into clinical trials. These agents were investigated in frontline MM in combination with lenalidomide and low-dose dexamethasone. These studies demonstrated positive efficacy and safety, and it is expected that they will be approved in near future. PMID:25016815

  17. Oxathiazolones Selectively Inhibit the Human Immunoproteasome over the Constitutive Proteasome

    PubMed Central

    2014-01-01

    Selective inhibitors for the human immunoproteasome LMP7 (β5i) subunit over the constitutive proteasome hold promise for the treatment of autoimmune and inflammatory diseases and hematologic malignancies. Here we report that oxathiazolones inhibit the immunoproteasome β5i with up to 4700-fold selectivity over the constitutive proteasome, are cell permeable, and inhibit proteasomes inside cells. PMID:24900849

  18. The ubiquitin proteasome system and efficacy of proteasome inhibitors in diseases.

    PubMed

    Chitra, Selvarajan; Nalini, Ganesan; Rajasekhar, Gopalakrishnan

    2012-06-01

    In eukaryotes the ubiquitin proteasome pathway plays an important role in cellular homeostasis and also it exerts a critical role in regulating a wide variety of cellular pathways, including cell growth and proliferation, apoptosis, DNA repair, transcription and immune response. Defects in these pathways have been implicated in a number of human pathologies. Inhibition of the ubiquitin proteasome pathway by proteasome inhibitors may be a rational therapeutic approach for various diseases, such as cancer and inflammatory diseases. Many of the critical cytokine and chemokine mediators of the progression of rheumatoid arthritis are regulated by nuclear factor kappa B (NF-κB). In peptidoglycan/polysaccharide-induced polyarthritis, proteasome inhibitors limit the overall inflammation, reduce NF-κB activation, decrease cellular adhesion molecule expression, inhibit nitric oxide synthase, attenuate circulating levels of proinflammatory cytokine interleukin-6 and reduce the arthritis index and swelling in the joints of the animals. Since proteasome inhibitors exhibit anti-inflammatory and anti proliferative effects, diseases characterized by both of these processes such as rheumatoid arthritis might also represent clinical opportunities for such drugs. The regulation of the proteasomal complex by proteasome inhibitors also has implications and potential benefits for the treatment of rheumatoid arthritis. This review summarizes the ubiquitin proteasome pathway, the structure of 26S proteasomes and types of proteasome inhibitors, with their actions, and clinical applications of proteasome inhibitors in various diseases. PMID:22709487

  19. Subpopulations of proteasomes in rat liver nuclei, microsomes and cytosol.

    PubMed Central

    Palmer, A; Rivett, A J; Thomson, S; Hendil, K B; Butcher, G W; Fuertes, G; Knecht, E

    1996-01-01

    Mammalian proteasomes are composed of 14-17 different types of subunits, some of which, including major-histocompatibility-complex-encoded subunits LMP2 and LMP7, are non-essential and present in variable amounts. We have investigated the distribution of total proteasomes and some individual subunits in rat liver by quantitative immunoblot analysis of purified subcellular fractions (nuclei, mitochondria, microsomes and cytosol). Proteasomes were mainly found in the cytosol but were also present in the purified nuclear and microsomal fractions. In the nuclei, proteasomes were soluble or loosely attached to the chromatin, since they could be easily extracted by treatment with nucleases or high concentrations of salt. In the microsomes, proteasomes were on the outside of the membranes. Further subfractionation of the microsomes showed that the proteasomes in this fraction were associated with the smooth endoplasmic reticulum and with the cis-Golgi but were practically absent from the rough endoplasmic reticulum. Using monospecific antibodies for some proteasomal subunits (C8, C9, LMP2 and Z), the composition of proteasomes in nuclei, microsomes and cytosol was investigated. Although there appear not to be differences in proteasome composition in the alpha subunits (C8 and C9) in the different locations, the relative amounts of some beta subunits varied. Subunit Z was enriched in nuclear proteasomes but low in microsome-associated proteasomes, whereas LMP2, which was relatively low in nuclei, showed a small enrichment in the microsomes. These differences in subunit composition of proteasomes probably reflect differences in the function of proteasomes in distinct cell compartments. PMID:8687380

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  1. 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

  2. An Archaeal Homolog of Proteasome Assembly Factor Functions as a Proteasome Activator

    PubMed Central

    Kumoi, Kentaro; Satoh, Tadashi; Murata, Kazuyoshi; Hiromoto, Takeshi; Mizushima, Tsunehiro; Kamiya, Yukiko; Noda, Masanori; Uchiyama, Susumu; Yagi, Hirokazu; Kato, Koichi

    2013-01-01

    Assembly of the eukaryotic 20S proteasome is an ordered process involving several proteins operating as proteasome assembly factors including PAC1-PAC2 but archaeal 20S proteasome subunits can spontaneously assemble into an active cylindrical architecture. Recent bioinformatic analysis identified archaeal PAC1-PAC2 homologs PbaA and PbaB. However, it remains unclear whether such assembly factor-like proteins play an indispensable role in orchestration of proteasome subunits in archaea. We revealed that PbaB forms a homotetramer and exerts a dual function as an ATP-independent proteasome activator and a molecular chaperone through its tentacle-like C-terminal segments. Our findings provide insights into molecular evolution relationships between proteasome activators and assembly factors. PMID:23555947

  3. In vivo pharmacodynamic imaging of proteasome inhibition.

    PubMed

    Kimbrel, Erin A; Davis, Tina N; Bradner, James E; Kung, Andrew L

    2009-01-01

    Inhibiting the proteolytic activity of the 26S proteasome has been shown to have selective apoptotic effects on cancer cells and to be clinically efficacious in certain malignancies. There is an unmet medical need for additional proteasome inhibitors, and their development will be facilitated by surrogate markers of proteasome function. Toward this end, ectopic fusion of the destruction domain from ornithine decarboxylase (ODC) to reporter proteins is often used for assessing proteasome function. For luciferase-based reporters, we hypothesized that the oxygen-dependent destruction domain (ODD) from hypoxia-inducible factor 1 alpha (HIF-1 alpha) may provide improved sensitivity over luciferase-ODC, owing to its extremely rapid turnover by the proteasome (HIF-1 alpha has a half-life of less than 5 minutes). In the current study, we show that ODD-luciferase affords a greater dynamic range and faster kinetics than luciferase-ODC in sensing proteasome inhibition in vitro. Importantly, ODD-luciferase also serves as an effective in vivo marker of proteasome function in xenograft tumor models, with inhibition being detected by noninvasive imaging within 3 hours of bortezomib administration. These data establish ODD-luciferase as a surrogate marker of proteasome function that can be used both in vitro and in vivo for the development of novel proteasome inhibitors. PMID:19723471

  4. 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. PMID:27462806

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

    PubMed Central

    Taylor, Allen

    2011-01-01

    The ubiquitin-proteasome pathway (UPP) is the primary cytosolic proteolytic machinery for the selective degradation of various forms of damaged proteins. Thus, the UPP is an important protein quality control mechanism. In the canonical UPP, both ubiquitin and the 26S proteasome are involved. Substrate proteins of the canonical UPP are first tagged by multiple ubiquitin molecules and then degraded by the 26S proteasome. However, in non-canonical UPP, proteins can be degraded by the 26S or the 20S proteasome without being ubiquitinated. It is clear that a proteasome is responsible for selective degradation of oxidized proteins, but the extent to which ubiquitination is involved in this process remains a subject of debate. While many publications suggest that the 20S proteasome degrades oxidized proteins independent of ubiquitin, there is also solid evidence indicating that ubiquitin and ubiquitination are involved in degradation of some forms of oxidized proteins. A fully functional UPP is required for cells to cope with oxidative stress and the activity of the UPP is also modulated by cellular redox status. Mild or transient oxidative stress up-regulates the ubiquitination system and proteasome activity in cells and tissues and transiently enhances intracellular proteolysis. Severe or sustained oxidative stress impairs the function of the UPP and decreases intracellular proteolysis. Both the ubiquitin conjugation enzymes and the proteasome can be inactivated by sustained oxidative stress, especially the 26S proteasome. Differential susceptibilities of the ubiquitin conjugation enzymes and the 26S proteasome to oxidative damage lead to an accumulation of ubiquitin conjugates in cells in response to mild oxidative stress. Thus, increased levels of ubiquitin conjugates in cells appear to be an indicator of mild oxidative stress. PMID:21530648

  6. Hyperglycemia Impairs Proteasome Function by Methylglyoxal

    PubMed Central

    Queisser, Markus A.; Yao, Dachun; Geisler, Sven; Hammes, Hans-Peter; Lochnit, Günter; Schleicher, Erwin D.; Brownlee, Michael; Preissner, Klaus T.

    2010-01-01

    OBJECTIVE The ubiquitin-proteasome system is the main degradation machinery for intracellularly altered proteins. Hyperglycemia has been shown to increase intracellular levels of the reactive dicarbonyl methylglyoxal (MGO) in cells damaged by diabetes, resulting in modification of proteins and alterations of their function. In this study, the influence of MGO-derived advanced glycation end product (AGE) formation on the activity of the proteasome was investigated in vitro and in vivo. RESEARCH DESIGN AND METHODS MGO-derived AGE modification of proteasome subunits was analyzed by mass spectrometry, immunoprecipitation, and Western blots. Proteasome activity was analyzed using proteasome-specific fluorogenic substrates. Experimental models included bovine retinal endothelial cells, diabetic Ins2Akita mice, glyoxalase 1 (GLO1) knockdown mice, and streptozotocin (STZ)-injected diabetic mice. RESULTS In vitro incubation with MGO caused adduct formation on several 20S proteasomal subunit proteins. In cultured endothelial cells, the expression level of the catalytic 20S proteasome subunit was not altered but proteasomal chymotrypsin-like activity was significantly reduced. In contrast, levels of regulatory 19S proteasomal proteins were decreased. In diabetic Ins2Akita, STZ diabetic, and nondiabetic and diabetic G101 knockdown mice, chymotrypsin-like activity was also reduced and MGO modification of the 20S-β2 subunit was increased. CONCLUSIONS Hyperglycemia-induced formation of MGO covalently modifies the 20S proteasome, decreasing its activity in the diabetic kidney and reducing the polyubiquitin receptor 19S-S5a. The results indicate a new link between hyperglycemia and impairment of cell functions. PMID:20009088

  7. Mutational analysis reveals a role for the C terminus of the proteasome subunit Rpt4p in spindle pole body duplication in Saccharomyces cerevisiae.

    PubMed Central

    McDonald, Heather B; Helfant, Astrid Hoes; Mahony, Erin M; Khosla, Shaun K; Goetsch, Loretta

    2002-01-01

    The ubiquitin/proteasome pathway plays a key role in regulating cell cycle progression. Previously, we reported that a conditional mutation in the Saccharomyces cerevisiae gene RPT4/PCS1, which encodes one of six ATPases in the proteasome 19S cap complex/regulatory particle (RP), causes failure of spindle pole body (SPB) duplication. To improve our understanding of Rpt4p, we created 58 new mutations, 53 of which convert clustered, charged residues to alanine. Virtually all mutations that affect the N-terminal region, which contains a putative nuclear localization signal and coiled-coil motif, result in a wild-type phenotype. Nine mutations that affect the central ATPase domain and the C-terminal region confer recessive lethality. The two conditional mutations identified, rpt4-145 and rpt4-150, affect the C terminus. After shift to high temperature, these mutations generally cause cells to progress slowly through the first cell cycle and to arrest in the second cycle with large buds, a G2 content of DNA, and monopolar spindles, although this phenotype can vary depending on the medium. Additionally, we describe a genetic interaction between RPT4 and the naturally polymorphic gene SSD1, which in wild-type form modifies the rpt4-145 phenotype such that cells arrest in G2 of the first cycle with complete bipolar spindles. PMID:12399382

  8. Multiple Sclerosis Autoantigen Myelin Basic Protein Escapes Control by Ubiquitination during Proteasomal Degradation*

    PubMed Central

    Belogurov, Alexey; Kudriaeva, Anna; Kuzina, Ekaterina; Smirnov, Ivan; Bobik, Tatyana; Ponomarenko, Natalia; Kravtsova-Ivantsiv, Yelena; Ciechanover, Aaron; Gabibov, Alexander

    2014-01-01

    The vast majority of cellular proteins are degraded by the 26S proteasome after their ubiquitination. Here, we report that the major component of the myelin multilayered membrane sheath, myelin basic protein (MBP), is hydrolyzed by the 26S proteasome in a ubiquitin-independent manner both in vitro and in mammalian cells. As a proteasomal substrate, MBP reveals a distinct and physiologically relevant concentration range for ubiquitin-independent proteolysis. Enzymatic deimination prevents hydrolysis of MBP by the proteasome, suggesting that an abnormally basic charge contributes to its susceptibility toward proteasome-mediated degradation. To our knowledge, our data reveal the first case of a pathophysiologically important autoantigen as a ubiquitin-independent substrate of the 26S proteasome. PMID:24739384

  9. The Proteasome Is a Molecular Target of Environmental Toxic Organotins

    PubMed Central

    Shi, Guoqing; Chen, Di; Zhai, Guangshu; Chen, Marina S.; Cui, Qiuzhi Cindy; Zhou, Qunfang; He, Bin; Dou, Q. Ping; Jiang, Guibin

    2009-01-01

    Background Because of the vital importance of the proteasome pathway, chemicals affecting proteasome activity could disrupt essential cellular processes. Although the toxicity of organotins to both invertebrates and vertebrates is well known, the essential cellular target of organotins has not been well identified. We hypothesize that the proteasome is a molecular target of environmental toxic organotins. Objectives Our goal was to test the above hypothesis by investigating whether organotins could inhibit the activity of purified and cellular proteasomes and, if so, the involved molecular mechanisms and downstream events. Results We found that some toxic organotins [e.g., triphenyltin (TPT)] can potently and preferentially inhibit the chymotrypsin-like activity of purified 20S proteasomes and human breast cancer cellular 26S proteasomes. Direct binding of tin atoms to cellular proteasomes is responsible for the observed irreversible inhibition. Inhibition of cellular proteasomes by TPT in several human cell lines results in the accumulation of ubiquitinated proteins and natural proteasome target proteins, accompanied by induction of cell death. Conclusions The proteasome is one of the molecular targets of environmental toxic organotins in human cells, and proteasome inhibition by organotins contributes to their cellular toxicity. PMID:19337512

  10. Fine-Tuning of FACT by the Ubiquitin Proteasome System in Regulation of Transcriptional Elongation.

    PubMed

    Sen, Rwik; Ferdoush, Jannatul; Kaja, Amala; Bhaumik, Sukesh R

    2016-06-01

    FACT (facilitates chromatin transcription), an evolutionarily conserved histone chaperone involved in transcription and other DNA transactions, is upregulated in cancers, and its downregulation is associated with cellular death. However, it is not clearly understood how FACT is fine-tuned for normal cellular functions. Here, we show that the FACT subunit Spt16 is ubiquitylated by San1 (an E3 ubiquitin ligase) and degraded by the 26S proteasome. Enhanced abundance of Spt16 in the absence of San1 impairs transcriptional elongation. Likewise, decreased abundance of Spt16 also reduces transcription. Thus, an optimal level of Spt16 is required for efficient transcriptional elongation, which is maintained by San1 via ubiquitylation and proteasomal degradation. Consistently, San1 associates with the coding sequences of active genes to regulate Spt16's abundance. Further, we found that enhanced abundance of Spt16 in the absence of San1 impairs chromatin reassembly at the coding sequence, similarly to the results seen following inactivation of Spt16. Efficient chromatin reassembly enhances the fidelity of transcriptional elongation. Taken together, our results demonstrate for the first time a fine-tuning of FACT by a ubiquitin proteasome system in promoting chromatin reassembly in the wake of elongating RNA polymerase II and transcriptional elongation, thus revealing novel regulatory mechanisms of gene expression. PMID:27044865

  11. Structural Basis for the Assembly and Gate Closure Mechanisms of the Mycobacterium tuberculosis 20S Proteasome

    SciTech Connect

    Lin, D.; Li, H; Wang, T; Pan, H; Lin, G; Li, H

    2010-01-01

    Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin-proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo-electron microscopy and X-ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the {beta}-subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven {alpha}-subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.

  12. Structural basis for the assembly and gate closure mechanisms of the Mycobacterium tuberculosis 20S proteasome

    SciTech Connect

    Li, D.; Li, H.; Li, H.; Wang, T.; Pan, H.; Lin, G.

    2010-06-16

    Mycobacterium tuberculosis (Mtb) possesses a proteasome system analogous to the eukaryotic ubiquitin-proteasome pathway. Mtb requires the proteasome to resist killing by the host immune system. The detailed assembly process and the gating mechanism of Mtb proteasome have remained unknown. Using cryo-electron microscopy and X-ray crystallography, we have obtained structures of three Mtb proteasome assembly intermediates, showing conformational changes during assembly, and explaining why the {beta}-subunit propeptide inhibits rather than promotes assembly. Although the eukaryotic proteasome core particles close their protein substrate entrance gates with different amino terminal peptides of the seven {alpha}-subunits, it has been unknown how a prokaryotic proteasome might close the gate at the symmetry axis with seven identical peptides. We found in the new Mtb proteasome crystal structure that the gate is tightly sealed by the seven identical peptides taking on three distinct conformations. Our work provides the structural bases for assembly and gating mechanisms of the Mtb proteasome.

  13. Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury

    PubMed Central

    Wang, Shaogui; Ni, Hong-Min; Dorko, Kenneth; Kumer, Sean C.; Schmitt, Timothy M.; Nawabi, Atta; Komatsu, Masaaki; Huang, Heqing; Ding, Wen-Xing

    2016-01-01

    Chronic alcohol exposure increased hepatic receptor-interacting protein kinase (RIP) 3 expression and necroptosis in the liver but its mechanisms are unclear. In the present study, we demonstrated that chronic alcohol feeding plus binge (Gao-binge) increased RIP3 but not RIP1 protein levels in mouse livers. RIP3 knockout mice had decreased serum alanine amino transferase activity and hepatic steatosis but had no effect on hepatic neutrophil infiltration compared with wild type mice after Gao-binge alcohol treatment. The hepatic mRNA levels of RIP3 did not change between Gao-binge and control mice, suggesting that alcohol-induced hepatic RIP3 proteins are regulated at the posttranslational level. We found that Gao-binge treatment decreased the levels of proteasome subunit alpha type-2 (PSMA2) and proteasome 26S subunit, ATPase 1 (PSMC1) and impaired hepatic proteasome function. Pharmacological or genetic inhibition of proteasome resulted in the accumulation of RIP3 in mouse livers. More importantly, human alcoholics had decreased expression of PSMA2 and PSMC1 but increased protein levels of RIP3 compared with healthy human livers. Moreover, pharmacological inhibition of RIP1 decreased Gao-binge-induced hepatic inflammation, neutrophil infiltration and NF-κB subunit (p65) nuclear translocation but failed to protect against steatosis and liver injury induced by Gao-binge alcohol. In conclusion, results from this study suggest that impaired hepatic proteasome function by alcohol exposure may contribute to hepatic accumulation of RIP3 resulting in necroptosis and steatosis while RIP1 kinase activity is important for alcohol-induced inflammation. PMID:26769846

  14. Cytoplasmic proteasomes are not indispensable for cell growth in Saccharomyces cerevisiae

    SciTech Connect

    Tsuchiya, Hikaru; Arai, Naoko; Tanaka, Keiji Saeki, Yasushi

    2013-07-05

    Highlights: •We succeeded to control the proteasome localization by the anchor-away technique. •Nuclear proteasome-depleted cells showed a lethal phenotype. •Cytoplasmic proteasomes are not indispensable for cell growth in dividing cells. -- Abstract: The 26S proteasome is an essential protease complex responsible for the degradation of ubiquitinated proteins in eukaryotic cells. In rapidly proliferating yeast cells, proteasomes are mainly localized in the nucleus, but the biological significance of the proteasome localization is still unclear. In this study, we investigated the relationship between the proteasome localization and the functions by the anchor-away technique, a ligand-dependent sequestration of a target protein into specific compartment(s). Anchoring of the proteasome to the plasma membrane or the ribosome resulted in conditional depletion of the nuclear proteasomes, whereas anchoring to histone resulted in the proteasome sequestration into the nucleus. We observed that the accumulation of ubiquitinated proteins in all the proteasome-targeted cells, suggesting that both the nuclear and cytoplasmic proteasomes have proteolytic functions and that the ubiquitinated proteins are produced and degraded in each compartment. Consistent with previous studies, the nuclear proteasome-depleted cells exhibited a lethal phenotype. In contrast, the nuclear sequestration of the proteasome resulted only in a mild growth defect, suggesting that the cytoplasmic proteasomes are not basically indispensable for cell growth in rapidly growing yeast cells.

  15. Visualization of prosomes (MCP-proteasomes), intermediate filament and actin networks by "instantaneous fixation" preserving the cytoskeleton.

    PubMed

    Arcangeletti, C; Sütterlin, R; Aebi, U; De Conto, F; Missorini, S; Chezzi, C; Scherrer, K

    1997-06-01

    A new "instantaneous" fixation/extraction procedure, yielding good preservation of intermediate filaments (IFs) and actin filaments when applied at 37 degrees C, has been explored to reexamine the relationships of the prosomes to the cytoskeleton. Prosomes are protein complexes of variable subunit composition, including occasionally a small RNA, which were originally observed as trans-acting factors in untranslated mRNPs. Constituting also the proteolytic core of the 26S proteasomes, they are also called "multicatalytic proteinase (MCP) complexes" or "20S-Proteasomes." In Triton X-100-extracted epithelial, fibroblastic, and muscle cells, prosome particles were found associated primarily with the IFs (Olink-Coux et al., 1994). Application of "instantaneous fixation" has now led to the new observation that a major fraction of prosome particles, composed of specific sets of subunits, is distributed in variable proportions between the IFs and the microfilament/ stress fiber system in PtK1 epithelial cells and human fibroblasts. Electron microscopy using gold-labeled antibodies confirms this dual localization on classical whole mounts and on cells exposed to instantaneous fixation. In contrast to the resistance of the prosome-IF association, a variable fraction of the prosome particles is released from the actin cytoskeleton by Triton X-100 when applied prior to fixation. Moreover, in vitro copolymerization of prosomes with G-actin made it possible to observe "ladder-like" filamentous structures in the electron microscope, in which the prosome particles, like the "rungs of a ladder," laterally crosslink two or more actin filaments in a regular pattern. These results demonstrate that prosomes are bound in the cell not only to IFs but also to the actin cytoskeleton and, furthermore, not only within large M(r) complexes (possibly mRNPs and/or 26S proteasomes), but also directly, as individual prosome particles. PMID:9216087

  16. The proteasome activator 11 S REG (PA28) and class I antigen presentation.

    PubMed Central

    Rechsteiner, M; Realini, C; Ustrell, V

    2000-01-01

    There are two immune responses in vertebrates: humoral immunity is mediated by circulating antibodies, whereas cytotoxic T lymphocytes (CTL) confer cellular immunity. CTL lyse infected cells upon recognition of cell-surface MHC Class I molecules complexed with foreign peptides. The displayed peptides are produced in the cytosol by degradation of host proteins or proteins from intracellular pathogens that might be present. Proteasomes are cylindrical multisubunit proteases that generate many of the peptides eventually transferred to the cell surface for immune surveillance. In mammalian proteasomes, six active sites face a central chamber. As this chamber is sealed off from the enzyme's surface, there must be mechanisms to promote entry of substrates. Two protein complexes have been found to bind the ends of the proteasome and activate it. One of the activators is the 19 S regulatory complex of the 26 S proteasome; the other activator is '11 S REG' [Dubiel, Pratt, Ferrell and Rechsteiner (1992) J. Biol. Chem. 267, 22369-22377] or 'PA28' [Ma, Slaughter and DeMartino (1992) J. Biol. Chem. 267, 10515-10523]. During the past 7 years, our understanding of the structure of REG molecules has increased significantly, but much less is known about their biological functions. There are three REG subunits, namely alpha, beta and gamma. Recombinant REGalpha forms a ring-shaped heptamer of known crystal structure. 11 S REG is a heteroheptamer of alpha and beta subunits. REGgamma is also presumably a heptameric ring, and it is found in the nuclei of the nematode work Caenorhabditis elegans and higher organisms, where it may couple proteasomes to other nuclear components. REGalpha and REGbeta, which are abundant in vertebrate immune tissues, are located mostly in the cytoplasm. Synthesis of REG alpha and beta subunits is induced by interferon-gamma, and this has led to the prevalent hypothesis that REG alpha/beta hetero-oligomers play an important role in Class I antigen

  17. Proteasome as a Molecular Target of Microcystin-LR

    PubMed Central

    Zhu, Zhu; Zhang, Li; Shi, Guoqing

    2015-01-01

    Proteasome degrades proteins in eukaryotic cells. As such, the proteasome is crucial in cell cycle and function. This study proved that microcystin-LR (MC-LR), which is a toxic by-product of algal bloom, can target cellular proteasome and selectively inhibit proteasome trypsin-like (TL) activity. MC-LR at 1 nM can inhibit up to 54% of the purified 20S proteasome TL activity and 43% of the proteasome TL activity in the liver of the cyprinid rare minnow (Gobiocypris rarus). Protein degradation was retarded in GFP-CL1-transfected PC-3 cells because MC-LR inhibited the proteasome TL activity. Docking studies indicated that MC-LR blocked the active site of the proteasome β2 subunit; thus, the proteasome TL activity was inhibited. In conclusion, MC-LR can target proteasome, selectively inhibit proteasome TL activity, and retard protein degradation. This study may be used as a reference of future research on the toxic mechanism of MC-LR. PMID:26090622

  18. Bacterial Proteasome Activator Bpa (Rv3780) Is a Novel Ring-Shaped Interactor of the Mycobacterial Proteasome

    PubMed Central

    Delley, Cyrille L.; Laederach, Juerg; Ziemski, Michal; Bolten, Marcel; Boehringer, Daniel; Weber-Ban, Eilika

    2014-01-01

    The occurrence of the proteasome in bacteria is limited to the phylum of actinobacteria, where it is maintained in parallel to the usual bacterial compartmentalizing proteases. The role it plays in these organisms is still not fully understood, but in the human pathogen Mycobacterium tuberculosis (Mtb) the proteasome supports persistence in the host. In complex with the ring-shaped ATPase Mpa (called ARC in other actinobacteria), the proteasome can degrade proteins that have been post-translationally modified with the prokaryotic ubiquitin-like protein Pup. Unlike for the eukaryotic proteasome core particle, no other bacterial proteasome interactors have been identified to date. Here we describe and characterize a novel bacterial proteasome activator of Mycobacterium tuberculosis we termed Bpa (Rv3780), using a combination of biochemical and biophysical methods. Bpa features a canonical C-terminal proteasome interaction motif referred to as the HbYX motif, and its orthologs are only found in those actinobacteria encoding the proteasomal subunits. Bpa can inhibit degradation of Pup-tagged substrates in vitro by competing with Mpa for association with the proteasome. Using negative-stain electron microscopy, we show that Bpa forms a ring-shaped homooligomer that can bind coaxially to the face of the proteasome cylinder. Interestingly, Bpa can stimulate the proteasomal degradation of the model substrate β-casein, which suggests it could play a role in the removal of non-native or damaged proteins. PMID:25469515

  19. Impaired proteasome function in sporadic amyotrophic lateral sclerosis.

    PubMed

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

    2012-06-01

    Abstract The ubiquitin-proteasome system, important for maintaining protein quality control, is compromised in experimental models of familial ALS. The objective of this study was to determine if proteasome function is impaired in sporadic ALS. Proteasomal activities and subunit composition were evaluated in homogenates of spinal cord samples obtained at autopsy from sporadic ALS and non-neurological control cases, compared to cerebellum as a clinically spared tissue. The level of 20S α structural proteasome subunits was assessed in motor neurons by immunohistochemistry. Catalysis of peptide substrates of the three major proteasomal activities was substantially reduced in ALS thoracic spinal cord, but not in cerebellum, accompanied by alterations in the constitutive proteasome machinery. Chymotrypsin-like activity was decreased to 60% and 65% of control in ventral and dorsal spinal cord, respectively, concomitant with reduction in the β5 subunit with this catalytic activity. Caspase- and trypsin-like activities were reduced to a similar extent (46% - 68% of control). Proteasome levels, although generally maintained, appeared reduced specifically in motor neurons by immunolabelling. In conclusion, there are commonalities of findings in sporadic ALS patients and presymptomatic SOD1-G93A transgenic mice and these implicate inadequate proteasome function in the pathogenesis of both familial and sporadic ALS. PMID:22632443

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

    PubMed

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

    2015-01-01

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

  1. Impaired methylation as a novel mechanism for proteasome suppression in liver cells

    SciTech Connect

    Osna, Natalia A.; White, Ronda L.; Donohue, Terrence M.; Beard, Michael R.; Tuma, Dean J.; Kharbanda, Kusum K.

    2010-01-08

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

  2. Activity and regulation of the centrosome-associated proteasome.

    PubMed

    Fabunmi, R P; Wigley, W C; Thomas, P J; DeMartino, G N

    2000-01-01

    Regulated proteolysis is important for maintaining appropriate cellular levels of many proteins. The bulk of intracellular protein degradation is catalyzed by the proteasome. Recently, the centrosome was identified as a novel site for concentration of the proteasome and associated regulatory proteins (Wigley, W. C., Fabunmi, R. P., Lee, M. G., Marino, C. R., Muallem, S., DeMartino, G. N., and Thomas, P. J. (1999) J. Cell Biol. 145, 481-490). Here we provide evidence that centrosomes contain the active 26 S proteasome that degrades ubiquitinated-protein and proteasome-specific peptide substrates. Moreover, the centrosomes contain an ubiquitin isopeptidase activity. The proteolytic activity is ATP-dependent and is inhibited by proteasome inhibitors. Notably, treatment of cells with inhibitors of proteasome activity promotes redistribution of the proteasome and associated regulatory proteins to the centrosome independent of an intact microtubule system. These data provide biochemical evidence for active proteasomal complexes at the centrosome, highlighting a novel function for this organizing structure. PMID:10617632

  3. Nuclear import of an intact preassembled proteasome particle

    PubMed Central

    Savulescu, Anca F.; Shorer, Hagai; Kleifeld, Oded; Cohen, Ilana; Gruber, Rita; Glickman, Michael H.; Harel, Amnon

    2011-01-01

    The 26S proteasome is a conserved 2.5 MDa protein degradation machine that localizes to different cellular compartments, including the nucleus. Little is known about the specific targeting mechanisms of proteasomes in eukaryotic cells. We used a cell-free nuclear reconstitution system to test for nuclear targeting and import of distinct proteasome species. Three types of stable, proteolytically active proteasomes particles were purified from Xenopus egg cytosol. Two of these, the 26S holoenzyme and the 20S core particle, were targeted to the nuclear periphery but did not reach the nucleoplasm. This targeting depends on the presence of mature nuclear pore complexes (NPCs) in the nuclear envelope. A third, novel form, designated here as 20S+, was actively imported through NPCs. The 20S+ proteasome particle resembles recently described structural intermediates from other systems. Nuclear import of this particle requires functional NPCs, but it is not directly regulated by the Ran GTPase cycle. The mere presence of the associated “+” factors is sufficient to reconstitute nuclear targeting and confer onto isolated 20S core particles the ability to be imported. Stable 20S+ particles found in unfertilized eggs may provide a means for quick mobilization of existing proteasome particles into newly formed nuclear compartments during early development. PMID:21289101

  4. Proteasome Inhibitors: An Expanding Army Attacking a Unique Target

    PubMed Central

    Kisselev, Alexei F.; van der Linden, Wouter A.; Overkleeft, Herman S.

    2012-01-01

    Proteasomes are large, multisubunit proteolytic complexes presenting multiple targets for therapeutic intervention. The 26S proteasome consists of a 20S proteolytic core and one or two 19S regulatory particles. The 20S core contains three types of active sites. Many structurally diverse inhibitors of these active sites, both natural product and synthetic, have been discovered in the last two decades. One, bortezomib, is used clinically for treatment of multiple myeloma, mantle cell lymphoma, and acute allograft rejection. Five more recently developed proteasome inhibitors are in trials for treatment of myeloma and other cancers. Proteasome inhibitors also have activity in animal models of autoimmune and inflammatory diseases, reperfusion injury, promote bone and hair growth, and can potentially be used as anti-infectives. In addition, inhibitors of ATPases and deubiquitinases of 19S regulatory particles have been discovered in the last decade. PMID:22284358

  5. CHIP promotes proteasomal degradation of familial ALS-linked mutant SOD1 by ubiquitinating Hsp/Hsc70.

    PubMed

    Urushitani, Makoto; Kurisu, Junko; Tateno, Minako; Hatakeyama, Shigetsugu; Nakayama, Kei-Ichi; Kato, Shinsuke; Takahashi, Ryosuke

    2004-07-01

    Over 100 mutants in superoxide dismutase 1 (SOD1) are reported in familial amyotrophic lateral sclerosis (ALS). However, the precise mechanism by which they are degraded through a ubiquitin-proteasomal pathway (UPP) remains unclear. Here, we report that heat-shock protein (Hsp) or heat-shock cognate (Hsc)70, and the carboxyl terminus of the Hsc70-interacting protein (CHIP), are involved in proteasomal degradation of mutant SOD1. Only mutant SOD1 interacted with Hsp/Hsc70 in vivo, and in vitro experiments revealed that Hsp/Hsc70 preferentially interacted with apo-SOD1 or dithiothreitol (DTT)-treated holo-SOD1, compared with metallated or oxidized forms. CHIP, a binding partner of Hsp/Hsc70, interacted only with mutant SOD1 and promoted its degradation. Both Hsp70 and CHIP promoted polyubiquitination of mutant SOD1-associated molecules, but not of mutant SOD1, indicating that mutant SOD1 is not a substrate of CHIP. Moreover, mutant SOD1-associated Hsp/Hsc70, a known substrate of CHIP, was polyubiquitinated in vivo, and polyubiquitinated Hsc70 by CHIP interacted with the S5a subunit of the 26S proteasome in vitro. Furthermore, CHIP was predominantly expressed in spinal neurons, and ubiquitinated inclusions in the spinal motor neurons of hSOD1(G93A) transgenic mice were CHIP-immunoreactive. Taken together, we propose a novel pathway in which ubiquitinated Hsp/Hsc70 might deliver mutant SOD1 to, and facilitate its degradation, at the proteasome. PMID:15198682

  6. Analysis of Myelin Basic Protein Fragmentation by Proteasome

    PubMed Central

    Bacheva, A. V.; Belogurov, A. A.; Ponomarenko, N. A.; Govorun, V. M.; Serebryakova, M. V.; Gabibov, A. G.

    2009-01-01

    The proteasome is a high molecular protein complex whose purpose is specific protein degradation in eukaryotic cells. One of the proteasome functions is to produce peptides, which will then be presented on the outer cell membrane using main histocompatibility complex (MHC) molecules of the first or second class. There are definite reasons to believe that proteasome directly takes part in the specific degradation of myelin basic protein (MBP), which make up to 30% of all proteins in the myelin sheath of neuronal axons. The details of the proteasomal degradation of MBP are still unclear. In this work, the features of specific MBP degradation by proteasome were studied. It was demonstrated that MBP (non-ubiquitinated) is a good substrate for 20S and for the 26S proteasome. This is the first work on detecting the sites of MBP proteolysis by proteasome from brains of SJL/J/J and Balb/C mice's lines. Substantial differences in the degradation pattern of this neuroantigen were found, which could indicate the better presentation MBP parts on MHC molecules in the case of mice predisposed to the development of experimental autoimmune encephalomyelitis. PMID:22649589

  7. Arabidopsis PROTEASOME REGULATOR1 is required for auxin-mediated suppression of proteasome activity and regulates auxin signalling.

    PubMed

    Yang, Bao-Jun; Han, Xin-Xin; Yin, Lin-Lin; Xing, Mei-Qing; Xu, Zhi-Hong; Xue, Hong-Wei

    2016-01-01

    The plant hormone auxin is perceived by the nuclear F-box protein TIR1 receptor family and regulates gene expression through degradation of Aux/IAA transcriptional repressors. Several studies have revealed the importance of the proteasome in auxin signalling, but details on how the proteolytic machinery is regulated and how this relates to degradation of Aux/IAA proteins remains unclear. Here we show that an Arabidopsis homologue of the proteasome inhibitor PI31, which we name PROTEASOME REGULATOR1 (PTRE1), is a positive regulator of the 26S proteasome. Loss-of-function ptre1 mutants are insensitive to auxin-mediated suppression of proteasome activity, show diminished auxin-induced degradation of Aux/IAA proteins and display auxin-related phenotypes. We found that auxin alters the subcellular localization of PTRE1, suggesting this may be part of the mechanism by which it reduces proteasome activity. Based on these results, we propose that auxin regulates proteasome activity via PTRE1 to fine-tune the homoeostasis of Aux/IAA repressor proteins thus modifying auxin activity. PMID:27109828

  8. Arabidopsis PROTEASOME REGULATOR1 is required for auxin-mediated suppression of proteasome activity and regulates auxin signalling

    PubMed Central

    Yang, Bao-Jun; Han, Xin-Xin; Yin, Lin-Lin; Xing, Mei-Qing; Xu, Zhi-Hong; Xue, Hong-Wei

    2016-01-01

    The plant hormone auxin is perceived by the nuclear F-box protein TIR1 receptor family and regulates gene expression through degradation of Aux/IAA transcriptional repressors. Several studies have revealed the importance of the proteasome in auxin signalling, but details on how the proteolytic machinery is regulated and how this relates to degradation of Aux/IAA proteins remains unclear. Here we show that an Arabidopsis homologue of the proteasome inhibitor PI31, which we name PROTEASOME REGULATOR1 (PTRE1), is a positive regulator of the 26S proteasome. Loss-of-function ptre1 mutants are insensitive to auxin-mediated suppression of proteasome activity, show diminished auxin-induced degradation of Aux/IAA proteins and display auxin-related phenotypes. We found that auxin alters the subcellular localization of PTRE1, suggesting this may be part of the mechanism by which it reduces proteasome activity. Based on these results, we propose that auxin regulates proteasome activity via PTRE1 to fine-tune the homoeostasis of Aux/IAA repressor proteins thus modifying auxin activity. PMID:27109828

  9. Proteasome activation as a novel anti-aging strategy.

    PubMed

    Gonos, Efstathios

    2014-10-01

    Aging and longevity are two multifactorial biological phenomena whose knowledge at molecular level is still limited. We have studied proteasome function in replicative senescence and cell survival (Mol Aspects Med 35, 1-71, 2014). We have observed reduced levels of proteasome content and activities in senescent cells due to the down-regulation of the catalytic subunits of the 20S complex (J Biol Chem 278, 28026-28037, 2003). In support, partial inhibition of proteasomes in young cells by specific inhibitors induces premature senescence which is p53 dependent (Aging Cell 7, 717-732, 2008). Stable over-expression of catalytic subunits or POMP resulted in enhanced proteasome assembly and activities and increased cell survival following treatments with various oxidants. Importantly, the developed "proteasome activated" human fibroblasts cell lines exhibit a delay of senescence by approximately 15% (J Biol Chem 280, 11840-11850, 2005; J Biol Chem 284, 30076-30086, 2009). Our current work proposes that proteasome activation is an evolutionary conserved mechanism, as it can delay aging in various in vivo systems. Moreover, additional findings indicate that the recorded proteasome activation by many inducers is Nrf2-dependent (J Biol Chem 285, 8171-8184, 2010). Finally, we have studied the proteolysis processes of various age-related proteins and we have identified that CHIP is a major p53 E3 ligase in senescent fibroblasts (Free Rad Biol Med 50, 157-165, 2011). PMID:26461417

  10. Low doses of single or combined agrichemicals induces α-synuclein aggregation in nigrostriatal system of mice through inhibition of proteasomal and autophagic pathways

    PubMed Central

    Su, Cen; Niu, Ping

    2015-01-01

    Alpha synuclein (SNCA) genes and environmental factors are important risk factors of Parkinson’s disease (PD). The agrichemicals paraquat, maneb and chlorpyrifos selectively target dopaminergic neurons, leading to parkinsonism, through ill-defined mechanisms. We analyzed the ability of low dose paraquat, maneb and chlorpyrifos, separately or combined together, to induce synucleinopathy in wild type mice. Paraquat and maneb applied together did not increase α-Synuclein (α-Syn) levels. By contrast, paraquat and chlorpyrifos together resulted in robust accumulation of α-Syn in striata in mice. Therefore, co-treatment with chlorpyrifos enhanced the effects of paraquat. Paraquat, and its co-treatment with maneb or chlorpyrifos, inhibited all soluble proteasomal expression of 26S proteasome subunits. Both paraquat and chlorpyrifos treatments increased levels of the autophagy inhibitor, mammalian target of rapamycin, mTOR, suggesting impaired axonal autophagy, despite increases in certain autophagic proteins, such as beclin 1 and Atg 12. Autophagic flux was also impaired, as ratios of LC3 II to LC3 I were reduced in all the treated animals. These results suggest that a combination of paraquat and chlorpyrifos is much more toxic than paraquat alone or combined with maneb. These effects are likely via inhibitory effects of these toxins on proteasomes and autophagy, which lead to accumulation of α-Syn. Our study provides a novel insight into the mechanisms of action of these agrichemicals. PMID:26884967

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

    NASA Technical Reports Server (NTRS)

    Weitman, D.; Etlinger, J. D.

    1992-01-01

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

  12. Halophilic 20S Proteasomes of the Archaeon Haloferax volcanii: Purification, Characterization, and Gene Sequence Analysis

    PubMed Central

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

    1999-01-01

    A 20S proteasome, composed of α1 and β subunits arranged in a barrel-shaped structure of four stacked rings, was purified from a halophilic archaeon Haloferax volcanii. The predominant peptide-hydrolyzing activity of the 600-kDa α1β-proteasome on synthetic substrates was cleavage carboxyl to hydrophobic residues (chymotrypsin-like [CL] activity) and was optimal at 2 M NaCl, pH 7.7 to 9.5, and 75°C. The α1β-proteasome also hydrolyzed insulin B-chain protein. Removal of NaCl inactivated the CL activity of the α1β-proteasome and dissociated the complex into monomers. Rapid equilibration of the monomers into buffer containing 2 M NaCl facilitated their reassociation into fully active α1β-proteasomes of 600 kDa. However, long-term incubation of the halophilic proteasome in the absence of salt resulted in hydrolysis and irreversible inactivation of the enzyme. Thus, the isolated proteasome has unusual salt requirements which distinguish it from any proteasome which has been described. Comparison of the β-subunit protein sequence with the sequence deduced from the gene revealed that a 49-residue propeptide is removed to expose a highly conserved N-terminal threonine which is proposed to serve as the catalytic nucleophile and primary proton acceptor during peptide bond hydrolysis. Consistent with this mechanism, the known proteasome inhibitors carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132) and N-acetyl-leucinyl-leucinyl-norleucinal (calpain inhibitor I) were found to inhibit the CL activity of the H. volcanii proteasome (Ki = 0.2 and 8 μM, respectively). In addition to the genes encoding the α1 and β subunits, a gene encoding a second α-type proteasome protein (α2) was identified. All three genes coding for the proteasome subunits were mapped in the chromosome and found to be unlinked. Modification of the methods used to purify the α1β-proteasome resulted in the copurification of the α2 protein with the α1 and β subunits in nonstoichometric ratios

  13. Crystal structure and versatile functional roles of the COP9 signalosome subunit 1

    PubMed Central

    Lee, Jung-Hoon; Yi, Lina; Li, Jixi; Schweitzer, Katrin; Borgmann, Marc; Naumann, Michael; Wu, Hao

    2013-01-01

    The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) plays key roles in many biological processes, such as repression of photomorphogenesis in plants and protein subcellular localization, DNA-damage response, and NF-κB activation in mammals. It is an evolutionarily conserved eight-protein complex with subunits CSN1 to CSN8 named following the descending order of molecular weights. Here, we report the crystal structure of the largest CSN subunit, CSN1 from Arabidopsis thaliana (atCSN1), which belongs to the Proteasome, COP9 signalosome, Initiation factor 3 (PCI) domain containing CSN subunit family, at 2.7 Å resolution. In contrast to previous predictions and distinct from the PCI-containing 26S proteasome regulatory particle subunit Rpn6 structure, the atCSN1 structure reveals an overall globular fold, with four domains consisting of helical repeat-I, linker helix, helical repeat-II, and the C-terminal PCI domain. Our small-angle X-ray scattering envelope of the CSN1–CSN7 complex agrees with the EM structure of the CSN alone (apo-CSN) and suggests that the PCI end of each molecule may mediate the interaction. Fitting of the CSN1 structure into the CSN–Skp1-Cul1-Fbox (SCF) EM structure shows that the PCI domain of CSN1 situates at the hub of the CSN for interaction with several other subunits whereas the linker helix and helical repeat-II of CSN1 contacts SCF using a conserved surface patch. Furthermore, we show that, in human, the C-terminal tail of CSN1, a segment not included in our crystal structure, interacts with IκBα in the NF-κB pathway. Therefore, the CSN complex uses multiple mechanisms to hinder NF-κB activation, a principle likely to hold true for its regulation of many other targets and pathways. PMID:23818606

  14. The novel β2-selective proteasome inhibitor LU-102 synergizes with bortezomib and carfilzomib to overcome proteasome inhibitor resistance of myeloma cells

    PubMed Central

    Kraus, Marianne; Bader, Juergen; Geurink, Paul P.; Weyburne, Emily S.; Mirabella, Anne C.; Silzle, Tobias; Shabaneh, Tamer B.; van der Linden, Wouter A.; de Bruin, Gerjan; Haile, Sarah R.; van Rooden, Eva; Appenzeller, Christina; Li, Nan; Kisselev, Alexei F.; Overkleeft, Herman; Driessen, Christoph

    2015-01-01

    Proteasome inhibitor resistance is a challenge for myeloma therapy. Bortezomib targets the β5 and β1 activity, but not the β2 activity of the proteasome. Bortezomib-resistant myeloma cells down-regulate the activation status of the unfolded protein response, and up-regulate β2 proteasome activity. To improve proteasome inhibition in bortezomib-resistant myeloma and to achieve more efficient UPR activation, we have developed LU-102, a selective inhibitor of the β2 proteasome activity. LU-102 inhibited the β2 activity in intact myeloma cells at low micromolar concentrations without relevant co-inhibition of β1 and β5 proteasome subunits. In proteasome inhibitor-resistant myeloma cells, significantly more potent proteasome inhibition was achieved by bortezomib or carfilzomib in combination with LU-102, compared to bortezomib/carfilzomib alone, resulting in highly synergistic cytotoxic activity of the drug combination via endoplasmatic reticulum stress-induced apoptosis. Combining bortezomib/carfilzomib with LU-102 significantly prolonged proteasome inhibition and increased activation of the unfolded protein response and IRE1-a activity. IRE1-α has recently been shown to control myeloma cell differentiation and bortezomib sensitivity (Leung-Hagesteijn, Cancer Cell 24:3, 289-304). Thus, β2-selective proteasome inhibition by LU-102 in combination with bortezomib or carfilzomib results in synergistic proteasome inhibition, activation of the unfolded protein response, and cytotoxicity, and overcomes bortezomib/carfilzomib resistance in myeloma cells in vitro PMID:26069288

  15. The novel β2-selective proteasome inhibitor LU-102 synergizes with bortezomib and carfilzomib to overcome proteasome inhibitor resistance of myeloma cells.

    PubMed

    Kraus, Marianne; Bader, Juergen; Geurink, Paul P; Weyburne, Emily S; Mirabella, Anne C; Silzle, Tobias; Shabaneh, Tamer B; van der Linden, Wouter A; de Bruin, Gerjan; Haile, Sarah R; van Rooden, Eva; Appenzeller, Christina; Li, Nan; Kisselev, Alexei F; Overkleeft, Herman; Driessen, Christoph

    2015-10-01

    Proteasome inhibitor resistance is a challenge for myeloma therapy. Bortezomib targets the β5 and β1 activity, but not the β2 activity of the proteasome. Bortezomib-resistant myeloma cells down-regulate the activation status of the unfolded protein response, and up-regulate β2 proteasome activity. To improve proteasome inhibition in bortezomib-resistant myeloma and to achieve more efficient UPR activation, we have developed LU-102, a selective inhibitor of the β2 proteasome activity. LU-102 inhibited the β2 activity in intact myeloma cells at low micromolar concentrations without relevant co-inhibition of β1 and β5 proteasome subunits. In proteasome inhibitor-resistant myeloma cells, significantly more potent proteasome inhibition was achieved by bortezomib or carfilzomib in combination with LU-102, compared to bortezomib/carfilzomib alone, resulting in highly synergistic cytotoxic activity of the drug combination via endoplasmatic reticulum stress-induced apoptosis. Combining bortezomib/carfilzomib with LU-102 significantly prolonged proteasome inhibition and increased activation of the unfolded protein response and IRE1-a activity. IRE1-α has recently been shown to control myeloma cell differentiation and bortezomib sensitivity (Leung-Hagesteijn, Cancer Cell 24:3, 289-304). Thus, β2-selective proteasome inhibition by LU-102 in combination with bortezomib or carfilzomib results in synergistic proteasome inhibition, activation of the unfolded protein response, and cytotoxicity, and overcomes bortezomib/carfilzomib resistance in myeloma cells in vitro. PMID:26069288

  16. The Pertussis Toxin S1 Subunit Is a Thermally Unstable Protein Susceptible to Degradation by the 20S Proteasome†

    PubMed Central

    Pande, Abhay H.; Moe, David; Jamnadas, Maneesha; Tatulian, Suren A.; Teter, Ken

    2008-01-01

    Pertussis toxin (PT) is an AB-type protein toxin that consists of a catalytic A subunit (PT S1) and an oligomeric, cell-binding B subunit. It belongs to a subset of AB toxins that move from the cell surface to the endoplasmic reticulum (ER) before A chain passage into the cytosol. Toxin translocation is thought to involve A chain unfolding in the ER and the quality control mechanism of ER-associated degradation (ERAD). The absence of lysine residues in PT S1 may allow the translocated toxin to avoid ubiquitin-dependent degradation by the 26S proteasome, which is the usual fate of exported ERAD substrates. As the conformation of PT S1 appears to play an important role in toxin translocation, we used biophysical and biochemical methods to examine the structural properties of PT S1. Our in vitro studies found that the isolated PT S1 subunit is a thermally unstable protein that can be degraded in a ubiquitin-independent fashion by the core 20S proteasome. The thermal denaturation of PT S1 was inhibited by its interaction with NAD, a donor molecule used by PT S1 for the ADP-ribosylation of target G proteins. These observations support a model of intoxication in which toxin translocation, degradation, and activity are all influenced by the heat-labile nature of the isolated toxin A chain. PMID:17105192

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

    PubMed Central

    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

    2016-01-01

    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. PMID:26957043

  18. 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

    2016-01-01

    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. PMID:26957043

  19. ATP-dependent incorporation of 20S protease into the 26S complex that degrades proteins conjugated to ubiquitin.

    PubMed Central

    Eytan, E; Ganoth, D; Armon, T; Hershko, A

    1989-01-01

    Previous studies have indicated that the ATP-dependent 26S protease complex that degrades proteins conjugated to ubiquitin is formed by the assembly of three factors in an ATP-requiring process. We now identify one of the factors as the 20S "multicatalytic" protease, a complex of low molecular weight subunits widely distributed in eukaryotic cells. Comparison of the subunit compositions of purified 20S and 26S complexes indicates that the former is an integral part of the latter. By the use of detergent treatment to activate latent protease activity, we show that the 20S protease becomes incorporated into the 26S complex in the ATP-dependent assembly process. It thus seems that the 20S protease is the "catalytic core" of the 26S complex of the ubiquitin proteolytic pathway. Images PMID:2554287

  20. 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

  1. A Single α Helix Drives Extensive Remodeling of the Proteasome Lid and Completion of Regulatory Particle Assembly

    PubMed Central

    Tomko, Robert J.; Taylor, David W.; Chen, Zhuo A.; Wang, Hong-Wei; Rappsilber, Juri; Hochstrasser, Mark

    2015-01-01

    Summary Most short-lived eukaryotic proteins are degraded by the proteasome. A proteolytic core particle (CP) capped by regulatory particles (RPs) constitutes the 26S proteasome complex. RP biogenesis culminates with the joining of two large subcomplexes, the lid and base. In yeast and mammals, the lid appears to assemble completely before attaching to the base, but how this hierarchical assembly is enforced has remained unclear. Using biochemical reconstitutions, quantitative cross-linking/mass spectrometry, and electron microscopy, we resolve the mechanistic basis for the linkage between lid biogenesis and lid-base joining. Assimilation of the final lid subunit, Rpn12, triggers a large-scale conformational remodeling of the nascent lid that drives RP assembly, in part by relieving steric clash with the base. Surprisingly, this remodeling is triggered by a single Rpn12 α helix. Such assembly-coupled conformational switching is reminiscent of viral particle maturation and may represent a commonly used mechanism to enforce hierarchical assembly in multisubunit complexes. PMID:26451487

  2. The N-terminal domain of Rpn4 serves as a portable ubiquitin-independent degron and is recognized by specific 19S RP subunits

    PubMed Central

    Ha, Seung-Wook; Ju, Donghong; Xie, Youming

    2014-01-01

    The number of proteasomal substrates that are degraded without prior ubiquitylation continues to grow. However, it remains poorly understood how the proteasome recognizes substrates lacking a ubiquitin (Ub) signal. Here we demonstrated that the Ub-independent degradation of Rpn4 requires the 19S regulatory particle (RP). The Ub-independent degron of Rpn4 was mapped to an N-terminal region including the first 80 residues. Inspection of its amino acid sequence revealed that the Ub-independent degron of Rpn4 consists of an intrinsically disordered domain followed by a folded segment. Using a photo-crosslinking-label transfer method, we captured three 19S RP subunits (Rpt1, Rpn2 and Rpn5) that bind the Ub-independent degron of Rpn4. This is the first time that specific 19S RP subunits have been identified interacting with a Ub-independent degron. This study provides insight into the mechanism by which Ub-independent substrates are recruited to the 26S proteasome. PMID:22349505

  3. The proteasome assembly line

    PubMed Central

    Madura, Kiran

    2013-01-01

    The assembly of the proteasome — the cellular machine that eliminates unwanted proteins — is a carefully choreographed affair, involving a complex sequence of steps overseen by dedicated protein chaperones. PMID:19516331

  4. Ni(II), Cu(II), and Zn(II) Diethyldithiocarbamate Complexes Show Various Activities Against the Proteasome in Breast Cancer Cells

    PubMed Central

    Cvek, Boris; Milacic, Vesna; Taraba, Jan; Dou, Q. Ping

    2008-01-01

    A series of three complexes with diethyldithiocarbamate ligand and three different metals (Ni, Cu, Zn) was prepared, confirmed by X-ray crystallography, and tested in human breast cancer MDA-MB-231 cells. Zinc and copper complexes, but not nickel complex, were found to be more active against cellular 26S proteasome than against purified 20S proteasome core particle. One of the possible explanations is inhibition of JAMM domain in the 19S proteasome lid. PMID:18816109

  5. Effects of aging and dietary restriction on ubiquitination, sumoylation, and the proteasome in the spleen

    PubMed Central

    Zhang, Le; Li, Feng; Dimayuga, Edgardo; Craddock, Jeffrey; Keller, Jeffrey N.

    2015-01-01

    In the present study we demonstrate for the first time that aging increases the levels of ubiquitinated protein in the spleen, and that dietary restriction (DR) significantly reduces these age-related increases in ubiquitinated protein. Sumoylated protein, proteasome subunits, and a protein essential for proteasome biogenesis (POMP1) were also increased with age in the spleen but were not significantly affected by DR. Chymotrypsin-like proteasome activity was elevated in the aged spleen, and was not significantly altered by DR. Together, these data demonstrate for the first time the multiple effects of aging and DR on ubiquitination, sumoylation, and the proteasome in the spleen. PMID:17991438

  6. The Regulatory Particle of the Saccharomyces cerevisiae Proteasome

    PubMed Central

    Glickman, Michael H.; Rubin, David M.; Fried, Victor A.; Finley, Daniel

    1998-01-01

    The proteasome is a multisubunit protease responsible for degrading proteins conjugated to ubiquitin. The 670-kDa core particle of the proteasome contains the proteolytic active sites, which face an interior chamber within the particle and are thus protected from the cytoplasm. The entry of substrates into this chamber is thought to be governed by the regulatory particle of the proteasome, which covers the presumed channels leading into the interior of the core particle. We have resolved native yeast proteasomes into two electrophoretic variants and have shown that these represent core particles capped with one or two regulatory particles. To determine the subunit composition of the regulatory particle, yeast proteasomes were purified and analyzed by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Resolution of the individual polypeptides revealed 17 distinct proteins, whose identities were determined by amino acid sequence analysis. Six of the subunits have sequence features of ATPases (Rpt1 to Rpt6). Affinity chromatography was used to purify regulatory particles from various strains, each of which expressed one of the ATPases tagged with hexahistidine. In all cases, multiple untagged ATPases copurified, indicating that the ATPases assembled together into a heteromeric complex. Of the remaining 11 subunits that we have identified (Rpn1 to Rpn3 and Rpn5 to Rpn12), 8 are encoded by previously described genes and 3 are encoded by genes not previously characterized for yeasts. One of the previously unidentified subunits exhibits limited sequence similarity with deubiquitinating enzymes. Overall, regulatory particles from yeasts and mammals are remarkably similar, suggesting that the specific mechanistic features of the proteasome have been closely conserved over the course of evolution. PMID:9584156

  7. Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis.

    PubMed

    Bai, Lin; Hu, Kuan; Wang, Tong; Jastrab, Jordan B; Darwin, K Heran; Li, Huilin

    2016-04-01

    The human pathogen Mycobacterium tuberculosis (Mtb) requires a proteasome system to cause lethal infections in mice. We recently found that proteasome accessory factor E (PafE, Rv3780) activates proteolysis by the Mtb proteasome independently of adenosine triphosphate (ATP). Moreover, PafE contributes to the heat-shock response and virulence of Mtb Here, we show that PafE subunits formed four-helix bundles similar to those of the eukaryotic ATP-independent proteasome activator subunits of PA26 and PA28. However, unlike any other known proteasome activator, PafE formed dodecamers with 12-fold symmetry, which required a glycine-XXX-glycine-XXX-glycine motif that is not found in previously described activators. Intriguingly, the truncation of the PafE carboxyl-terminus resulted in the robust binding of PafE rings to native proteasome core particles and substantially increased proteasomal activity, suggesting that the extended carboxyl-terminus of this cofactor confers suboptimal binding to the proteasome core particle. Collectively, our data show that proteasomal activation is not limited to hexameric ATPases in bacteria. PMID:27001842

  8. Structural analysis of the dodecameric proteasome activator PafE in Mycobacterium tuberculosis

    DOE PAGESBeta

    Bai, Lin; Hu, Kuan; Wang, Tong; Jastrab, Jordan B.; Darwin, K. Heran; Li, Huilin

    2016-03-21

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

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

    PubMed

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

    2015-10-01

    Aging is the progressive loss of cellular function which inevitably leads to death. Failure of proteostasis including the decrease in proteasome function is one hallmark of aging. In the lung, proteasome activity was shown to be impaired in age-related diseases such as chronic obstructive pulmonary disease. However, little is known on proteasome function during healthy aging. Here, we comprehensively analyzed healthy lung aging and proteasome function in wildtype, proteasome reporter and immunoproteasome knockout mice. Wildtype mice spontaneously developed senile lung emphysema while expression and activity of proteasome complexes and turnover of ubiquitinated substrates was not grossly altered in lungs of aged mice. Immunoproteasome subunits were specifically upregulated in the aged lung and the caspase-like proteasome activity concomitantly decreased. Aged knockout mice for the LMP2 or LMP7 immunoproteasome subunits showed no alteration in proteasome activities but exhibited typical lung aging phenotypes suggesting that immunoproteasome function is dispensable for physiological lung aging in mice. Our results indicate that healthy aging of the lung does not involve impairment of proteasome function. Apparently, the reserve capacity of the proteostasis systems in the lung is sufficient to avoid severe proteostasis imbalance during healthy aging. PMID:26540298

  10. Assembly of an Evolutionarily Conserved Alternative Proteasome Isoform in Human Cells

    PubMed Central

    Padmanabhan, Achuth; Vuong, Simone Anh-Thu; Hochstrasser, Mark

    2016-01-01

    Summary Targeted intracellular protein degradation in eukaryotes is largely mediated by the proteasome. Here we report formation of an alternative proteasome isoform in human cells, previously found only in budding yeast, which bears an altered subunit arrangement in the outer ring of the proteasome core particle. These proteasomes result from incorporation of an additional α4 (PSMA7) subunit in the position normally occupied by α3 (PSMA4). Assembly of ‘α4-α4’ proteasomes depends on the relative cellular levels of α4 and α3, and on the proteasome assembly chaperone PAC3. The oncogenic tyrosine kinases ABL and ARG and the tumor suppressor BRCA1 regulate cellular α4 levels and formation of α4-α4 proteasomes. Cells primed to assemble α4-α4 proteasomes exhibit enhanced resistance to toxic metal ions. Taken together, our results establish the existence of a novel mammalian proteasome isoform and suggest a potential role in enabling cells to adapt to environmental stresses. PMID:26997268

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

    PubMed Central

    Lukas, Christina; Yildirim, Ali Ö.; Eickelberg, Oliver; Meiners, Silke

    2015-01-01

    Aging is the progressive loss of cellular function which inevitably leads to death. Failure of proteostasis including the decrease in proteasome function is one hallmark of aging. In the lung, proteasome activity was shown to be impaired in age‐related diseases such as chronic obstructive pulmonary disease. However, little is known on proteasome function during healthy aging. Here, we comprehensively analyzed healthy lung aging and proteasome function in wildtype, proteasome reporter and immunoproteasome knockout mice. Wildtype mice spontaneously developed senile lung emphysema while expression and activity of proteasome complexes and turnover of ubiquitinated substrates was not grossly altered in lungs of aged mice. Immunoproteasome subunits were specifically upregulated in the aged lung and the caspase‐like proteasome activity concomitantly decreased. Aged knockout mice for the LMP2 or LMP7 immunoproteasome subunits showed no alteration in proteasome activities but exhibited typical lung aging phenotypes suggesting that immunoproteasome function is dispensable for physiological lung aging in mice. Our results indicate that healthy aging of the lung does not involve impairment of proteasome function. Apparently, the reserve capacity of the proteostasis systems in the lung is sufficient to avoid severe proteostasis imbalance during healthy aging. PMID:26540298

  12. Assembly of an Evolutionarily Conserved Alternative Proteasome Isoform in Human Cells.

    PubMed

    Padmanabhan, Achuth; Vuong, Simone Anh-Thu; Hochstrasser, Mark

    2016-03-29

    Targeted intracellular protein degradation in eukaryotes is largely mediated by the proteasome. Here, we report the formation of an alternative proteasome isoform in human cells, previously found only in budding yeast, that bears an altered subunit arrangement in the outer ring of the proteasome core particle. These proteasomes result from incorporation of an additional α4 (PSMA7) subunit in the position normally occupied by α3 (PSMA4). Assembly of "α4-α4" proteasomes depends on the relative cellular levels of α4 and α3 and on the proteasome assembly chaperone PAC3. The oncogenic tyrosine kinases ABL and ARG and the tumor suppressor BRCA1 regulate cellular α4 levels and formation of α4-α4 proteasomes. Cells primed to assemble α4-α4 proteasomes exhibit enhanced resistance to toxic metal ions. Taken together, our results establish the existence of an alternative mammalian proteasome isoform and suggest a potential role in enabling cells to adapt to environmental stresses. PMID:26997268

  13. The Archaeal Proteasome Is Regulated by a Network of AAA ATPases*

    PubMed Central

    Forouzan, Dara; Ammelburg, Moritz; Hobel, Cedric F.; Ströh, Luisa J.; Sessler, Nicole; Martin, Jörg; Lupas, Andrei N.

    2012-01-01

    The proteasome is the central machinery for targeted protein degradation in archaea, Actinobacteria, and eukaryotes. In its basic form, it consists of a regulatory ATPase complex and a proteolytic core particle. The interaction between the two is governed by an HbYX motif (where Hb is a hydrophobic residue, Y is tyrosine, and X is any amino acid) at the C terminus of the ATPase subunits, which stimulates gate opening of the proteasomal α-subunits. In archaea, the proteasome-interacting motif is not only found in canonical proteasome-activating nucleotidases of the PAN/ARC/Rpt group, which are absent in major archaeal lineages, but also in proteins of the CDC48/p97/VAT and AMA groups, suggesting a regulatory network of proteasomal ATPases. Indeed, Thermoplasma acidophilum, which lacks PAN, encodes one CDC48 protein that interacts with the 20S proteasome and activates the degradation of model substrates. In contrast, Methanosarcina mazei contains seven AAA proteins, five of which, both PAN proteins, two out of three CDC48 proteins, and the AMA protein, function as proteasomal gatekeepers. The prevalent presence of multiple, distinct proteasomal ATPases in archaea thus results in a network of regulatory ATPases that may widen the substrate spectrum of proteasomal protein degradation. PMID:22992741

  14. Combined 3D-QSAR, Molecular Docking and Molecular Dynamics Study on Derivatives of Peptide Epoxyketone and Tyropeptin-Boronic Acid as Inhibitors Against the β5 Subunit of Human 20S Proteasome

    PubMed Central

    Liu, Jianling; Zhang, Hong; Xiao, Zhengtao; Wang, Fangfang; Wang, Xia; Wang, Yonghua

    2011-01-01

    An abnormal ubiquitin-proteasome is found in many human diseases, especially in cancer, and has received extensive attention as a promising therapeutic target in recent years. In this work, several in silico models have been built with two classes of proteasome inhibitors (PIs) by using 3D-QSAR, homology modeling, molecular docking and molecular dynamics (MD) simulations. The study resulted in two types of satisfactory 3D-QSAR models, i.e., the CoMFA model (Q2 = 0.462, R2pred = 0.820) for epoxyketone inhibitors (EPK) and the CoMSIA model (Q2 = 0.622, R2pred = 0.821) for tyropeptin-boronic acid derivatives (TBA). From the contour maps, some key structural factors responsible for the activity of these two series of PIs are revealed. For EPK inhibitors, the N-cap part should have higher electropositivity; a large substituent such as a benzene ring is favored at the C6-position. In terms of TBA inhibitors, hydrophobic substituents with a larger size anisole group are preferential at the C8-position; higher electropositive substituents like a naphthalene group at the C3-position can enhance the activity of the drug by providing hydrogen bond interaction with the protein target. Molecular docking disclosed that residues Thr60, Thr80, Gly106 and Ser189 play a pivotal role in maintaining the drug-target interactions, which are consistent with the contour maps. MD simulations further indicated that the binding modes of each conformation derived from docking is stable and in accord with the corresponding structure extracted from MD simulation overall. These results can offer useful theoretical references for designing more potent PIs. PMID:21673924

  15. Enhanced proteasome degradation extends Caenorhabditis elegans lifespan and alleviates aggregation-related pathologies.

    PubMed

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

    2014-10-01

    Collapse of proteostasis and accumulation of damaged macromolecules have been recognized as hallmarks of aging and age-related diseases. The proteasome is the major cellular protease responsible for intracellular protein degradation, having an impaired function during aging. We have previously shown that proteasome activation through overexpression of β5 proteasome subunit delays replicative senescence and confers resistance to oxidative stress in primary fibroblasts. Herein, we have investigated the impact of enhanced proteasome function on organismal longevity and aggregation-related pathologies by employing Caenorhabditis elegans as a model system. We have found that overexpression of a core 20S proteasome subunit in wild type worms extends lifespan, healthspan and survival under proteotoxic conditions. The longevity prolonging effect of the proteasome subunit overexpression was found to depend on the FOXO transcription factor DAF-16 and was associated with its elevated transcriptional activity. We have also uncovered a major role of enhanced proteasome activity in aggregation-related pathologies underlying neurodegenerative diseases. Genetic activation of the proteasome minimized the detrimental effect of polyglutamine-induced toxicity mimicking Huntington's disease, whereas knock-down of the proteasome component exaggerated the disease phenotypes. Similar results were obtained by using a C.elegans model of Amyloid beta (Αβ) -induced toxicity mimicking Alzheimer's disease. Collectively, these findings demonstrate that enhanced proteasome function alleviates proteotoxicity and promotes longevity in synergy with other nodes of lifespan regulation in C.elegans. Understanding the mechanism by which preservation of proteostasis via enhancement of proteasome function, decelerates the aging process and alleviates age-related pathologies may assist in the rational design of therapeutic and anti-aging interventions. PMID:26461298

  16. The transition zone protein Rpgrip1l regulates proteasomal activity at the primary cilium

    PubMed Central

    Lier, Johanna Maria; Burmühl, Stephan; Struchtrup, Andreas; Deutschmann, Kathleen; Vetter, Maik; Leu, Tristan; Reeg, Sandra; Grune, Tilman; Rüther, Ulrich

    2015-01-01

    Mutations in RPGRIP1L result in severe human diseases called ciliopathies. To unravel the molecular function of RPGRIP1L, we analyzed Rpgrip1l−/− mouse embryos, which display a ciliopathy phenotype and die, at the latest, around birth. In these embryos, cilia-mediated signaling was severely disturbed. Defects in Shh signaling suggested that the Rpgrip1l deficiency causes an impairment of protein degradation and protein processing. Indeed, we detected a cilia-dependent decreased proteasomal activity in the absence of Rpgrip1l. We found different proteasomal components localized to cilia and identified Psmd2, a component of the regulatory proteasomal 19S subunit, as an interaction partner for Rpgrip1l. Quantifications of proteasomal substrates demonstrated that Rpgrip1l regulates proteasomal activity specifically at the basal body. Our study suggests that Rpgrip1l controls ciliary signaling by regulating the activity of the ciliary proteasome via Psmd2. PMID:26150391

  17. The proteasome stress regulon is controlled by a pair of NAC transcription factors in arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteotoxic stress is mitigated by a variety of mechanisms, including activation of the unfolded protein response and coordinated increases in protein chaperones and activities that direct proteolysis such as the 26S proteasome. Using RNA-seq analyses combined with either chemical inhibitors or mut...

  18. Immunoproteasome Assembly: Cooperative Incorporation of Interferon γ (IFN-γ)–inducible Subunits

    PubMed Central

    Griffin, Thomas A.; Nandi, Dipankar; Cruz, Miguel; Fehling, Hans Jörg; Kaer, Luc Van; Monaco, John J.; Colbert, Robert A.

    1998-01-01

    LMP2, LMP7, and MECL are interferon γ–inducible catalytic subunits of vertebrate 20S proteasomes, which can replace constitutive catalytic subunits (delta, X, and Z, respectively) during proteasome biogenesis. We demonstrate that MECL requires LMP2 for efficient incorporation into preproteasomes, and preproteasomes containing LMP2 and MECL require LMP7 for efficient maturation. The latter effect depends on the presequence of LMP7, but not on LMP7 catalytic activity. This cooperative mechanism favors the assembly of homogeneous “immunoproteasomes” containing all three inducible subunits, suggesting that these subunits act in concert to enhance proteasomal generation of major histocompatibility complex class I–binding peptides. PMID:9419215

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

    PubMed Central

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

    2014-01-01

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

  20. Induction of caspase-3-like activity in rice following release of cytochrome-f from the chloroplast and subsequent interaction with the ubiquitin-proteasome system.

    PubMed

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

    2014-01-01

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

  1. Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate

    SciTech Connect

    Hu,G.; Lin, G.; Wang, M.; Dick, L.; Xu, R.; Nathan, C.; Li, H.

    2006-01-01

    Mycobacterium tuberculosis (Mtb) has the remarkable ability to resist killing by human macrophages. The 750 kDa proteasome, not available in most eubacteria except Actinomycetes, appears to contribute to Mtb's resistance. The crystal structure of the Mtb proteasome at 3.0 Angstroms resolution reveals a substrate-binding pocket with composite features of the distinct {beta}1, {beta}2 and {beta}5 substrate binding sites of eukaryotic proteasomes, accounting for the broad specificity of the Mtb proteasome towards oligopeptides described in the companion article [Lin et al. (2006), Mol Microbiol doi:10.1111/j.1365-2958.2005.05035.x]. The substrate entrance at the end of the cylindrical proteasome appears open in the crystal structure due to partial disorder of the a-subunit N-terminal residues. However, cryo-electron microscopy of the core particle reveals a closed end, compatible with the density observed in negative-staining electron microscopy that depended on the presence of the N-terminal octapeptides of the a-subunits in the companion article, suggesting that the Mtb proteasome has a gated structure. We determine for the first time the proteasomal inhibition mechanism of the dipeptidyl boronate N-(4-morpholine)carbonyl-{beta}-(1-naphthyl)-l-alanine-l-leucine boronic acid (MLN-273), an analogue of the antimyeloma drug bortezomib. The structure improves prospects for designing Mtb-specific proteasomal inhibitors as a novel approach to chemotherapy of tuberculosis.

  2. Structure of the Mycobacterium tuberculosis proteasome and mechanism of inhibition by a peptidyl boronate.

    PubMed

    Hu, Guiqing; Lin, Gang; Wang, Ming; Dick, Lawrence; Xu, Rui-Ming; Nathan, Carl; Li, Huilin

    2006-03-01

    Mycobacterium tuberculosis (Mtb) has the remarkable ability to resist killing by human macrophages. The 750 kDa proteasome, not available in most eubacteria except Actinomycetes, appears to contribute to Mtb's resistance. The crystal structure of the Mtb proteasome at 3.0 A resolution reveals a substrate-binding pocket with composite features of the distinct beta1, beta2 and beta5 substrate binding sites of eukaryotic proteasomes, accounting for the broad specificity of the Mtb proteasome towards oligopeptides described in the companion article [Lin et al. (2006), Mol Microbiol doi:10.1111/j.1365-2958.2005.05035.x]. The substrate entrance at the end of the cylindrical proteasome appears open in the crystal structure due to partial disorder of the alpha-subunit N-terminal residues. However, cryo-electron microscopy of the core particle reveals a closed end, compatible with the density observed in negative-staining electron microscopy that depended on the presence of the N-terminal octapetides of the alpha-subunits in the companion article, suggesting that the Mtb proteasome has a gated structure. We determine for the first time the proteasomal inhibition mechanism of the dipeptidyl boronate N-(4-morpholine)carbonyl-beta-(1-naphthyl)-L-alanine-L-leucine boronic acid (MLN-273), an analogue of the antimyeloma drug bortezomib. The structure improves prospects for designing Mtb-specific proteasomal inhibitors as a novel approach to chemotherapy of tuberculosis. PMID:16468986

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

    PubMed Central

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

    2014-01-01

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

  4. Starvation Induces Proteasome Autophagy with Different Pathways for Core and Regulatory Particles*

    PubMed Central

    Waite, Kenrick A.; Mota-Peynado, Alina De-La; Vontz, Gabrielle; Roelofs, Jeroen

    2016-01-01

    The proteasome is responsible for the degradation of many cellular proteins. If and how this abundant and normally stable complex is degraded by cells is largely unknown. Here we show that in yeast, upon nitrogen starvation, proteasomes are targeted for vacuolar degradation through autophagy. Using GFP-tagged proteasome subunits, we observed that autophagy of a core particle (CP) subunit depends on the deubiquitinating enzyme Ubp3, although a regulatory particle (RP) subunit does not. Furthermore, upon blocking of autophagy, RP remained largely nuclear, although CP largely localized to the cytosol as well as granular structures within the cytosol. In all, our data reveal a regulated process for the removal of proteasomes upon nitrogen starvation. This process involves CP and RP dissociation, nuclear export, and independent vacuolar targeting of CP and RP. Thus, in addition to the well characterized transcriptional up-regulation of genes encoding proteasome subunits, cells are also capable of down-regulating cellular levels of proteasomes through proteaphagy. PMID:26670610

  5. The genes for the {alpha}-type HC3 (PMSA2) and {beta}-type HC5 (PMSB1) subunits of human proteasomes map to chromosomes 6q27 and 7p12-p13 by fluorescence in situ hybridization

    SciTech Connect

    Okumura, Katsuzumi; Nogami, Masahiro; Taguchi, Hiroshi

    1995-05-20

    The authors have determined the locations of the genes for the two subunits, HC3 and HC5, by fluorescence in situ hybridization (FISH). Chromosome spreads were obtained from phytohemagglutinin-stimulated blood lymphocytes of a healthy donor after thymidine synchronization and bromodeoxyuridine incorporation by the method of Takahashi et al. Genomic DNA fragments of HC3 (4.3 kb, including exons 3, 4, and 5) and HC5 (7.5 kb including exons 1 and 2) (11) were labeled with biotin-16-dUTP by nick-translation. In situ hybridization was performed according to Lichter et al. in the presence of COT-1 DNA as a competitor. Hybridized probe was detected with FITC-conjugated avidin without further signal amplification. Comparison of the fluorescence signals and the banding patterns of the chromosomes indicated that the HC3 and HC5 genes were located on chromosome band 6q27 and 7p12-p13, respectively.

  6. Structural basis for proteasome formation controlled by an assembly chaperone nas2.

    PubMed

    Satoh, Tadashi; Saeki, Yasushi; Hiromoto, Takeshi; Wang, Ying-Hui; Uekusa, Yoshinori; Yagi, Hirokazu; Yoshihara, Hidehito; Yagi-Utsumi, Maho; Mizushima, Tsunehiro; Tanaka, Keiji; Kato, Koichi

    2014-05-01

    Proteasome formation does not occur due to spontaneous self-organization but results from a highly ordered process assisted by several assembly chaperones. The assembly of the proteasome ATPase subunits is assisted by four client-specific chaperones, of which three have been structurally resolved. Here, we provide the structural basis for the working mechanisms of the last, hereto structurally uncharacterized assembly chaperone, Nas2. We revealed that Nas2 binds to the Rpt5 subunit in a bivalent mode: the N-terminal helical domain of Nas2 masks the Rpt1-interacting surface of Rpt5, whereas its C-terminal PDZ domain caps the C-terminal proteasome-activating motif. Thus, Nas2 operates as a proteasome activation blocker, offering a checkpoint during the formation of the 19S ATPase prior to its docking onto the proteolytic 20S core particle. PMID:24685148

  7. Characterisation of 20S Proteasome in Tritrichomonas foetus and Its Role during the Cell Cycle and Transformation into Endoflagellar Form

    PubMed Central

    Pereira-Neves, Antonio; Gonzaga, Luiz; Menna-Barreto, Rubem F. S.; Benchimol, Marlene

    2015-01-01

    Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in

  8. Characterisation of 20S Proteasome in Tritrichomonas foetus and Its Role during the Cell Cycle and Transformation into Endoflagellar Form.

    PubMed

    Pereira-Neves, Antonio; Gonzaga, Luiz; Menna-Barreto, Rubem F S; Benchimol, Marlene

    2015-01-01

    Proteasomes are intracellular complexes that control selective protein degradation in organisms ranging from Archaea to higher eukaryotes. These structures have multiple proteolytic activities that are required for cell differentiation, replication and maintaining cellular homeostasis. Here, we document the presence of the 20S proteasome in the protist parasite Tritrichomonas foetus. Complementary techniques, such as a combination of whole genome sequencing technologies, bioinformatics algorithms, cell fractionation and biochemistry and microscopy approaches were used to characterise the 20S proteasome of T. foetus. The 14 homologues of the typical eukaryotic proteasome subunits were identified in the T. foetus genome. Alignment analyses showed that the main regulatory and catalytic domains of the proteasome were conserved in the predicted amino acid sequences from T. foetus-proteasome subunits. Immunofluorescence assays using an anti-proteasome antibody revealed a labelling distributed throughout the cytosol as punctate cytoplasmic structures and in the perinuclear region. Electron microscopy of a T. foetus-proteasome-enriched fraction confirmed the presence of particles that resembled the typical eukaryotic 20S proteasome. Fluorogenic assays using specific peptidyl substrates detected presence of the three typical peptidase activities of eukaryotic proteasomes in T. foetus. As expected, these peptidase activities were inhibited by lactacystin, a well-known specific proteasome inhibitor, and were not affected by inhibitors of serine or cysteine proteases. During the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, we observed correlations between the EFF formation rates, increases in the proteasome activities and reduced levels of ubiquitin-protein conjugates. The growth, cell cycle and EFF transformation of T. foetus were inhibited after treatment with lactacystin in a dose-dependent manner. Lactacystin treatment also resulted in

  9. The ubiquitin-proteasome system regulates plant hormone signaling

    PubMed Central

    Santner, Aaron; Estelle, Mark

    2011-01-01

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

  10. Implications for proteasome nuclear localization revealed by the structure of the nuclear proteasome tether protein Cut8

    PubMed Central

    Takeda, Kojiro; Tonthat, Nam K.; Glover, Tiffany; Xu, Weijun; Koonin, Eugene V.; Yanagida, Mitsuhiro; Schumacher, Maria A.

    2011-01-01

    Degradation of nuclear proteins by the 26S proteasome is essential for cell viability. In yeast, the nuclear envelope protein Cut8 mediates nuclear proteasomal sequestration by an uncharacterized mechanism. Here we describe structures of Schizosaccharomyces pombe Cut8, which shows that it contains a unique, modular fold composed of an extended N-terminal, lysine-rich segment that when ubiquitinated binds the proteasome, a dimer domain followed by a six-helix bundle connected to a flexible C tail. The Cut8 six-helix bundle shows structural similarity to 14-3-3 phosphoprotein-binding domains, and binding assays show that this domain is necessary and sufficient for liposome and cholesterol binding. Moreover, specific mutations in the 14-3-3 regions corresponding to putative cholesterol recognition/interaction amino acid consensus motifs abrogate cholesterol binding. In vivo studies confirmed that the 14-3-3 region is necessary for Cut8 membrane localization and that dimerization is critical for its function. Thus, the data reveal the Cut8 organization at the nuclear envelope. Reconstruction of Cut8 evolution suggests that it was present in the last common ancestor of extant eukaryotes and accordingly that nuclear proteasomal sequestration is an ancestral eukaryotic feature. The importance of Cut8 for cell viability and its absence in humans suggests it as a possible target for the development of specific chemotherapeutics against invasive fungal infections. PMID:21976488

  11. Ubiquitinated proteins activate the proteasomal ATPases by binding to Usp14 or Uch37 homologs.

    PubMed

    Peth, Andreas; Kukushkin, Nikolay; Bossé, Marc; Goldberg, Alfred L

    2013-03-15

    Degradation of ubiquitinated proteins by 26 S proteasomes requires ATP hydrolysis, but it is unclear how the proteasomal ATPases are regulated and how proteolysis, substrate deubiquitination, degradation, and ATP hydrolysis are coordinated. Polyubiquitinated proteins were shown to stimulate ATP hydrolysis by purified proteasomes, but only if the proteins contain a loosely folded domain. If they were not ubiquitinated, such proteins did not increase ATPase activity. However, they did so upon addition of ubiquitin aldehyde, which mimics the ubiquitin chain and binds to 26 S-associated deubiquitinating enzymes (DUBs): in yeast to Ubp6, which is essential for the ATPase activation, and in mammalian 26 S to the Ubp6 homolog, Usp14, and Uch37. Occupancy of either DUB by a ubiquitin conjugate leads to ATPase stimulation, thereby coupling deubiquitination and ATP hydrolysis. Thus, ubiquitinated loosely folded proteins, after becoming bound to the 26 S, interact with Ubp6/Usp14 or Uch37 to activate ATP hydrolysis and enhance their own destruction. PMID:23341450

  12. Profiling human protein degradome delineates cellular responses to proteasomal inhibition and reveals a feedback mechanism in regulating proteasome homeostasis.

    PubMed

    Yu, Tao; Tao, Yonghui; Yang, Meiqiang; Chen, Peng; Gao, Xiaobo; Zhang, Yanbo; Zhang, Tao; Chen, Zi; Hou, Jian; Zhang, Yan; Ruan, Kangcheng; Wang, Hongyan; Hu, Ronggui

    2014-10-01

    Global change in protein turnover (protein degradome) constitutes a central part of cellular responses to intrinsic or extrinsic stimuli. However, profiling protein degradome remains technically challenging. Recently, inhibition of the proteasome, e.g., by using bortezomib (BTZ), has emerged as a major chemotherapeutic strategy for treating multiple myeloma and other human malignancies, but systematic understanding of the mechanisms for BTZ drug action and tumor drug resistance is yet to be achieved. Here we developed and applied a dual-fluorescence-based Protein Turnover Assay (ProTA) to quantitatively profile global changes in human protein degradome upon BTZ-induced proteasomal inhibition. ProTA and subsequent network analyses delineate potential molecular basis for BTZ action and tumor drug resistance in BTZ chemotherapy. Finally, combined use of BTZ with drugs targeting the ProTA-identified key genes or pathways in BTZ action reduced BTZ resistance in multiple myeloma cells. Remarkably, BTZ stabilizes proteasome subunit PSMC1 and proteasome assembly factor PSMD10, suggesting a previously under-appreciated mechanism for regulating proteasome homeostasis. Therefore, ProTA is a novel tool for profiling human protein degradome to elucidate potential mechanisms of drug action and resistance, which might facilitate therapeutic development targeting proteostasis to treat human disorders. PMID:25223703

  13. Plant ubiquitin-proteasome pathway and its role in gibberellin signaling

    PubMed Central

    Wang, Feng; Deng, Xing Wang

    2011-01-01

    The ubiquitin-proteasome system (UPS) in plants, like in other eukaryotes, targets numerous intracellular regulators and thus modulates almost every aspect of growth and development. The well-known and best-characterized outcome of ubiquitination is mediating target protein degradation via the 26S proteasome, which represents the major selective protein degradation pathway conserved among eukaryotes. In this review, we will discuss the molecular composition, regulation and function of plant UPS, with a major focus on how DELLA protein degradation acts as a key in gibberellin signal transduction and its implication in the regulation of plant growth. PMID:21788985

  14. A Set of Activity-Based Probes to Visualize Human (Immuno)proteasome Activities.

    PubMed

    de Bruin, Gerjan; Xin, Bo Tao; Kraus, Marianne; van der Stelt, Mario; van der Marel, Gijsbert A; Kisselev, Alexei F; Driessen, Christoph; Florea, Bogdan I; Overkleeft, Herman S

    2016-03-18

    Proteasomes are therapeutic targets for various cancers and autoimmune diseases. Constitutively expressed proteasomes have three active sites, β1c, β2c, and β5c. Lymphoid tissues also express the immunoproteasome subunits β1i, β2i, and β5i. Rapid and simultaneous measurement of the activity of these catalytic subunits would assist in the discovery of new inhibitors, improve analysis of proteasome inhibitors in clinical trials, and simplify analysis of subunit expression. In this work, we present a cocktail of activity-based probes that enables simultaneous gel-based detection of all six catalytic human proteasome subunits. We used this cocktail to develop specific inhibitors for β1c, β2c, β5c, and β2i, to compare the active-site specificity of clinical proteasome inhibitors, and to demonstrate that many hematologic malignancies predominantly express immunoproteasomes. Furthermore, we show that selective and complete inhibition of β5i and β1i is cytotoxic to primary cells from acute lymphocytic leukemia (ALL) patients. PMID:26511210

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

    PubMed

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

    2016-02-11

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

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

    PubMed Central

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

    2016-01-01

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

  17. Activation of Chymotrypsin-Like Activity of the Proteasome during Ischemia Induces Myocardial Dysfunction and Death.

    PubMed

    Sanchez, Gina; Berrios, Daniela; Olmedo, Ivonne; Pezoa, Javier; Riquelme, Jaime A; Montecinos, Luis; Pedrozo, Zully; Donoso, Paulina

    2016-01-01

    Inhibitors of the ubiquitin-proteasome system improve hemodynamic parameters and decrease the infarct size after ischemia reperfusion. The molecular basis of this protection is not fully understood since most available data report inhibition of the 26 proteasome after ischemia reperfusion. The decrease in cellular ATP levels during ischemia leads to the dissociation of the 26S proteasome into the 19S regulatory complex and the 20S catalytic core, which results in protein degradation independently of ubiquitination. There is scarce information on the activity of the 20S proteasome during cardiac ischemia. Accordingly, the aim of this work was to determine the effects of 30 minutes of ischemia, or 30 min of ischemia followed by 60 minutes of reperfusion on the three main peptidase activities of the 20S proteasome in Langendorff perfused rat hearts. We found that 30 min of ischemia produced a significant increase in the chymotrypsin-like activity of the proteasome, without changes in its caspase-like or trypsin-like activities. In contrast, all three activities were decreased upon reperfusion. Ixazomib, perfused before ischemia at a concentration that reduced the chymotrypsin-like activity to 50% of the control values, without affecting the other proteasomal activities, improved the hemodynamic parameters upon reperfusion and decreased the infarct size. Ixazomib also prevented the 50% reduction in RyR2 content observed after ischemia. The protection was lost, however, when simultaneous inhibition of chymotrypsin-like and caspase-like activities of the proteasome was achieved at higher concentration of ixazomib. Our results suggest that selective inhibition of chymotrypsin-like activity of the proteasome during ischemia preserves key proteins for cardiomyocyte function and exerts a positive impact on cardiac performance after reperfusion. PMID:27529620

  18. Activation of Chymotrypsin-Like Activity of the Proteasome during Ischemia Induces Myocardial Dysfunction and Death

    PubMed Central

    Sanchez, Gina; Berrios, Daniela; Olmedo, Ivonne; Pezoa, Javier; Riquelme, Jaime A.; Montecinos, Luis; Pedrozo, Zully; Donoso, Paulina

    2016-01-01

    Inhibitors of the ubiquitin-proteasome system improve hemodynamic parameters and decrease the infarct size after ischemia reperfusion. The molecular basis of this protection is not fully understood since most available data report inhibition of the 26 proteasome after ischemia reperfusion. The decrease in cellular ATP levels during ischemia leads to the dissociation of the 26S proteasome into the 19S regulatory complex and the 20S catalytic core, which results in protein degradation independently of ubiquitination. There is scarce information on the activity of the 20S proteasome during cardiac ischemia. Accordingly, the aim of this work was to determine the effects of 30 minutes of ischemia, or 30 min of ischemia followed by 60 minutes of reperfusion on the three main peptidase activities of the 20S proteasome in Langendorff perfused rat hearts. We found that 30 min of ischemia produced a significant increase in the chymotrypsin-like activity of the proteasome, without changes in its caspase-like or trypsin-like activities. In contrast, all three activities were decreased upon reperfusion. Ixazomib, perfused before ischemia at a concentration that reduced the chymotrypsin-like activity to 50% of the control values, without affecting the other proteasomal activities, improved the hemodynamic parameters upon reperfusion and decreased the infarct size. Ixazomib also prevented the 50% reduction in RyR2 content observed after ischemia. The protection was lost, however, when simultaneous inhibition of chymotrypsin-like and caspase-like activities of the proteasome was achieved at higher concentration of ixazomib. Our results suggest that selective inhibition of chymotrypsin-like activity of the proteasome during ischemia preserves key proteins for cardiomyocyte function and exerts a positive impact on cardiac performance after reperfusion. PMID:27529620

  19. [Proteasome inhibitors in cancer therapy].

    PubMed

    Romaniuk, Wioletta; Ołdziej, Agnieszka Ewa; Zińczuk, Justyna; Kłoczko, Janusz

    2015-01-01

    Proteasomes are multisubunit enzyme complexes. They contain three enzymatic active sites which are termed chymotrypsin-like, trypsin-like, and caspase-like. The elementary function of the proteasomes is degradation of damaged proteins. Proteasome inhibition leads to accumulation of damaged protein, which leads to caspase activation and cell death. This relationship is used in cancer therapy. Bortezomib is the first proteasome inhibitor approved by the US Food and Drug Administration for the treatment of relapsed/refractory multiple myeloma. Carfilzomib belongs to the second generation of drugs, which was approved by the US FDA in 2012. Currently in the study phase there are four new inhibitors: ixazomib (MLN9780/MLN2238), delanzomib (CEP-18770), oprozomib (ONX0912/PR-047) and marizomib (NPI-0052). PMID:27259216

  20. Proteasome dysfunction triggers activation of SKN-1A/Nrf1 by the aspartic protease DDI-1.

    PubMed

    Lehrbach, Nicolas J; Ruvkun, Gary

    2016-01-01

    Proteasomes are essential for protein homeostasis in eukaryotes. To preserve cellular function, transcription of proteasome subunit genes is induced in response to proteasome dysfunction caused by pathogen attacks or proteasome inhibitor drugs. In Caenorhabditis elegans, this response requires SKN-1, a transcription factor related to mammalian Nrf1/2. Here, we use comprehensive genetic analyses to identify the pathway required for C. elegans to detect proteasome dysfunction and activate SKN-1. Genes required for SKN-1 activation encode regulators of ER traffic, a peptide N-glycanase, and DDI-1, a conserved aspartic protease. DDI-1 expression is induced by proteasome dysfunction, and we show that DDI-1 is required to cleave and activate an ER-associated isoform of SKN-1. Mammalian Nrf1 is also ER-associated and subject to proteolytic cleavage, suggesting a conserved mechanism of proteasome surveillance. Targeting mammalian DDI1 protease could mitigate effects of proteasome dysfunction in aging and protein aggregation disorders, or increase effectiveness of proteasome inhibitor cancer chemotherapies. PMID:27528192

  1. Proteasome dysfunction triggers activation of SKN-1A/Nrf1 by the aspartic protease DDI-1

    PubMed Central

    Lehrbach, Nicolas J; Ruvkun, Gary

    2016-01-01

    Proteasomes are essential for protein homeostasis in eukaryotes. To preserve cellular function, transcription of proteasome subunit genes is induced in response to proteasome dysfunction caused by pathogen attacks or proteasome inhibitor drugs. In Caenorhabditis elegans, this response requires SKN-1, a transcription factor related to mammalian Nrf1/2. Here, we use comprehensive genetic analyses to identify the pathway required for C. elegans to detect proteasome dysfunction and activate SKN-1. Genes required for SKN-1 activation encode regulators of ER traffic, a peptide N-glycanase, and DDI-1, a conserved aspartic protease. DDI-1 expression is induced by proteasome dysfunction, and we show that DDI-1 is required to cleave and activate an ER-associated isoform of SKN-1. Mammalian Nrf1 is also ER-associated and subject to proteolytic cleavage, suggesting a conserved mechanism of proteasome surveillance. Targeting mammalian DDI1 protease could mitigate effects of proteasome dysfunction in aging and protein aggregation disorders, or increase effectiveness of proteasome inhibitor cancer chemotherapies. DOI: http://dx.doi.org/10.7554/eLife.17721.001 PMID:27528192

  2. Negatively Charged Metal Oxide Nanoparticles Interact with the 20S Proteasome and Differentially Modulate Its Biologic Functional Effects

    PubMed Central

    Falaschetti, Christine A.; Paunesku, Tatjana; Kurepa, Jasmina; Nanavati, Dhaval; Chou, Stanley S.; De, Mrinmoy; Song, MinHa; Jang, Jung-tak; Wu, Aiguo; Dravid, Vinayak P.; Cheon, Jinwoo; Smalle, Jan; Woloschak, Gayle E.

    2013-01-01

    The multicatalytic ubiquitin-proteasome system (UPS) carries out proteolysis in a highly orchestrated way and regulates a large number of cellular processes. Deregulation of the UPS in many disorders has been documented. In some cases, e.g. carcinogenesis, elevated proteasome activity has been implicated in disease development, while the etiology of other diseases, e.g. neurodegeneration, includes decreased UPS activity. Therefore, agents that alter proteasome activity could suppress as well as enhance a multitude of diseases. Metal oxide nanoparticles, often developed as diagnostic tools, have not previously been tested as modulators of proteasome activity. Here, several types of metal oxide nanoparticles were found to adsorb to the proteasome and show variable preferential binding for particular proteasome subunits with several peptide binding “hotspots” possible. These interactions depend on the size, charge, and concentration of the nanoparticles and affect proteasome activity in a time-dependent manner. Should metal oxide nanoparticles increase proteasome activity in cells, as they do in vitro, unintended effects related to changes in proteasome function can be expected. PMID:23930940

  3. Proteasome Assay in Cell Lysates

    PubMed Central

    Maher, Pamela

    2016-01-01

    The ubiquitin-proteasome system (UPS) mediates the majority of the proteolysis seen in the cytoplasm and nucleus of mammalian cells. As such it plays an important role in the regulation of a variety of physiological and pathophysiological processes including tumorigenesis, inflammation and cell death (Ciechanover, 2005; Kisselev and Goldberg, 2001). A number of recent studies have shown that proteasome activity is decreased in a variety of neurological disorders including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and stroke as well as during normal aging (Chung et al., 2001; Ciechanover and Brundin, 2003; Betarbet et al., 2005). This decrease in proteasome activity is thought to play a critical role in the accumulation of abnormal and oxidized proteins. Protein clearance by the UPS involves two sequential reactions. The first is the tagging of protein lysine residues with ubiquitin (Ub) and the second is the subsequent degradation of the tagged proteins by the proteasome. We herein describe an assay for the second of these two reactions (Valera et al., 2013). This assay uses fluorogenic substrates for each of the three activities of the proteasome: chymotrypsin-like activity, trypsin-like activity and caspase-like activity. Cleavage of the fluorophore from the substrate by the proteasome results in fluorescence that can be detected with a fluorescent plate reader.

  4. Coupling caspase cleavage and proteasomal degradation of proteins carrying PEST motif.

    PubMed

    Belizario, José E; Alves, Juliano; Garay-Malpartida, Miguel; Occhiucci, João Marcelo

    2008-06-01

    The degradation is critical to activation and deactivation of regulatory proteins involved in signaling pathways to cell growth, differentiation, stress responses and physiological cell death. Proteins carry domains and sequence motifs that function as prerequisite for their proteolysis by either individual proteases or the 26S multicomplex proteasomes. Two models for entry of substrates into the proteasomes have been considered. In one model, it is proposed that the ubiquitin chain attached to the protein serves as recognition element to drag them into the 19S regulatory particle, which promotes the unfolding required to its access into the 20S catalytic chamber. In second model, it is proposed that an unstructured tail located at amino or carboxyl terminus directly track proteins into the 26S/20S proteasomes. Caspases are cysteinyl aspartate proteases that control diverse signaling pathways, promoting the cleavage at one or two sites of hundreds of structural and regulatory protein substrates. Caspase cleavage sites are commonly found within PEST motifs, which are segments rich in proline (P), glutamic acid (D), aspartic acid (E) and serine (S) or threonine (T) residues. Considering that N- and C- terminal peptide carrying PEST motifs form disordered loops in the globular proteins after caspase cleavage, it is postulated here that these exposed termini serve as unstructured initiation site, coupling caspase cleavage and ubiquitin-proteasome dependent and independent degradation of short-lived proteins. This could explain the inherent susceptibility to proteolysis among proteins containing PEST motif. PMID:18537676

  5. Synthesis and Biological Evaluation of Naphthoquinone Analogs as a Novel Class of Proteasome Inhibitors

    PubMed Central

    Lawrence, Harshani R.; Kazi, Aslamuzzaman; Luo, Yunting; Kendig, Robert; Ge, Yiyu; Jain, Sanjula; Daniel, Kenyon; Santiago, Daniel; Guida, Wayne C.; Sebti, Saïd M.

    2012-01-01

    Screening of the NCI Diversity Set-1 identified PI-083 (NSC-45382) a proteasome inhibitor selective for cancer over normal cells. Focused libraries of novel compounds based on PI-083 chloronaphthoquinone and sulfonamide moieties were synthesized to gain a better understanding of the structure activity relationship responsible for chymotrypsin-like proteasome inhibitory activity. This led to the demonstration that the chloronaphthoquinone and the sulfonamide moieties are critical for inhibitory activity. The pyridyl group in PI-083 can be replaced with other heterocyclic groups without significant loss of activity. Molecular modeling studies were also performed to explore the detailed interactions of PI-083 and its derivatives with the β5 and β6 subunits of the 20S proteasome. The refined model showed an H-bond interaction between the Asp-114 and the sulfonamide moiety of the PI-083 in the β6 subunit. PMID:20621484

  6. Proteasome regulates turnover of toxic human amylin in pancreatic cells.

    PubMed

    Singh, Sanghamitra; Trikha, Saurabh; Sarkar, Anjali; Jeremic, Aleksandar M

    2016-09-01

    Toxic human amylin (hA) oligomers and aggregates are implicated in the pathogenesis of type 2 diabetes mellitus (T2DM). Although recent studies demonstrated a causal connection between hA uptake and toxicity in pancreatic cells, the mechanism of amylin's clearance following its internalization and its relationship to toxicity is yet to be determined, and hence was investigated here. Using pancreatic rat insulinoma β-cells and human islets as model systems, we show that hA, following its internalization, first accumulates in the cytosol followed by its translocation into nucleus, and to a lesser extent lysosomes, keeping the net cytosolic amylin content low. An increase in hA accumulation in the nucleus of pancreatic cells correlated with its cytotoxicity, suggesting that its excessive accumulation in the nucleus is detrimental. hA interacted with 20S core and 19S lid subunits of the β-cell proteasomal complex, as suggested by immunoprecipitation and confocal microscopy studies, which subsequently resulted in a decrease in the proteasome's proteolytic activity in these cells. In vitro binding and activity assays confirmed an intrinsic and potent ability of amylin to interact with the 20S core complex thereby modulating its proteolytic activity. Interestingly, less toxic and aggregation incapable rat amylin (rA) showed a comparable inhibitory effect on proteasome activity and protein ubiquitination, decoupling amylin aggregation/ toxicity and amylin-induced protein stress. In agreement with these studies, inhibition of proteasomal proteolytic activity significantly increased intracellular amylin content and toxicity. Taken together, our results suggest a pivotal role of proteasomes in amylin's turnover and detoxification in pancreatic cells. PMID:27340132

  7. Proteasome inhibition improves fractionated radiation treatment against non-small cell lung cancer: an antioxidant connection.

    PubMed

    Grimes, Kristopher Ray; Daosukho, Chotiros; Zhao, Yunfeng; Meigooni, Ali; St Clair, William

    2005-10-01

    Non-small cell lung cancer frequently presents as a locally advanced disease. In this setting, radiation has a prominent role in cancer therapy. However, tumor adaptation to oxidative stress may lessen the efficacy of radiation therapy. Recent studies demonstrate that proteasome inhibitors increase the efficacy of radiation against a range of tumors. Although proteasome inhibition impacts on NF-kappaB translocation, the precise mechanism through which proteasome inhibitors induce tumor cell death and promote radiation efficacy remains unclear. The purpose of this study is to evaluate the potential of the proteasome inhibitor, MG-132, to improve the efficacy of radiation therapy and to determine whether its effect is linked to the suppression of the antioxidant enzyme, manganese superoxide dismutase (MnSOD). Human NSCLC (A549) cells were utilized both in vivo and in vitro to evaluate proteasome inhibition on radiation response. In vivo, mice that received combined treatments of 2.5 microg/g body weight MG-132 and 30 Gy demonstrated a delay in tumor regrowth in comparison to the 30 Gy control group. In vitro, clonegenic survival assays confirmed a dose-dependent enhancement of radiation sensitivity in combination with MG-132 and a significant interaction between the two. The levels of IkappaB-alpha, a NF-kappaB target gene and also an inhibitor of NF-kappaB nuclear translocation, decreased in a time-dependent manner following administration of MG-132 confirming the inhibition of the 26S proteasome. The MnSOD protein level was increased consistent with lower levels of IkappaB-alpha, confirming a NF-kappaB-mediated effect. Cells treated with radiation demonstrated an induction of MnSOD; however, the administration of MG-132 suppressed this induction These results support the hypothesis that proteasome inhibitors such as MG-132 can increase the efficacy of radiation therapy, in part, by suppression of cytoprotective NF-kappaB-mediated MnSOD expression. PMID:16142322

  8. PROTEASOME ACTIVITY DECLINES IN AGED MACROPHAGES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome pathway is involved in regulation of a variety of biologically important processes including antigen presentation by macrophages. Age-related decrease in proteasome activity has been reported in other tissues. However, the effect of aging on the ubiquitin-proteasome pathway ...

  9. PROTEASOME ACTIVITY DECLINES IN AGED MACROPHAGES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ubiquitin-proteasome pathway is involved in regulation of a variety of biologically important processes including antigen presentation by macrophages (Mf). Age-related decrease in proteasome activity has been reported in other tissues. However, the effect of aging on the ubiquitin-proteasome pat...

  10. 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

  11. 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

    2011-01-01

    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

  12. 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.

  13. Prolonged Proteasome Inhibition Cyclically Upregulates Oct3/4 and Nanog Gene Expression, but Reduces Induced Pluripotent Stem Cell Colony Formation

    PubMed Central

    Floyd, Elizabeth Z.; Staszkiewicz, Jaroslaw; Power, Rachel A.; Kilroy, Gail; Kirk-Ballard, Heather; Barnes, Christian W.; Strickler, Karen L.; Rim, Jong S.; Harkins, Lettie L.; Gao, Ru; Kim, Jeong

    2015-01-01

    Abstract There is ample evidence that the ubiquitin–proteasome system is an important regulator of transcription and its activity is necessary for maintaining pluripotency and promoting cellular reprogramming. Moreover, proteasome activity contributes to maintaining the open chromatin structure found in pluripotent stem cells, acting as a transcriptional inhibitor at specific gene loci generally associated with differentiation. The current study was designed to understand further the role of proteasome inhibition in reprogramming and its ability to modulate endogenous expression of pluripotency-related genes and induced pluripotent stem cells (iPSCs) colony formation. Herein, we demonstrate that acute combinatorial treatment with the proteasome inhibitors MG101 or MG132 and the histone deacetylase (HDAC) inhibitor valproic acid (VPA) increases gene expression of the pluripotency marker Oct3/4, and that MG101 alone is as effective as VPA in the induction of Oct3/4 mRNA expression in fibroblasts. Prolonged proteasome inhibition cyclically upregulates gene expression of Oct3/4 and Nanog, but reduces colony formation in the presence of the iPSC induction cocktail. In conclusion, our results demonstrate that the 26S proteasome is an essential modulator in the reprogramming process. Its inhibition enhances expression of pluripotency-related genes; however, efficient colony formation requires proteasome activity. Therefore, discovery of small molecules that increase proteasome activity might lead to more efficient cell reprogramming and generation of pluripotent cells. PMID:25826722

  14. Proteolysis, proteasomes and antigen presentation

    NASA Technical Reports Server (NTRS)

    Goldberg, A. L.; Rock, K. L.

    1992-01-01

    Proteins presented to the immune system must first be cleaved to small peptides by intracellular proteinases. Proteasomes are proteolytic complexes that degrade cytosolic and nuclear proteins. These particles have been implicated in ATP-ubiquitin-dependent proteolysis and in the processing of intracellular antigens for cytolytic immune responses.

  15. Suppression of BRCA1 sensitizes cells to proteasome inhibitors

    PubMed Central

    Gu, Y; Bouwman, P; Greco, D; Saarela, J; Yadav, B; Jonkers, J; Kuznetsov, S G

    2014-01-01

    BRCA1 is a multifunctional protein best known for its role in DNA repair and association with breast and ovarian cancers. To uncover novel biologically significant molecular functions of BRCA1, we tested a panel of 198 approved and experimental drugs to inhibit growth of MDA-MB-231 breast cancer cells depleted for BRCA1 by siRNA. 26S proteasome inhibitors bortezomib and carfilzomib emerged as a new class of selective BRCA1-targeting agents. The effect was confirmed in HeLa and U2OS cancer cell lines using two independent siRNAs, and in mouse embryonic stem (ES) cells with inducible deletion of Brca1. Bortezomib treatment did not cause any increase in nuclear foci containing phosphorylated histone H2AX, and knockdown of BRCA2 did not entail sensitivity to bortezomib, suggesting that the DNA repair function of BRCA1 may not be directly involved. We found that a toxic effect of bortezomib on BRCA1-depleted cells is mostly due to deregulated cell cycle checkpoints mediated by RB1-E2F pathway and 53BP1. Similar to BRCA1, depletion of RB1 also conferred sensitivity to bortezomib, whereas suppression of E2F1 or 53BP1 together with BRCA1 reduced induction of apoptosis after bortezomib treatment. A gene expression microarray study identified additional genes activated by bortezomib treatment only in the context of inactivation of BRCA1 including a critical involvement of the ERN1-mediated unfolded protein response. Our data indicate that BRCA1 has a novel molecular function affecting cell cycle checkpoints in a manner dependent on the 26S proteasome activity. PMID:25522274

  16. Suppression of BRCA1 sensitizes cells to proteasome inhibitors.

    PubMed

    Gu, Y; Bouwman, P; Greco, D; Saarela, J; Yadav, B; Jonkers, J; Kuznetsov, S G

    2014-01-01

    BRCA1 is a multifunctional protein best known for its role in DNA repair and association with breast and ovarian cancers. To uncover novel biologically significant molecular functions of BRCA1, we tested a panel of 198 approved and experimental drugs to inhibit growth of MDA-MB-231 breast cancer cells depleted for BRCA1 by siRNA. 26S proteasome inhibitors bortezomib and carfilzomib emerged as a new class of selective BRCA1-targeting agents. The effect was confirmed in HeLa and U2OS cancer cell lines using two independent siRNAs, and in mouse embryonic stem (ES) cells with inducible deletion of Brca1. Bortezomib treatment did not cause any increase in nuclear foci containing phosphorylated histone H2AX, and knockdown of BRCA2 did not entail sensitivity to bortezomib, suggesting that the DNA repair function of BRCA1 may not be directly involved. We found that a toxic effect of bortezomib on BRCA1-depleted cells is mostly due to deregulated cell cycle checkpoints mediated by RB1-E2F pathway and 53BP1. Similar to BRCA1, depletion of RB1 also conferred sensitivity to bortezomib, whereas suppression of E2F1 or 53BP1 together with BRCA1 reduced induction of apoptosis after bortezomib treatment. A gene expression microarray study identified additional genes activated by bortezomib treatment only in the context of inactivation of BRCA1 including a critical involvement of the ERN1-mediated unfolded protein response. Our data indicate that BRCA1 has a novel molecular function affecting cell cycle checkpoints in a manner dependent on the 26S proteasome activity. PMID:25522274

  17. Cellular and computational studies of proteasome inhibition and apoptosis induction in human cancer cells by amino acid Schiff base–copper complexes

    PubMed Central

    Zuo, Jian; Bi, Caifeng; Fan, Yuhua; Buac, Daniela; Nardon, Chiara; Daniel, Kenyon G.; Dou, Q. Ping

    2013-01-01

    Proliferation and apoptosis pathways are tightly regulated in a cell by the ubiquitin–proteasome system (UPS) and alterations in the UPS may result in cellular transformation or other pathological conditions. Indeed, the proteasome is often found to be overactive in cancer cells. It has also been found that cancer cells are more sensitive to proteasome inhibition than normal cells, and therefore proteasome inhibitors are pursued as antitumor drugs. The use of the proteasome inhibitor Bortezomib for treatment of multiple myeloma and mantle cell lymphoma has proved this principle. Recent studies have suggested that copper complexes can inhibit proteasome activity and induce apoptosis in some human cancer cells. However, the involved molecular mechanism is unknown. In this study, we investigated the biological activities of four amino acid Schiff base–copper(II) complexes by using human breast (MDA-MB-231 and MCF-7) and prostate (PC-3) cancer cells. The complexes C1 and C3, but not their counterparts C2 and C4, inhibit the chymotrypsin-like activity of purified 20S proteasome and human cancer cellular 26S proteasome, cause accumulation of proteasome target proteins Bax and IκB-α, and induce growth inhibition and apoptosis in concentration- and time-dependent manners. Docking analysis shows that C1, but not C2 has hydrophobic, pi–pi, pi–cation and hydrogen bond interactions with the proteasomal chymotrypsin-like pocket and could stably fit into the S3 region, leading to specific inhibition. Our study has identified the mechanism of action of these copper complexes on inhibiting tumor cell proteasome and suggested their great potential as novel anticancer agents. PMID:23142973

  18. Inhibition of Tumor Proteasome Activity by Gold Dithiocarbamato Complexes via both Redox-Dependent and –Independent Processes

    PubMed Central

    Milacic, Vesna; Ronconi, Luca; Fan, Yuhua; Bi, Caifeng; Fregona, Dolores; Dou, Q Ping

    2013-01-01

    We have previously reported on a gold(III) complex, namely [AuBr2(DMDT)] (N,N-dimethyldithiocarbamate) showing potent in vitro and in vivo growth inhibitory activities toward human cancer cells and identifying the cellular proteasome as one of the major targets. However, the importance of the oxidation state of the gold center and the involved mechanism of action has yet to be established. Here we show that both gold(III)- and gold(I)-dithiocarbamato species, namely [AuBr2(ESDT)] (AUL12) and [Au(ESDT)]2 (AUL15), could inhibit the chymotrypsin-like activity of purified 20S proteasome and 26S proteasome in human breast cancer MDA-MB-231 cells, resulting in accumulation of ubiquitinated proteins and proteasome target proteins, and induction of cell death, but at significantly different levels. Gold(I) and gold(III) compounds-mediated proteasome inhibition and cell death induction were completely reversed by the addition of a reducing agent, dithiothreitol or N-acetyl-l-cysteine, suggesting the involvement of redox processes. Furthermore, treatment of MDA-MB-231 cells with gold(III) compound (AUL12), but not the gold(I) analogue (AUL15), resulted in the production of significant level of reactive oxygen species. Our study provides strong evidence that the cellular proteasome is an imporant target of both gold(I) and gold(III) dithiocarbamates, but distinct cellular mechanisms of action are responsible for their different overall effect. PMID:19911377

  19. Proteasome activity or expression is not altered by activation of the heat shock transcription factor Hsf1 in cultured fibroblasts or myoblasts.

    PubMed

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

    2005-01-01

    Heat shock proteins (Hsps) with chaperoning function work together with the ubiquitin-proteasome pathway to prevent the accumulation of misfolded, potentially toxic proteins, as well as to control catabolism of the bulk of cytoplasmic, cellular protein. There is evidence for the involvement of both systems in neurodegenerative disease, and a therapeutic target is the heat shock transcription factor, Hsf1, which mediates upregulation of Hsps in response to cellular stress. The mechanisms regulating expression of proteasomal proteins in mammalian cells are less well defined. To assess any direct effect of Hsf1 on expression of proteasomal subunits and activity in mammalian cells, a plasmid encoding a constitutively active form of Hsf1 (Hsf1act) was expressed in mouse embryonic fibroblasts lacking Hsf1 and in cultured human myoblasts. Plasmid encoding an inactivatible form of Hsf1 (Hsf1inact) served as control. In cultures transfected with plasmid hsf1act, robust expression of the major stress-inducible Hsp, Hsp70, occurred but not in cultures transfected with hsf1inact. No significant changes in the level of expression of representative proteasomal proteins (structural [20Salpha], a nonpeptidase beta subunit [20Sbeta3], or 2 regulatory subunits [19S subunit 6b, 11 Salpha]) or in chymotrypsin-, trypsin-, and caspaselike activities of the proteasome were measured. Thus, stress-induced or pharmacological activation of Hsf1 in mammalian cells would upregulate Hsps but not directly affect expression or activity of proteasomes. PMID:16184768

  20. The ubiquitin ligase HERC3 attenuates NF-κB-dependent transcription independently of its enzymatic activity by delivering the RelA subunit for degradation

    PubMed Central

    Hochrainer, Karin; Pejanovic, Nadja; Olaseun, Victoria A.; Zhang, Sheng; Iadecola, Costantino; Anrather, Josef

    2015-01-01

    Activation of NF-κB-dependent transcription represents an important hallmark of inflammation. While the acute inflammatory response is per se beneficial, it can become deleterious if its spatial and temporal profile is not tightly controlled. Classically, NF-κB activity is limited by cytoplasmic retention of the NF-κB dimer through binding to inhibitory IκB proteins. However, increasing evidence suggests that NF-κB activity can also be efficiently contained by direct ubiquitination of NF-κB subunits. Here, we identify the HECT-domain ubiquitin ligase HERC3 as novel negative regulator of NF-κB activity. We find that HERC3 restricts NF-κB nuclear import and DNA binding without affecting IκBα degradation. Instead HERC3 indirectly binds to the NF-κB RelA subunit after liberation from IκBα inhibitor leading to its ubiquitination and protein destabilization. Remarkably, the regulation of RelA activity by HERC3 is independent of its inherent ubiquitin ligase activity. Rather, we show that HERC3 and RelA are part of a multi-protein complex containing the proteasome as well as the ubiquitin-like protein ubiquilin-1 (UBQLN1). We present evidence that HERC3 and UBQLN1 provide a link between NF-κB RelA and the 26S proteasome, thereby facilitating RelA protein degradation. Our findings establish HERC3 as novel candidate regulating the inflammatory response initiated by NF-κB. PMID:26476452

  1. The ubiquitin ligase HERC3 attenuates NF-κB-dependent transcription independently of its enzymatic activity by delivering the RelA subunit for degradation.

    PubMed

    Hochrainer, Karin; Pejanovic, Nadja; Olaseun, Victoria A; Zhang, Sheng; Iadecola, Costantino; Anrather, Josef

    2015-11-16

    Activation of NF-κB-dependent transcription represents an important hallmark of inflammation. While the acute inflammatory response is per se beneficial, it can become deleterious if its spatial and temporal profile is not tightly controlled. Classically, NF-κB activity is limited by cytoplasmic retention of the NF-κB dimer through binding to inhibitory IκB proteins. However, increasing evidence suggests that NF-κB activity can also be efficiently contained by direct ubiquitination of NF-κB subunits. Here, we identify the HECT-domain ubiquitin ligase HERC3 as novel negative regulator of NF-κB activity. We find that HERC3 restricts NF-κB nuclear import and DNA binding without affecting IκBα degradation. Instead HERC3 indirectly binds to the NF-κB RelA subunit after liberation from IκBα inhibitor leading to its ubiquitination and protein destabilization. Remarkably, the regulation of RelA activity by HERC3 is independent of its inherent ubiquitin ligase activity. Rather, we show that HERC3 and RelA are part of a multi-protein complex containing the proteasome as well as the ubiquitin-like protein ubiquilin-1 (UBQLN1). We present evidence that HERC3 and UBQLN1 provide a link between NF-κB RelA and the 26S proteasome, thereby facilitating RelA protein degradation. Our findings establish HERC3 as novel candidate regulating the inflammatory response initiated by NF-κB. PMID:26476452

  2. 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

  3. A reversible and highly selective inhibitor of the proteasomal ubiquitin receptor rpn13 is toxic to multiple myeloma cells.

    PubMed

    Trader, Darci J; Simanski, Scott; Kodadek, Thomas

    2015-05-20

    The proteasome is a multisubunit complex responsible for most nonlysosomal turnover of proteins in eukaryotic cells. Proteasome inhibitors are of great interest clinically, particularly for the treatment of multiple myeloma (MM). Unfortunately, resistance arises almost inevitably to these active site-targeted drugs. One strategy to overcome this resistance is to inhibit other steps in the protein turnover cascade mediated by the proteasome. Previously, Anchoori et al. identified Rpn13 as the target of an electrophilic compound (RA-190) that was selectively toxic to MM cells (Cancer Cell 2013, 24, 791-805), suggesting that this subunit of the proteasome is also a viable cancer drug target. Here we describe the discovery of the first highly selective, reversible Rpn13 ligands and show that they are also selectively toxic to MM cells. These data strongly support the hypothesis that Rpn13 is a viable target for the development of drugs to treat MM and other cancers. PMID:25914958

  4. ahg12 is a dominant proteasome mutant that affects multiple regulatory systems for germination of Arabidopsis.

    PubMed

    Hayashi, Shimpei; Hirayama, Takashi

    2016-01-01

    The ubiquitin-proteasome system is fundamentally involved in myriad biological phenomena of eukaryotes. In plants, this regulated protein degradation system has a pivotal role in the cellular response mechanisms for both internal and external stimuli, such as plant hormones and environmental stresses. Information about substrate selection by the ubiquitination machinery has accumulated, but there is very little information about selectivity for substrates at the proteasome. Here, we report characterization of a novel abscisic acid (ABA)-hypersensitive mutant named ABA hypersensitive germination12 (ahg12) in Arabidopsis. The ahg12 mutant showed a unique pleiotropic phenotype, including hypersensitivity to ABA and ethylene, and hyposensitivity to light. Map-based cloning identified the ahg12 mutation to cause an amino acid conversion in the L23 loop of RPT5a, which is predicted to form the pore structure of the 19S RP complex of the proteasome. Transient expression assays demonstrated that some plant-specific signaling components accumulated at higher levels in the ahg12 mutant. These results suggest that the ahg12 mutation led to changes in the substrate preference of the 26S proteasome. The discovery of the ahg12 mutation thus will contribute to elucidate the characteristics of the regulated protein degradation system. PMID:27139926

  5. Proteasome targeting of proteins in Arabidopsis leaf mesophyll, epidermal and vascular tissues

    PubMed Central

    Svozil, Julia; Gruissem, Wilhelm; Baerenfaller, Katja

    2015-01-01

    Protein and transcript levels are partly decoupled as a function of translation efficiency and protein degradation. Selective protein degradation via the Ubiquitin-26S proteasome system (UPS) ensures protein homeostasis and facilitates adjustment of protein abundance during changing environmental conditions. Since individual leaf tissues have specialized functions, their protein composition is different and hence also protein level regulation is expected to differ. To understand UPS function in a tissue-specific context we developed a method termed Meselect to effectively and rapidly separate Arabidopsis thaliana leaf epidermal, vascular and mesophyll tissues. Epidermal and vascular tissue cells are separated mechanically, while mesophyll cells are obtained after rapid protoplasting. The high yield of proteins was sufficient for tissue-specific proteome analyses after inhibition of the proteasome with the specific inhibitor Syringolin A (SylA) and affinity enrichment of ubiquitylated proteins. SylA treatment of leaves resulted in the accumulation of 225 proteins and identification of 519 ubiquitylated proteins. Proteins that were exclusively identified in the three different tissue types are consistent with specific cellular functions. Mesophyll cell proteins were enriched for plastid membrane translocation complexes as targets of the UPS. Epidermis enzymes of the TCA cycle and cell wall biosynthesis specifically accumulated after proteasome inhibition, and in the vascular tissue several enzymes involved in glucosinolate biosynthesis were found to be ubiquitylated. Our results demonstrate that protein level changes and UPS protein targets are characteristic of the individual leaf tissues and that the proteasome is relevant for tissue-specific functions. PMID:26074939

  6. The poxvirus encoded ubiquitin ligase, p28, is regulated by proteasomal degradation and autoubiquitination.

    PubMed

    Mottet, Kelly; Bareiss, Bettina; Milne, Craig D; Barry, Michele

    2014-11-01

    Virus manipulation of the ubiquitin-proteasome system has become increasingly apparent. Ubiquitin is a 76 amino acid protein that is post-translationally conjugated to target proteins, while poly-ubiquitination subsequently leads to degradation via the 26S proteasome. Target specificity is determined by a large family of ubiquitin ligases. Poxviruses encode p28, a highly conserved ubiquitin ligase expressed in a wide range of poxviruses (J. Virol. 79:597). Here we investigate the relationship between p28 and ubiquitination. Confocal microscopy indicated that orthologs of p28 co-localized with ubiquitin at the virus factory. Flow cytometry assays further demonstrated that p28 was regulated by proteasomal degradation. Moreover, when the ubiquitin ligase activity of p28 was disrupted by mutating the RING domain conjugated ubiquitin still localized to the viral factories, indicating that an unknown ubiquitin ligase(s) was responsible for regulating p28. Our observations indicate that p28 is a ubiquitin ligase that is regulated by ubiquitination and proteasomal degradation. PMID:25240226

  7. Dss1 associating with the proteasome functions in selective nuclear mRNA export in yeast

    SciTech Connect

    Mannen, Taro; Andoh, Tomoko; Tani, Tokio

    2008-01-25

    Dss1p is an evolutionarily conserved small protein that interacts with BRCA2, a tumor suppressor protein, in humans. The Schizosaccharomyces pombe strain lacking the dss1{sup +} gene ({delta}dss1) shows a temperature-sensitive growth defect and accumulation of bulk poly(A){sup +} RNA in the nucleus at a nonpermissive temperature. In situ hybridization using probes for several specific mRNAs, however, revealed that the analyzed mRNAs were exported normally to the cytoplasm in {delta}dss1, suggesting that Dss1p is required for export of some subsets of mRNAs. We identified the pad1{sup +} gene, which encodes a component of the 26S proteasome, as a suppressor for the ts{sup -} phenotype of {delta}dss1. Unexpectedly, overexpression of Pad1p could suppress neither the defect in nuclear mRNA export nor a defect in proteasome function. In addition, loss of proteasome functions does not cause defective nuclear mRNA export. Dss1p seems to be a multifunctional protein involved in nuclear export of specific sets of mRNAs and the ubiquitin-proteasome pathway in fission yeast.

  8. ahg12 is a dominant proteasome mutant that affects multiple regulatory systems for germination of Arabidopsis

    PubMed Central

    Hayashi, Shimpei; Hirayama, Takashi

    2016-01-01

    The ubiquitin-proteasome system is fundamentally involved in myriad biological phenomena of eukaryotes. In plants, this regulated protein degradation system has a pivotal role in the cellular response mechanisms for both internal and external stimuli, such as plant hormones and environmental stresses. Information about substrate selection by the ubiquitination machinery has accumulated, but there is very little information about selectivity for substrates at the proteasome. Here, we report characterization of a novel abscisic acid (ABA)-hypersensitive mutant named ABA hypersensitive germination12 (ahg12) in Arabidopsis. The ahg12 mutant showed a unique pleiotropic phenotype, including hypersensitivity to ABA and ethylene, and hyposensitivity to light. Map-based cloning identified the ahg12 mutation to cause an amino acid conversion in the L23 loop of RPT5a, which is predicted to form the pore structure of the 19S RP complex of the proteasome. Transient expression assays demonstrated that some plant-specific signaling components accumulated at higher levels in the ahg12 mutant. These results suggest that the ahg12 mutation led to changes in the substrate preference of the 26S proteasome. The discovery of the ahg12 mutation thus will contribute to elucidate the characteristics of the regulated protein degradation system. PMID:27139926

  9. Genomic organization and mapping of the mouse P26s4 ATPase gene: A member of the remarkably conserved AAA gene family

    SciTech Connect

    Hoyle, J.; Fisher, E.M.C.

    1996-01-01

    The eukaryotic genome contains a large family of ATPases in which each member has at least one highly conserved domain of approximately 200 amino acids with an ATP binding motif (the {open_quotes}AAA{close_quotes} domain). AAA ATPases play diverse roles in the cell and are of considerable interest to researchers investigating a number of different phenomena, including control of the cell cycle. We have characterized the mouse P26s4 AAA ATPase gene that encodes a subunit of the 26S protease, a multimeric complex that is responsible for the ubiquitin- and ATP-dependent degradation of specific proteins. The normal functioning of eukaryotic cells depends on this pathway to remove regulatory proteins such as cyclins or signal transduction molecules from the intracellular environment, with the appropriate timing to allow normal cell division and development. We have isolated mouse P26s4 cDNAs and mapped the P26s4 gene to chromosome 12. We have analyzed the intron-exon structure of the P26s4 genomic locus and have determined that the gene contains at least 10 introns, the first of which separates the start methionine from the rest of the coding sequence. 18 refs., 2 figs.

  10. Modeling proteasome dynamics in Parkinson's disease.

    PubMed

    Sneppen, Kim; Lizana, Ludvig; Jensen, Mogens H; Pigolotti, Simone; Otzen, Daniel

    2009-01-01

    In Parkinson's disease (PD), there is evidence that alpha-synuclein (alphaSN) 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 alphaSN 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 alphaSN 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 alphaSN 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. PMID:19411740

  11. 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.

  12. Autophagy and ubiquitin-proteasome system contribute to sperm mitophagy after mammalian fertilization.

    PubMed

    Song, Won-Hee; Yi, Young-Joo; Sutovsky, Miriam; Meyers, Stuart; Sutovsky, Peter

    2016-09-01

    Maternal inheritance of mitochondria and mtDNA is a universal principle in human and animal development, guided by selective ubiquitin-dependent degradation of the sperm-borne mitochondria after fertilization. However, it is not clear how the 26S proteasome, the ubiquitin-dependent protease that is only capable of degrading one protein molecule at a time, can dispose of a whole sperm mitochondrial sheath. We hypothesized that the canonical ubiquitin-like autophagy receptors [sequestosome 1 (SQSTM1), microtubule-associated protein 1 light chain 3 (LC3), gamma-aminobutyric acid receptor-associated protein (GABARAP)] and the nontraditional mitophagy pathways involving ubiquitin-proteasome system and the ubiquitin-binding protein dislocase, valosin-containing protein (VCP), may act in concert during mammalian sperm mitophagy. We found that the SQSTM1, but not GABARAP or LC3, associated with sperm mitochondria after fertilization in pig and rhesus monkey zygotes. Three sperm mitochondrial proteins copurified with the recombinant, ubiquitin-associated domain of SQSTM1. The accumulation of GABARAP-containing protein aggregates was observed in the vicinity of sperm mitochondrial sheaths in the zygotes and increased in the embryos treated with proteasomal inhibitor MG132, in which intact sperm mitochondrial sheaths were observed. Pharmacological inhibition of VCP significantly delayed the process of sperm mitophagy and completely prevented it when combined with microinjection of autophagy-targeting antibodies specific to SQSTM1 and/or GABARAP. Sperm mitophagy in higher mammals thus relies on a combined action of SQSTM1-dependent autophagy and VCP-mediated dislocation and presentation of ubiquitinated sperm mitochondrial proteins to the 26S proteasome, explaining how the whole sperm mitochondria are degraded inside the fertilized mammalian oocytes by a protein recycling system involved in degradation of single protein molecules. PMID:27551072

  13. Induction of Tumor Cell Apoptosis by a Proteasome Deubiquitinase Inhibitor Is Associated with Oxidative Stress

    PubMed Central

    Brnjic, Slavica; Mazurkiewicz, Magdalena; Fryknäs, Mårten; Sun, Chao; Zhang, Xiaonan; Larsson, Rolf

    2014-01-01

    Abstract Aims: b-AP15 is a recently described inhibitor of the USP14/UCHL5 deubiquitinases (DUBs) of the 19S proteasome. Exposure to b-AP15 results in blocking of proteasome function and accumulation of polyubiquitinated protein substrates in cells. This novel mechanism of proteasome inhibition may potentially be exploited for cancer therapy, in particular for treatment of malignancies resistant to currently used proteasome inhibitors. The aim of the present study was to characterize the cellular response to b-AP15-mediated proteasome DUB inhibition. Results: We report that b-AP15 elicits a similar, but yet distinct, cellular response as the clinically used proteasome inhibitor bortezomib. b-AP15 induces a rapid apoptotic response, associated with enhanced induction of oxidative stress and rapid activation of Jun-N-terminal kinase 1/2 (JNK)/activating protein-1 signaling. Scavenging of reactive oxygen species and pharmacological inhibition of JNK reduced b-AP15-induced apoptosis. We further report that endoplasmic reticulum (ER) stress is induced by b-AP15 and is involved in apoptosis induction. In contrast to bortezomib, ER stress is associated with induction of α-subunit of eukaryotic initiation factor 2 phosphorylation. Innovation: The findings establish that different modes of proteasome inhibition result in distinct cellular responses, a finding of potential therapeutic importance. Conclusion: Our data show that enhanced oxidative stress and ER stress are major determinants of the strong apoptotic response elicited by the 19S DUB inhibitor b-AP15. Antioxid. Redox Signal. 21, 2271–2285. PMID:24011031

  14. Phosphorylation and Methylation of Proteasomal Proteins of the Haloarcheon Haloferax volcanii

    DOE PAGESBeta

    Humbard, Matthew A.; Reuter, Christopher J.; Zuobi-Hasona, Kheir; Zhou, Guangyin; Maupin-Furlow, Julie A.

    2010-01-01

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

  15. Proteasome Inhibitors Block DNA Repair and Radiosensitize Non-Small Cell Lung Cancer

    PubMed Central

    Kushwaha, Deepa S.; Hsieh, Grace; Merzon, Dmitry; Rameseder, Jonathan; Chen, Clark C.; D’Andrea, Alan D.; Kozono, David

    2013-01-01

    Despite optimal radiation therapy (RT), chemotherapy and/or surgery, a majority of patients with locally advanced non-small cell lung cancer (NSCLC) fail treatment. To identify novel gene targets for improved tumor control, we performed whole genome RNAi screens to identify knockdowns that most reproducibly increase NSCLC cytotoxicity. These screens identified several proteasome subunits among top hits, including the topmost hit PSMA1, a component of the core 20 S proteasome. Radiation and proteasome inhibition showed synergistic effects. Proteasome inhibition resulted in an 80–90% decrease in homologous recombination (HR), a 50% decrease in expression of NF-κB-inducible HR genes BRCA1 and FANCD2, and a reduction of BRCA1, FANCD2 and RAD51 ionizing radiation-induced foci. IκBα RNAi knockdown rescued NSCLC radioresistance. Irradiation of mice with NCI-H460 xenografts after inducible PSMA1 shRNA knockdown markedly increased murine survival compared to either treatment alone. Proteasome inhibition is a promising strategy for NSCLC radiosensitization via inhibition of NF-κB-mediated expression of Fanconi Anemia/HR DNA repair genes. PMID:24040035

  16. Role of ubiquitin-proteasome system (UPS) in left ventricular hypertrophy (LVH)

    PubMed Central

    Cacciapuoti, Federico

    2014-01-01

    Cardiac hypertrophy is a key compensatory mechanism acting in response to pressure or volume overload, involving some alterations in signaling transduction pathways and transcription factors-regulation. These changes result in enhanced proteins’ synthesis leading to Left Ventricular Hypertrophy (LVH). It is known that the main function of Ubiquitin-Proteasome System (UPS) is to prevent accumulation of damaged, misfolded and mutant proteins by proteolysis. But emerging evidences suggest that UPS also attends to the cells’ growth, favoring proteins’ synthesis, subsequently evolving in LVH. The role of the proteasome in to favor cellular hypertrophy consists in upregulation of the catalytic proteasome subunit, with prevalence of proteins-synthesis on proteins degradation. It is also evident that UPS inhibition may prevent cells’ growth opposing to the hypertrophy. In fact in several experimental models, UPS inhibition demonstrated to be able to prevent or reverse cardiac hypertrophy induced by abdominal aortic banding (AAB). That can happen with several proteasome inhibitors acting by multifactorial mechanisms. These evidences induce to hypothesize that, in the future, in patients with the increased volume overload by systemic hypertension, some proteasome-inhibitors could be used to antagonize or prevent LVH without reducing peripheral high blood pressure levels too. PMID:24551479

  17. Surface induced dissociation yields substructure of Methanosarcina thermophila 20S proteasome complexes

    PubMed Central

    Ma, Xin; Loo, Joseph A.; Wysocki, Vicki H.

    2015-01-01

    Native mass spectrometry (MS) and surface induced dissociation (SID) have been applied to study the stoichiometry and quaternary structure of non-covalent protein complexes. In this study, Methanosarcina thermophila 20S proteasome, which consists of four stacked heptameric rings (α7β7β7α7 symmetry), has been selected to explore the SID dissociation pattern of a complicated stacked ring protein complex. SID produces both α and β subunits while collision induced dissociation (CID) produces only highly charged α subunit. In addition, the charge reduced 20S proteasome produces the α7β7 fragment, reflecting the stacked ring topology of the complex. The combination of SID and charge reduction is shown to be a powerful tool for the study of protein complex structure. PMID:26005366

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

    PubMed Central

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

    2016-01-01

    Structured Abstract INTRODUCTION The ubiquitin-proteasome system comprises hundreds of distinct pathways of degradation, which converge at the step of ubiquitin recognition by the proteasome. Five proteasomal ubiquitin receptors have been identified, two that are intrinsic to the proteasome (Rpn10 and Rpn13) and three reversibly associated proteasomal ubiquitin receptors (Rad23, Dsk2, and Ddi1). RATIONALE We found that the five known proteasomal ubiquitin receptors of yeast are collectively nonessential for ubiquitin recognition by the proteasome. We therefore screened for additional ubiquitin receptors in the proteasome and identified subunit Rpn1 as a candidate. We used nuclear magnetic resonance (NMR) spectroscopy to characterize the structure of the binding site within Rpn1, which we term the T1 site. Mutational analysis of this site showed its functional importance within the context of intact proteasomes. T1 binds both ubiquitin and ubiquitin-like (UBL) proteins, in particular the substrate-delivering shuttle factor Rad23. A second site within the Rpn1 toroid, T2, recognizes the UBL domain of deubiquitinating enzyme Ubp6, as determined by hydrogen-deuterium exchange mass spectrometry analysis and validated by amino acid substitution and functional assays. The Rpn1 toroid thus serves a critical scaffolding role within the proteasome, helping to assemble multiple proteasome cofactors as well as substrates. RESULTS Our results indicate that proteasome subunit Rpn1 can recognize both ubiquitin and UBL domains of substrate shuttling factors that themselves bind ubiquitin and function as reversibly-associated proteasomal ubiquitin receptors. Recognition is mediated by the T1 site within the Rpn1 toroid, which supports proteasome function in vivo. We found that the capacity of T1 to recognize both ubiquitin and UBL proteins was shared with Rpn10 and Rpn13. The surprising multiplicity of ubiquitin-recognition domains within the proteasome may promote enhanced

  19. Pleiotropic defects caused by loss of the proteasome-interacting factors Rad23 and Rpn10 of Saccharomyces cerevisiae.

    PubMed Central

    Lambertson, D; Chen, L; Madura, K

    1999-01-01

    Rad23 is a member of a novel class of proteins that contain unprocessed ubiquitin-like (UbL) domains. We showed recently that a small fraction of Rad23 can form an interaction with the 26S proteasome. Similarly, a small fraction of Rpn10 is a component of the proteasome. Rpn10 can bind multiubiquitin chains in vitro, but genetic studies have not clarified its role in vivo. We report here that the loss of both Rad23 and Rpn10 results in pleiotropic defects that are not observed in either single mutant. rad23Delta rpn10Delta displays slow growth, cold sensitivity, and a pronounced G2/M phase delay, implicating overlapping roles for Rad23 and Rpn10. Although rad23Delta rpn10Delta displays similar sensitivity to DNA damage as a rad23Delta single mutant, deletion of RAD23 in rpn10Delta significantly increased sensitivity to canavanine, a phenotype associated with an rpn10Delta single mutant. A mutant Rad23 that is unable to bind the proteasome ((DeltaUbL)rad23) does not suppress the canavanine or cold-sensitive defects of rad23Delta rpn10Delta, demonstrating that Rad23/proteasome interaction is related to these effects. Finally, the accumulation of multiubiquitinated proteins and the stabilization of a specific proteolytic substrate in rad23Delta rpn10Delta suggest that proteasome function is altered. PMID:10471701

  20. Differential Influence on Cytotoxic T Lymphocyte Epitope Presentation by Controlled Expression of Either Proteasome Immunosubunits or Pa28

    PubMed Central

    van Hall, Thorbald; Sijts, Alice; Camps, Marcel; Offringa, Rienk; Melief, Cornelis; Kloetzel, Peter-M.; Ossendorp, Ferry

    2000-01-01

    The proteasome is the principal provider of major histocompatibility complex (MHC) class I–presented peptides. Interferon (IFN)-γ induces expression of three catalytically active proteasome subunits (LMP2, LMP7, and MECL-1) and the proteasome-associated activator PA28. These molecules are thought to optimize the generation of MHC class I–presented peptides. However, known information on their contribution in vivo is very limited. Here, we examined the antigen processing of two murine leukemia virus-encoded cytotoxic T lymphocyte (CTL) epitopes in murine cell lines equipped with a tetracycline-controlled, IFN-γ–independent expression system. We thus were able to segregate the role of the immunosubunits from the role of PA28. The presence of either immunosubunits or PA28 did not alter the presentation of a subdominant murine leukemia virus (MuLV)-derived CTL epitope. However, the presentation of the immunodominant MuLV-derived epitope was markedly enhanced upon induction of each of these two sets of genes. Thus, the IFN-γ–inducible proteasome subunits and PA28 can independently enhance antigen presentation of some CTL epitopes. Our data show that tetracycline-regulated expression of PA28 increases CTL epitope generation without affecting the 20S proteasome composition or half-life. The differential effect of these IFN-γ–inducible proteins on MHC class I processing may have a decisive influence on the quality of the CTL immune response. PMID:10952718

  1. New proteasome inhibitors in myeloma.

    PubMed

    Lawasut, Panisinee; Chauhan, Dharminder; Laubach, Jacob; Hayes, Catriona; Fabre, Claire; Maglio, Michelle; Mitsiades, Constantine; Hideshima, Teru; Anderson, Kenneth C; Richardson, Paul G

    2012-12-01

    Proteasome inhibition has a validated role in cancer therapy since the successful introduction of bortezomib for the treatment of multiple myeloma (MM) and mantle cell lymphoma, leading to the development of second-generation proteasome inhibitors (PI) for MM patients in whom currently approved therapies have failed. Five PIs have reached clinical evaluation, with the goals of improving efficacy and limiting toxicity, including peripheral neuropathy (PN). Carfilzomib, an epoxyketone with specific chymothrypsin-like activity, acts as an irreversible inhibitor and was recently FDA approved for the response benefit seen in relapsed and refractory MM patients previously treated with bortezomib, thalidomide and lenalidomide. ONX-0912 is now under evaluation as an oral form with similar activity. The boronate peptides MLN9708 and CEP-18770 are orally bioactive bortezomib analogs with prolonged activity and greater tissue penetration. NPI-0052 (marizomib) is a unique, beta-lactone non-selective PI that has been shown to potently overcome bortezomib resistance in vitro. All of these second-generation PIs demonstrate encouraging anti-MM activity and appear to reduce the incidence of PN, with clinical trials ongoing. PMID:23065395

  2. The Mycobacterium tuberculosis Proteasome Active Site Threonine Is Essential for Persistence Yet Dispensable for Replication and Resistance to Nitric Oxide

    PubMed Central

    Gandotra, Sheetal; Lebron, Maria B.; Ehrt, Sabine

    2010-01-01

    Previous work revealed that conditional depletion of the core proteasome subunits PrcB and PrcA impaired growth of Mycobacterium tuberculosis in vitro and in mouse lungs, caused hypersusceptibility to nitric oxide (NO) and impaired persistence of the bacilli during chronic mouse infections. Here, we show that genetic deletion of prcBA led to similar phenotypes. Surprisingly, however, an active site mutant proteasome complemented the in vitro and in vivo growth defects of the prcBA knockout (ΔprcBA) as well as its NO hypersensitivity. In contrast, long-term survival of M. tuberculosis in stationary phase and during starvation in vitro and in the chronic phase of mouse infection required a proteolytically active proteasome. Inhibition of inducible nitric oxide synthase did not rescue survival of ΔprcBA, revealing a function beyond NO defense, by which the proteasome contributes to M. tuberculosis fitness during chronic mouse infections. These findings suggest that proteasomal proteolysis facilitates mycobacterial persistence, that M. tuberculosis faces starvation during chronic mouse infections and that the proteasome serves a proteolysis-independent function. PMID:20711362

  3. Proteasome-mediated degradation of IκBα and processing of p105 in Crohn disease and ulcerative colitis

    PubMed Central

    Visekruna, Alexander; Joeris, Thorsten; Seidel, Daniel; Kroesen, Anjo; Loddenkemper, Christoph; Zeitz, Martin; Kaufmann, Stefan H.E.; Schmidt-Ullrich, Ruth; Steinhoff, Ulrich

    2006-01-01

    Enhanced NF-κB activity is involved in the pathology of both forms of inflammatory bowel disease (IBD), Crohn disease (CD) and ulcerative colitis (UC). Here we analyzed the mechanism of proteasome-mediated NF-κB activation in CD and UC. Our studies demonstrate that the subunit composition and the proteolytic function of proteasomes differ between UC and CD. High expression of the immunoproteasome subunits β1i and β2i is characteristic of the inflamed mucosa of CD. In line with this, we found enhanced processing of NF-κB precursor p105 and degradation of inhibitor of NF-κB, IκBα, by immunoproteasomes isolated from the mucosa of CD patients. In comparison with healthy controls and CD patients, UC patients exhibited an intermediate phenotype regarding the proteasome-mediated processing/degradation of NF-κB components. Finally, increased expression of the NF-κB family member c-Rel in the inflamed mucosa of CD patients suggests that p50/c-Rel is important for IFN-γ–mediated induction of immunoproteasomes via IL-12–driven Th1 responses. These findings suggest that distinct proteasome subunits influence the intensity of NF-κB–mediated inflammation in IBD patients. PMID:17124531

  4. The therapeutic potential of microbial proteasome inhibitors.

    PubMed

    Momose, Isao; Kawada, Manabu

    2016-08-01

    The proteasome influences cellular homeostasis through the degradation of regulatory proteins, many of which are also involved in disease pathogenesis. In particular, numerous regulatory proteins associated with tumor growth, such as cyclins, cyclin-dependent kinase inhibitors, tumor suppressors, and NF-κB inhibitors are degraded by the proteasome. Proteasome inhibitors can stabilize these regulatory proteins, resulting in the suppression of tumor development and the regulation of immune responses. Thus, proteasome inhibitors are promising candidate antitumor agents and immune-regulatory agents. Bortezomib is the first-in-class proteasome inhibitor approved for the treatment of multiple myeloma. Despite its high efficiency, however, a large proportion of patients do not attain sufficient clinical response due to toxicity and drug resistance. Therefore, the development of new proteasome inhibitors with improved pharmacological properties is needed. Natural products produced by microorganisms are a promising source of such compounds. This review provides an overview of proteasome inhibitors produced by microorganisms, with special focus on inhibitors isolated from actinomycetes. PMID:26589840

  5. 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

  6. Antileukemic Activity and Mechanism of Drug Resistance to the Marine Salinispora tropica Proteasome Inhibitor Salinosporamide A (Marizomib)

    PubMed Central

    Niewerth, Denise; Jansen, Gerrit; Riethoff, Lesley F. V.; van Meerloo, Johan; Kale, Andrew J.; Moore, Bradley S.; Assaraf, Yehuda G.; Anderl, Janet L.; Zweegman, Sonja; Kaspers, Gertjan J. L.

    2014-01-01

    Salinosporamide A (NPI-0052, marizomib) is a naturally occurring proteasome inhibitor derived from the marine actinobacterium Salinispora tropica, and represents a promising clinical agent in the treatment of hematologic malignancies. Recently, these actinobacteria were shown to harbor self-resistance properties to salinosporamide A by expressing redundant catalytically active mutants of the 20S proteasome β-subunit, reminiscent of PSMB5 mutations identified in cancer cells with acquired resistance to the founding proteasome inhibitor bortezomib (BTZ). Here, we assessed the growth inhibitory potential of salinosporamide A in human acute lymphocytic leukemia CCRF-CEM cells, and its 10-fold (CEM/BTZ7) and 123-fold (CEM/BTZ200) bortezomib-resistant sublines harboring PSMB5 mutations. Parental cells displayed sensitivity to salinosporamide A (IC50 = 5.1 nM), whereas their bortezomib-resistant sublines were 9- and 17-fold cross-resistant to salinosporamide A, respectively. Notably, combination experiments of salinosporamide A and bortezomib showed synergistic activity in CEM/BTZ200 cells. CEM cells gradually exposed to 20 nM salinosporamide A (CEM/S20) displayed stable 5-fold acquired resistance to salinosporamide A and were 3-fold cross-resistant to bortezomib. Consistent with the acquisition of a PSMB5 point mutation (M45V) in CEM/S20 cells, salinosporamide A displayed a markedly impaired capacity to inhibit β5-associated catalytic activity. Last, compared with parental CEM cells, CEM/S20 cells exhibited up to 2.5-fold upregulation of constitutive proteasome subunits, while retaining unaltered immunoproteasome subunit expression. In conclusion, salinosporamide A displayed potent antileukemic activity against bortezomib-resistant leukemia cells. β-Subunit point mutations as a common feature of acquired resistance to salinosporamide A and bortezomib in hematologic cells and S. tropica suggest an evolutionarily conserved mechanism of resistance to proteasome

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

    PubMed

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

    2016-01-01

    It is well known that the ubiquitin-proteasome system is activated in response to skeletal muscle wasting and functions to degrade contractile proteins. The loss of these proteins inevitably reduces skeletal muscle size (i.e., quantity). However, it is currently unknown whether activation of this pathway also affects function by impairing the muscle's intrinsic ability to produce force (i.e., quality). Therefore, the purpose of this study was twofold, (1) document how the ubiquitin-proteasome system responds to denervation and (2) identify the physiological consequences of these changes. To induce soleus muscle atrophy, C57BL6 mice underwent tibial nerve transection of the left hindlimb for 7 or 14 days (n = 6-8 per group). At these time points, content of several proteins within the ubiquitin-proteasome system were determined via Western blot, while ex vivo whole muscle contractility was specifically analyzed at day 14. Denervation temporarily increased several key proteins within the ubiquitin-proteasome system, including the E3 ligase MuRF1 and the proteasome subunits 19S, α7 and β5. These changes were accompanied by reductions in absolute peak force and power, which were offset when expressed relative to physiological cross-sectional area. Contrary to peak force, absolute and relative forces at submaximal stimulation frequencies were significantly greater following 14 days of denervation. Taken together, these data represent two keys findings. First, activation of the ubiquitin-proteasome system is associated with reductions in skeletal muscle quantity rather than quality. Second, shortly after denervation, it appears the muscle remodels to compensate for the loss of neural activity via changes in Ca2+ handling. PMID:27513942

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

    PubMed Central

    Liu, Haiming M.; Thompson, LaDora V.

    2016-01-01

    It is well known that the ubiquitin-proteasome system is activated in response to skeletal muscle wasting and functions to degrade contractile proteins. The loss of these proteins inevitably reduces skeletal muscle size (i.e., quantity). However, it is currently unknown whether activation of this pathway also affects function by impairing the muscle’s intrinsic ability to produce force (i.e., quality). Therefore, the purpose of this study was twofold, (1) document how the ubiquitin-proteasome system responds to denervation and (2) identify the physiological consequences of these changes. To induce soleus muscle atrophy, C57BL6 mice underwent tibial nerve transection of the left hindlimb for 7 or 14 days (n = 6–8 per group). At these time points, content of several proteins within the ubiquitin-proteasome system were determined via Western blot, while ex vivo whole muscle contractility was specifically analyzed at day 14. Denervation temporarily increased several key proteins within the ubiquitin-proteasome system, including the E3 ligase MuRF1 and the proteasome subunits 19S, α7 and β5. These changes were accompanied by reductions in absolute peak force and power, which were offset when expressed relative to physiological cross-sectional area. Contrary to peak force, absolute and relative forces at submaximal stimulation frequencies were significantly greater following 14 days of denervation. Taken together, these data represent two keys findings. First, activation of the ubiquitin-proteasome system is associated with reductions in skeletal muscle quantity rather than quality. Second, shortly after denervation, it appears the muscle remodels to compensate for the loss of neural activity via changes in Ca2+ handling. PMID:27513942

  9. Substituted quinolines as noncovalent proteasome inhibitors.

    PubMed

    McDaniel, Tanner J; Lansdell, Theresa A; Dissanayake, Amila A; Azevedo, Lauren M; Claes, Jacob; Odom, Aaron L; Tepe, Jetze J

    2016-06-01

    Screening of a library of diverse heterocyclic scaffolds identified substituted quinolines as inhibitors of the human proteasome. The heterocyclic library was prepared via a novel titanium-catalyzed multicomponent coupling reaction, which rendered a diverse set of isoxazoles, pyrimidines, pyrroles, pyrazoles and quinolines. SAR of the parent lead compound indicated that hydrophobic residues on the benzo-moiety significantly improved potency. Lead compound 25 inhibits the chymotryptic-like proteolytic activity of the proteasome (IC50 5.4μM), representing a new class of nonpeptidic, noncovalent proteasome inhibitors. PMID:27112450

  10. Survival of mycobacteria depends on proteasome-mediated amino acid recycling under nutrient limitation

    PubMed Central

    Elharar, Yifat; Roth, Ziv; Hermelin, Inna; Moon, Alexandra; Peretz, Gabriella; Shenkerman, Yael; Vishkautzan, Marina; Khalaila, Isam; Gur, Eyal

    2014-01-01

    Intracellular protein degradation is an essential process in all life domains. While in all eukaryotes regulated protein degradation involves ubiquitin tagging and the 26S-proteasome, bacterial prokaryotic ubiquitin-like protein (Pup) tagging and proteasomes are conserved only in species belonging to the phyla Actinobacteria and Nitrospira. In Mycobacterium tuberculosis, the Pup-proteasome system (PPS) is important for virulence, yet its physiological role in non-pathogenic species has remained an enigma. We now report, using Mycobacterium smegmatis as a model organism, that the PPS is essential for survival under starvation. Upon nitrogen limitation, PPS activity is induced, leading to accelerated tagging and degradation of many cytoplasmic proteins. We suggest a model in which the PPS functions to recycle amino acids under nitrogen starvation, thereby enabling the cell to maintain basal metabolic activities. We also find that the PPS auto-regulates its own activity via pupylation and degradation of its components in a manner that promotes the oscillatory expression of PPS components. As such, the destructive activity of the PPS is carefully balanced to maintain cellular functions during starvation. PMID:24986881

  11. 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

  12. Protein Abundance Changes and Ubiquitylation Targets Identified after Inhibition of the Proteasome with Syringolin A*

    PubMed Central

    Svozil, Julia; Hirsch-Hoffmann, Matthias; Dudler, Robert; Gruissem, Wilhelm; Baerenfaller, Katja

    2014-01-01

    As proteins are the main effectors inside cells, their levels need to be tightly regulated. This is partly achieved by specific protein degradation via the Ubiquitin-26S proteasome system (UPS). In plants, an exceptionally high number of proteins are involved in Ubiquitin-26S proteasome system-mediated protein degradation and it is known to regulate most, if not all, important cellular processes. Here, we investigated the response to the inhibition of the proteasome at the protein level treating leaves with the specific inhibitor Syringolin A (SylA) in a daytime specific manner and found 109 accumulated and 140 decreased proteins. The patterns of protein level changes indicate that the accumulating proteins cause proteotoxic stress that triggers various responses. Comparing protein level changes in SylA treated with those in a transgenic line over-expressing a mutated ubiquitin unable to form polyubiquitylated proteins produced little overlap pointing to different response pathways. To distinguish between direct and indirect targets of the UPS we also enriched and identified ubiquitylated proteins after inhibition of the proteasome, revealing a total of 1791 ubiquitylated proteins in leaves and roots and 1209 that were uniquely identified in our study. The comparison of the ubiquitylated proteins with those changing in abundance after SylA-mediated inhibition of the proteasome confirmed the complexity of the response and revealed that some proteins are regulated both at transcriptional and post-transcriptional level. For the ubiquitylated proteins that accumulate in the cytoplasm but are targeted to the plastid or the mitochondrion, we often found peptides in their target sequences, demonstrating that the UPS is involved in controlling organellar protein levels. Attempts to identify the sites of ubiquitylation revealed that the specific properties of this post-translational modification can lead to incorrect peptide spectrum assignments in complex peptide mixtures

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

    PubMed

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

    2016-01-01

    Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100(mel)47-52/40-42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100(mel)47-52/40-42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8(+) T cell response. Importantly, we demonstrate that different gp100(mel)-derived spliced epitopes are generated and presented to CD8(+) T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100(mel)-derived spliced epitopes trigger activation of CD8(+) T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes. PMID:27049119

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

    PubMed Central

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

    2016-01-01

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

  15. Serendipity in discovery of proteasome inhibitors.

    PubMed

    Dunn, Derek; Iqbal, Mohamed; Husten, Jean; Ator, Mark A; Chatterjee, Sankar

    2012-05-15

    Among its various catalytic activities, the 'chymotrypsin-like' activity of the proteasome, a large multicatalytic proteinase complex has emerged as the focus of drug discovery efforts in cancer therapy. Herein, a series of first generation (2S, 3R)-2-amino-3-hydroxybutyric acid derived proteasome inhibitors that were discovered serendipitously en route to original goal of generating a series of sterically constrained oxazoline derivatives has been reported. PMID:22503349

  16. 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

  17. Inhibitors Selective for Mycobacterial Versus Human Proteasomes

    SciTech Connect

    Lin, G.; Li, D; Sorio de Carvalho, L; Deng, H; Tao, H; Vogt, G; Wu, K; Schneider, J; Chidawanyika, T; et. al.

    2009-01-01

    Many anti-infectives inhibit the synthesis of bacterial proteins, but none selectively inhibits their degradation. Most anti-infectives kill replicating pathogens, but few preferentially kill pathogens that have been forced into a non-replicating state by conditions in the host. To explore these alternative approaches we sought selective inhibitors of the proteasome of Mycobacterium tuberculosis. Given that the proteasome structure is extensively conserved, it is not surprising that inhibitors of all chemical classes tested have blocked both eukaryotic and prokaryotic proteasomes, and no inhibitor has proved substantially more potent on proteasomes of pathogens than of their hosts. Here we show that certain oxathiazol-2-one compounds kill non-replicating M.?tuberculosis and act as selective suicide-substrate inhibitors of the M.?tuberculosis proteasome by cyclocarbonylating its active site threonine. Major conformational changes protect the inhibitor-enzyme intermediate from hydrolysis, allowing formation of an oxazolidin-2-one and preventing regeneration of active protease. Residues outside the active site whose hydrogen bonds stabilize the critical loop before and after it moves are extensively non-conserved. This may account for the ability of oxathiazol-2-one compounds to inhibit the mycobacterial proteasome potently and irreversibly while largely sparing the human homologue.

  18. Angelman syndrome-associated ubiquitin ligase UBE3A/E6AP mutants interfere with the proteolytic activity of the proteasome

    PubMed Central

    Tomaić, V; Banks, L

    2015-01-01

    Angelman syndrome, a severe neurodevelopmental disease, occurs primarily due to genetic defects, which cause lack of expression or mutations in the wild-type E6AP/UBE3A protein. A proportion of the Angelman syndrome patients bear UBE3A point mutations, which do not interfere with the expression of the full-length protein, however, these individuals still develop physiological conditions of the disease. Interestingly, most of these mutations are catalytically defective, thereby indicating the importance of UBE3A enzymatic activity role in the Angelman syndrome pathology. In this study, we show that Angelman syndrome-associated mutants interact strongly with the proteasome via the S5a proteasomal subunit, resulting in an overall inhibitory effect on the proteolytic activity of the proteasome. Our results suggest that mutated catalytically inactive forms of UBE3A may cause defects in overall proteasome function, which could have an important role in the Angelman syndrome pathology. PMID:25633294

  19. Genetic variants at PSMD3 interact with dietary fat and carbohydrate to modulate insulin resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Proteasome (prosome, macropain) 26S subunit, non-ATPase, 3 (PSMD3) encodes subunit 3 of the 26S proteasome, which is involved in regulating insulin signal transduction. We aimed to investigate the associations of PSMD3 variants with glucose-related traits and the interactions of those variants with ...

  20. Lipopolysaccharide Induces Degradation of Connexin43 in Rat Astrocytes via the Ubiquitin-Proteasome Proteolytic Pathway

    PubMed Central

    Liao, Chih-Kai; Jeng, Chung-Jiuan; Wang, Hwai-Shi; Wang, Shu-Huei; Wu, Jiahn-Chun

    2013-01-01

    The astrocytic syncytium plays a critical role in maintaining the homeostasis of the brain through the regulation of gap junction intercellular communication (GJIC). Changes to GJIC in response to inflammatory stimuli in astrocytes may have serious effects on the brain. We have previously shown that lipopolysaccharide (LPS) reduces connexin43 (Cx43) expression and GJIC in cultured rat astrocytes via a toll-like receptor 4-mediated signaling pathway. In the present study, treatment of astrocytes with LPS resulted in a significant increase in levels of the phosphorylated forms of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) -1, -2, and -3 for up to 18 h. An increase in nuclear transcription factor NF-κB levels was also observed after 8 h of LPS treatment and was sustained for up to 18 h. The LPS-induced decrease in Cx43 protein levels and inhibition of GJIC were blocked by the SAPK/JNK inhibitor SP600125, but not by the NF-κB inhibitor BAY11-7082. Following blockade of de novo protein synthesis by cycloheximide, LPS accelerated Cx43 degradation. Moreover, the LPS-induced downregulation of Cx43 was blocked following inhibition of 26S proteasome activity using the reversible proteasome inhibitor MG132 or the irreversible proteasome inhibitor lactacystin. Immunoprecipitation analyses revealed an increased association of Cx43 with both ubiquitin and E3 ubiquitin ligase Nedd4 in astrocytes after LPS stimulation for 6 h and this effect was prevented by SP600125. Taken together, these results suggest that LPS stimulation leads to downregulation of Cx43 expression and GJIC in rat astrocytes by activation of SAPK/JNK and the ubiquitin-proteasome proteolytic pathway. PMID:24236122

  1. Interactions of PAN's C-termini with archaeal 20S proteasome and implications for the eukaryotic proteasome–ATPase interactions

    PubMed Central

    Yu, Yadong; Smith, David M; Kim, Ho Min; Rodriguez, Victor; Goldberg, Alfred L; Cheng, Yifan

    2010-01-01

    Protein degradation in the 20S proteasome is regulated in eukaryotes by the 19S ATPase complex and in archaea by the homologous PAN ATPase ring complex. Subunits of these hexameric ATPases contain on their C-termini a conserved hydrophobic-tyrosine-X (HbYX) motif that docks into pockets in the 20S to stimulate the opening of a gated substrate entry channel. Here, we report the crystal structure of the archaeal 20S proteasome in complex with the C-terminus of the archaeal proteasome regulatory ATPase, PAN. This structure defines the detailed interactions between the critical C-terminal HbYX motif and the 20S α-subunits and indicates that the intersubunit pocket in the 20S undergoes an induced-fit conformational change on binding of the HbYX motif. This structure together with related mutagenesis data suggest how in eukaryotes certain proteasomal ATPases bind to specific pockets in an asymmetrical manner to regulate gate opening. PMID:20019667

  2. Syrbactin Structural Analog TIR-199 Blocks Proteasome Activity and Induces Tumor Cell Death.

    PubMed

    Bachmann, André S; Opoku-Ansah, John; Ibarra-Rivera, Tannya R; Yco, Lisette P; Ambadi, Sudhakar; Roberts, Christopher C; Chang, Chia-En A; Pirrung, Michael C

    2016-04-15

    Multiple myeloma is an aggressive hematopoietic cancer of plasma cells. The recent emergence of three effective FDA-approved proteasome-inhibiting drugs, bortezomib (Velcade®), carfilzomib (Kyprolis®), and ixazomib (Ninlaro®), confirms that proteasome inhibitors are therapeutically useful against neoplastic disease, in particular refractory multiple myeloma and mantle cell lymphoma. This study describes the synthesis, computational affinity assessment, and preclinical evaluation of TIR-199, a natural product-derived syrbactin structural analog. Molecular modeling and simulation suggested that TIR-199 covalently binds each of the three catalytic subunits (β1, β2, and β5) and revealed key interaction sites. In vitro and cell culture-based proteasome activity measurements confirmed that TIR-199 inhibits the proteasome in a dose-dependent manner and induces tumor cell death in multiple myeloma and neuroblastoma cells as well as other cancer types in the NCI-60 cell panel. It is particularly effective against kidney tumor cell lines, with >250-fold higher anti-tumor activities than observed with the natural product syringolin A. In vivo studies in mice revealed a maximum tolerated dose of TIR-199 at 25 mg/kg. The anti-tumor activity of TIR-199 was confirmed in hollow fiber assays in mice. Adverse drug reaction screens in a kidney panel revealed no off-targets of concern. This is the first study to examine the efficacy of a syrbactin in animals. Taken together, the results suggest that TIR-199 is a potent new proteasome inhibitor with promise for further development into a clinical drug for the treatment of multiple myeloma and other forms of cancer. PMID:26907687

  3. Proteasome inhibitor associated thrombotic microangiopathy.

    PubMed

    Yui, Jennifer C; Van Keer, Jan; Weiss, Brendan M; Waxman, Adam J; Palmer, Matthew B; D'Agati, Vivette D; Kastritis, Efstathios; Dimopoulos, Meletios A; Vij, Ravi; Bansal, Dhruv; Dingli, David; Nasr, Samih H; Leung, Nelson

    2016-09-01

    A variety of medications have been implicated in the causation of thrombotic microangiopathy (TMA). Recently, a few case reports have emerged of TMA attributed to the proteasome inhibitors (PI) bortezomib and carfilzomib in patients with multiple myeloma. The aim of this case series was to better characterize the role of PI in the etiology of drug-induced TMA. We describe eleven patients from six medical centers from around the world who developed TMA while being treated with PI. The median time between medication initiation and diagnosis of TMA was 21 days (range 5 days to 17 months). Median laboratory values at diagnosis included hemoglobin-7.5 g dL(-1) , platelet count-20 × 10(9) /L, LDH-698 U L(-1) , creatinine-3.12 mg dL(-1) . No patient had any other cause of TMA, including ADAMTS13 inhibition, other malignancy or use of any other medication previously associated with TMA. Nine patients had resolution of TMA without evidence of hemolysis after withdrawal of PI. Two patients had stabilization of laboratory values but persistent evidence of hemolysis despite medication withdrawal. One patient had recurrence of TMA with rechallenge of PI. There is a strong level of evidence that PI can cause DITMA. In evaluating patients with suspected TMA, PI use should be recognized as a potential etiology, and these medications should be discontinued promptly if thought to be the cause of TMA. Am. J. Hematol. 91:E348-E352, 2016. © 2016 Wiley Periodicals, Inc. PMID:27286661

  4. Clinical and marketed proteasome inhibitors for cancer treatment.

    PubMed

    Zhang, Jiankang; Wu, Peng; Hu, Yongzhou

    2013-01-01

    The ubiquitin-proteasome pathway (UPP), which influences essential cellular functions including cell growth, differentiation, apoptosis, signal transduction, antigen processing and inflammatory responses, has been considered as one of the most important cellular protein degradation approaches. Proteasome functions as a gatekeeper, which controls the execution of protein degradation and plays a critical role in the ubiquitin-proteasome pathway. The unfolding of the close connection between proteasome and cancer provides a potential strategy for cancer treatment by using proteasome inhibitors. Small molecular inhibitors of varied structures and potency against proteasome have been discovered in recent years, with bortezomib and carfilzomib having been successfully approved for clinical application while some other promising candidates are currently under clinical trials. Herein, we review the development history of drugs and candidates that target the 20S proteasome, structure-activity relationships (SARs) of various proteasome inhibitors, and related completed or ongoing clinical trials. PMID:23531219

  5. Trial Watch: Proteasomal inhibitors for anticancer therapy

    PubMed Central

    Obrist, Florine; Manic, Gwenola; Kroemer, Guido; Vitale, Ilio; Galluzzi, Lorenzo

    2015-01-01

    The so-called “ubiquitin-proteasome system” (UPS) is a multicomponent molecular apparatus that catalyzes the covalent attachment of several copies of the small protein ubiquitin to other proteins that are generally (but not always) destined to proteasomal degradation. This enzymatic cascade is crucial for the maintenance of intracellular protein homeostasis (both in physiological conditions and in the course of adaptive stress responses), and regulates a wide array of signaling pathways. In line with this notion, defects in the UPS have been associated with aging as well as with several pathological conditions including cardiac, neurodegenerative, and neoplastic disorders. As transformed cells often experience a constant state of stress (as a result of the hyperactivation of oncogenic signaling pathways and/or adverse microenvironmental conditions), their survival and proliferation are highly dependent on the integrity of the UPS. This rationale has driven an intense wave of preclinical and clinical investigation culminating in 2003 with the approval of the proteasomal inhibitor bortezomib by the US Food and Drug Administration for use in multiple myeloma patients. Another proteasomal inhibitor, carfilzomib, is now licensed by international regulatory agencies for use in multiple myeloma patients, and the approved indications for bortezomib have been extended to mantle cell lymphoma. This said, the clinical activity of bortezomib and carfilzomib is often limited by off-target effects, innate/acquired resistance, and the absence of validated predictive biomarkers. Moreover, the antineoplastic activity of proteasome inhibitors against solid tumors is poor. In this Trial Watch we discuss the contribution of the UPS to oncogenesis and tumor progression and summarize the design and/or results of recent clinical studies evaluating the therapeutic profile of proteasome inhibitors in cancer patients. PMID:27308423

  6. Trial Watch: Proteasomal inhibitors for anticancer therapy.

    PubMed

    Obrist, Florine; Manic, Gwenola; Kroemer, Guido; Vitale, Ilio; Galluzzi, Lorenzo

    2015-01-01

    The so-called "ubiquitin-proteasome system" (UPS) is a multicomponent molecular apparatus that catalyzes the covalent attachment of several copies of the small protein ubiquitin to other proteins that are generally (but not always) destined to proteasomal degradation. This enzymatic cascade is crucial for the maintenance of intracellular protein homeostasis (both in physiological conditions and in the course of adaptive stress responses), and regulates a wide array of signaling pathways. In line with this notion, defects in the UPS have been associated with aging as well as with several pathological conditions including cardiac, neurodegenerative, and neoplastic disorders. As transformed cells often experience a constant state of stress (as a result of the hyperactivation of oncogenic signaling pathways and/or adverse microenvironmental conditions), their survival and proliferation are highly dependent on the integrity of the UPS. This rationale has driven an intense wave of preclinical and clinical investigation culminating in 2003 with the approval of the proteasomal inhibitor bortezomib by the US Food and Drug Administration for use in multiple myeloma patients. Another proteasomal inhibitor, carfilzomib, is now licensed by international regulatory agencies for use in multiple myeloma patients, and the approved indications for bortezomib have been extended to mantle cell lymphoma. This said, the clinical activity of bortezomib and carfilzomib is often limited by off-target effects, innate/acquired resistance, and the absence of validated predictive biomarkers. Moreover, the antineoplastic activity of proteasome inhibitors against solid tumors is poor. In this Trial Watch we discuss the contribution of the UPS to oncogenesis and tumor progression and summarize the design and/or results of recent clinical studies evaluating the therapeutic profile of proteasome inhibitors in cancer patients. PMID:27308423

  7. Characterization of the Last Subunit of the Arabidopsis COP9 Signalosome

    PubMed Central

    Serino, Giovanna; Su, Hongwen; Peng, Zhaohua; Tsuge, Tomohiko; Wei, Ning; Gu, Hongya; Deng, Xing Wang

    2003-01-01

    The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that resembles the lid subcomplex of proteasomes. Through its ability to regulate specific proteasome-mediated protein degradation events, CSN controls multiple aspects of development. Here, we report the cloning and characterization of AtCSN2, the last uncharacterized CSN subunit from Arabidopsis. We show that the AtCSN2 gene corresponds to the previously identified FUS12 locus and that AtCSN2 copurifies with CSN, confirming that AtCSN2 is an integral component of CSN. AtCSN2 is not only able to interact with the SCFTIR1 subunit AtCUL1, which is partially responsible for the regulatory interaction between CSN and SCFTIR1, but also interacts with AtCUL3, suggesting that CSN is able to regulate the activity of other cullin-based E3 ligases through conserved interactions. Phylogenetic analysis indicated that the duplication and subsequent divergence events that led to the genes that encode CSN and lid subunits occurred before the divergence of unicellular and multicellular eukaryotic organisms and that the CSN subunits were more conserved than the lid subunits during evolution. Comparative analyses of the subunit interaction of CSN revealed a set of conserved subunit contacts and resulted in a model of CSN subunit topology, some aspects of which were substantiated by in vivo cross-link tests. PMID:12615944

  8. Intracellular Dynamics of the Ubiquitin-Proteasome-System.

    PubMed

    Chowdhury, Maisha; Enenkel, Cordula

    2015-01-01

    The ubiquitin-proteasome system is the major degradation pathway for short-lived proteins in eukaryotic cells. Targets of the ubiquitin-proteasome-system are proteins regulating a broad range of cellular processes including cell cycle progression, gene expression, the quality control of proteostasis and the response to geno- and proteotoxic stress. Prior to degradation, the proteasomal substrate is marked with a poly-ubiquitin chain. The key protease of the ubiquitin system is the proteasome. In dividing cells, proteasomes exist as holo-enzymes composed of regulatory and core particles. The regulatory complex confers ubiquitin-recognition and ATP dependence on proteasomal protein degradation. The catalytic sites are located in the proteasome core particle. Proteasome holo-enzymes are predominantly nuclear suggesting a major requirement for proteasomal proteolysis in the nucleus. In cell cycle arrested mammalian or quiescent yeast cells, proteasomes deplete from the nucleus and accumulate in granules at the nuclear envelope (NE) / endoplasmic reticulum (ER) membranes. In prolonged quiescence, proteasome granules drop off the NE / ER membranes and migrate as stable organelles throughout the cytoplasm, as thoroughly investigated in yeast. When quiescence yeast cells are allowed to resume growth, proteasome granules clear and proteasomes are rapidly imported into the nucleus. Here, we summarize our knowledge about the enigmatic structure of proteasome storage granules and the trafficking of proteasomes and their substrates between the cyto- and nucleoplasm. Most of our current knowledge is based on studies in yeast. Their translation to mammalian cells promises to provide keen insight into protein degradation in non-dividing cells which comprise the majority of our body's cells. PMID:26339477

  9. Inhibition of Proteasome Activity by Low-dose Bortezomib Attenuates Angiotensin II-induced Abdominal Aortic Aneurysm in Apo E−/− Mice

    PubMed Central

    Ren, Hualiang; Li, Fangda; Tian, Cui; Nie, Hao; Wang, Lei; Li, Hui-Hua; Zheng, Yuehong

    2015-01-01

    Abdominal aortic aneurysm (AAA) is a leading cause of sudden death in aged people. Activation of ubiquitin proteasome system (UPS) plays a critical role in the protein quality control and various diseases. However, the functional role of UPS in AAA formation remains unclear. In this study, we found that the proteasome activities and subunit expressions in AAA tissues from human and angiotensin II (Ang II)-infused apolipoprotein E knockout (Apo E−/−) mice were significantly increased. To investigate the effect of proteasome activation on the AAA formation, Apo E−/− mice were cotreated with bortezomib (BTZ) (a proteasome inhibitor, 50 μg/kg, 2 times per week) and Ang II (1000 ng/kg/min) up to 28 days. Ang II infusion significantly increased the incidence and severity of AAA in Apo E−/− mice, whereas BTZ treatment markedly inhibited proteasome activities and prevented AAA formation. Furthermore, BTZ treatment significantly reduced the inflammation, inhibited the metal matrix metalloprotease activity, and reversed the phenotypic SMC modulation in AAA tissue. In conclusion, these results provide a new evidence that proteasome activation plays a critical role in AAA formation through multiple mechanisms, and suggest that BTZ might be a novel therapeutic target for treatment of AAA formation. PMID:26508670

  10. Proteasome Regulation of ULBP1 Transcription

    PubMed Central

    Butler, James E.; Moore, Mikel B.; Presnell, Steven R.; Chan, Huei-Wei; Chalupny, N. Jan; Lutz, Charles T.

    2009-01-01

    Killer lymphocytes recognize stress-activated NKG2D ligands on tumors. We examined NKG2D ligand expression in head and neck squamous cell carcinoma (HNSCC) cells and other cell lines. HNSCC cells typically expressed MHC class I chain-related gene A (MICA), MICB, UL16-binding protein (ULBP)2, and ULBP3, but they were uniformly negative for cell surface ULBP1 and ULBP4. We then studied how cancer treatments affected NKG2D ligand expression. NKG2D ligand expression was not changed by most cancer-relevant treatments. However, bortezomib and other proteasome inhibitor drugs with distinct mechanisms of action dramatically and specifically up-regulated HNSCC ULBP1 mRNA and cell surface protein. Proteasome inhibition also increased RNA for ULBP1 and other NKG2D ligands in nontransformed human keratinocytes. Proteasome inhibitor drugs increased ULBP1 transcription by acting at a site in the 522-bp ULBP1 promoter. Although the DNA damage response pathways mediated by ATM (ataxia-telangiectasia, mutated) and ATR (ATM and Rad3-related) signaling had been reported to up-regulate NKG2D ligand expression, we found that ULBP1 up-regulation was not inhibited by caffeine and wortmannin, inhibitors of ATM/ATR signaling. ULBP1 expression in HNSCC cells was not increased by several ATM/ATR activating treatments, including bleomycin, cisplatin, aphidicolin, and hydroxyurea. Ionizing radiation caused ATM activation in HNSCC cells, but high-level ULBP1 expression was not induced by gamma radiation or UV radiation. Thus, ATM/ATR signaling was neither necessary nor sufficient for high-level ULBP1 expression in human HNSCC cell lines and could not account for the proteasome effect. The selective induction of ULBP1 expression by proteasome inhibitor drugs, along with variable NKG2D ligand expression by human tumor cells, indicates that NKG2D ligand genes are independently regulated. PMID:19414815

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

    PubMed Central

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

    2013-01-01

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

  12. Dynamic recruitment of active proteasomes into polyglutamine initiated inclusion bodies.

    PubMed

    Schipper-Krom, Sabine; Juenemann, Katrin; Jansen, Anne H; Wiemhoefer, Anne; van den Nieuwendijk, Rianne; Smith, Donna L; Hink, Mark A; Bates, Gillian P; Overkleeft, Hermen; Ovaa, Huib; Reits, Eric

    2014-01-01

    Neurodegenerative disorders such as Huntington's disease are hallmarked by neuronal intracellular inclusion body formation. Whether proteasomes are irreversibly recruited into inclusion bodies in these protein misfolding disorders is a controversial subject. In addition, it has been proposed that the proteasomes may become clogged by the aggregated protein fragments, leading to impairment of the ubiquitin-proteasome system. Here, we show by fluorescence pulse-chase experiments in living cells that proteasomes are dynamically and reversibly recruited into inclusion bodies. As these recruited proteasomes remain catalytically active and accessible to substrates, our results challenge the concept of proteasome sequestration and impairment in Huntington's disease, and support the reported absence of proteasome impairment in mouse models of Huntington's disease. PMID:24291262

  13. Potential role of 20S proteasome in maintaining stem cell integrity of human bone marrow stromal cells in prolonged culture expansion

    SciTech Connect

    Lu, Li; Song, Hui-Fang; Zhang, Wei-Guo; Liu, Xue-Qin; Zhu, Qian; Cheng, Xiao-Long; Yang, Gui-Jiao; Li, Ang; Xiao, Zhi-Cheng

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer Prolonged culture expansion retards proliferation and induces senescence of hBMSCs. Black-Right-Pointing-Pointer Reduced 20S proteasomal activity and expression potentially contribute to cell aging. Black-Right-Pointing-Pointer MG132-mediated 20S proteasomal inhibition induces senescence-like phenotype. Black-Right-Pointing-Pointer 18{alpha}-GA stimulates proteasomal activity and restores replicative senescence. Black-Right-Pointing-Pointer 18{alpha}-GA retains differentiation without affecting stem cell characterizations. -- Abstract: Human bone marrow stromal cells (hBMSCs) could be used in clinics as precursors of multiple cell lineages following proper induction. Such application is impeded by their characteristically short lifespan, together with the increasing loss of proliferation capability and progressive reduction of differentiation potential after the prolonged culture expansion. In the current study, we addressed the possible role of 20S proteasomes in this process. Consistent with prior reports, long-term in vitro expansion of hBMSCs decreased cell proliferation and increased replicative senescence, accompanied by reduced activity and expression of the catalytic subunits PSMB5 and PSMB1, and the 20S proteasome overall. Application of the proteasome inhibitor MG132 produced a senescence-like phenotype in early passages, whereas treating late-passage cells with 18{alpha}-glycyrrhetinic acid (18{alpha}-GA), an agonist of 20S proteasomes, delayed the senescence progress, enhancing the proliferation and recovering the capability of differentiation. The data demonstrate that activation of 20S proteasomes assists in counteracting replicative senescence of hBMSCs expanded in vitro.

  14. LPS-Induced Formation of Immunoproteasomes: TNF-α and Nitric Oxide Production are Regulated by Altered Composition of Proteasome-Active Sites

    PubMed Central

    Reis, Julia; Guan, Xiu Qin; Kisselev, Alexei F.; Papasian, Christopher J.; Qureshi, Asaf A.; Morrison, David C.; Van Way, Charles W.; Vogel, Stefanie N.

    2011-01-01

    Stimulation of mouse macrophages with LPS leads to tumor necrosis factor (TNF-α) secretion and nitric oxide (NO) release at different times through independent signaling pathways. While the precise regulatory mechanisms responsible for these distinct phenotypic responses have not been fully delineated, results of our recent studies strongly implicate the cellular cytoplasmic ubiquitin–proteasome pathway as a key regulator of LPS-induced macrophage inflammatory responses. Our objective in this study was to define the relative contribution of specific proteasomal active-sites in induction of TNF-α and NO after LPS treatment of RAW 264.7 macrophages using selective inhibitors of these active sites. Our data provide evidence that LPS stimulation of mouse macrophages triggers a selective increase in the levels of gene and protein expression of the immunoproteasomes, resulting in a modulation of specific functional activities of the proteasome and a corresponding increase in NO production as compared to untreated controls. These findings suggest the LPS-dependent induction of immunoproteasome. In contrast, we also demonstrate that TNF-α expression is primarily dependent on both the chymotrypsin- and the trypsin-like activities of X, Y, Z subunits of the proteasome. Proteasome-associated post-acidic activity alone also contributes to LPS-induced expression of TNF-α. Taken together; our results indicate that LPS-induced TNF-α in macrophages is differentially regulated by each of the three proteasome activities. Since addition of proteasome inhibitors to mouse macrophages profoundly affects the degradation of proteins involved in signal transduction, we conclude that proteasome-specific degradation of several signaling proteins is likely involved in differential regulation of LPS-dependent secretion of proinflammatory mediators. PMID:21455682

  15. New Difluoro Knoevenagel Condensates of Curcumin, Their Schiff Bases and Copper Complexes as Proteasome Inhibitors and Apoptosis Inducers in Cancer Cells

    PubMed Central

    Padhye, Subhash; Yang, Huanjie; Jamadar, Abeda; Cui, Qiuzhi Cindy; Chavan, Deepak; Dominiak, Kristin; McKinney, Jaclyn; Banerjee, Sanjeev; Dou, Q. Ping; Sarkar, Fazlul H.

    2013-01-01

    Purpose Emerging evidence clearly suggests the potential chemopreventive and anti-tumor activity of a well known “natural agent” curcumin. However, studies have shown that curcumin is not readily bioavailable, and thus the tissue bioavailability of curcumin is also poor except for gastrointestinal track. Because of the potential biological activity of curcumin, many studies have attempted for making a better analog of cucumin that is equally effective or better with increased bioavailability, which was the purpose of our current study. Methods We have designed and synthesized new difluoro Knoevenagel condensates of curcumin and Schiff bases along with their copper (II) complexes and evaluated their biological activities with respect to the inhibitory effects on purified rabbit 26S proteasome, and growth inhibition and induction of apoptosis in colon and pancreatic cancer cell lines. Results All copper complexes possess distorted square planar geometries with 1:1 metal to ligand stoichiometry with reversible copper redox couple. The difluoro compound CDF exhibited inhibitory effects on purified rabbit 20S proteasome or cellular 26S proteasome, and caused both growth inhibition of cancer cell lines and induced apoptotic cell death in our preliminary assessment. Conclusion Our results suggest that our newly synthesized classes of curcumin analogs could be useful as chemopreventive and/or therapeutic agents against cancers. PMID:19421843

  16. Hepatitis B Virus HBx Protein Interactions with the Ubiquitin Proteasome System

    PubMed Central

    Minor, Marissa M.; Slagle, Betty L.

    2014-01-01

    The hepatitis B virus (HBV) causes acute and chronic hepatitis, and the latter is a major risk factor for the development of hepatocellular carcinoma (HCC). HBV encodes a 17-kDa regulatory protein, HBx, which is required for virus replication. Although the precise contribution(s) of HBx to virus replication is unknown, many viruses target cellular pathways to create an environment favorable for virus replication. The ubiquitin proteasome system (UPS) is a major conserved cellular pathway that controls several critical processes in the cell by regulating the levels of proteins involved in cell cycle, DNA repair, innate immunity, and other processes. We summarize here the interactions of HBx with components of the UPS, including the CUL4 adaptor DDB1, the cullin regulatory complex CSN, and the 26S proteasome. Understanding how these protein interactions benefit virus replication remains a challenge due to limited models in which to study HBV replication. However, studies from other viral systems that similarly target the UPS provide insight into possible strategies used by HBV. PMID:25421893

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

    SciTech Connect

    Lee, Sanghwan; Hur, Eu-gene; Ryoo, In-geun; Jung, Kyeong-Ah; Kwak, Jiyeon; Kwak, Mi-Kyoung

    2012-11-01

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

  18. The ubiquitin-proteasome pathway mediates the regulated degradation of mammalian 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

    PubMed

    Ravid, T; Doolman, R; Avner, R; Harats, D; Roitelman, J

    2000-11-17

    3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the key regulatory enzyme in the mevalonate (MVA) pathway, is rapidly degraded in mammalian cells supplemented with sterols or MVA. This accelerated turnover was blocked by N-acetyl-leucyl-leucyl-norleucinal (ALLN), MG-132, and lactacystin, and to a lesser extent by N-acetyl-leucyl-leucyl-methional (ALLM), indicating the involvement of the 26 S proteasome. Proteasome inhibition led to enhanced accumulation of high molecular weight polyubiquitin conjugates of HMGR and of HMGal, a chimera between the membrane domain of HMGR and beta-galactosidase. Importantly, increased amounts of polyubiquitinated HMGR and HMGal were observed upon treating cells with sterols or MVA. Cycloheximide inhibited the sterol-stimulated degradation of HMGR concomitantly with a marked reduction in polyubiquitination of the enzyme. Inhibition of squalene synthase with zaragozic acid blocked the MVA- but not sterol-stimulated ubiquitination and degradation of HMGR. Thus, similar to yeast, the ubiquitin-proteasome pathway is involved in the metabolically regulated turnover of mammalian HMGR. Yet, the data indicate divergence between yeast and mammals and suggest distinct roles for sterol and nonsterol metabolic signals in the regulated ubiquitination and degradation of mammalian HMGR. PMID:10964918

  19. ASB2 targets filamins A and B to proteasomal degradation

    PubMed Central

    Heuzé, Mélina L.; Lamsoul, Isabelle; Baldassarre, Massimiliano; Lad, Yatish; Lévêque, Sophie; Razinia, Ziba; Moog-Lutz, Christel; Calderwood, David A.

    2008-01-01

    The ordered series of proliferation and differentiation from hematopoietic progenitor cells is disrupted in leukemia, resulting in arrest of differentiation at immature proliferative stages. Characterizing the molecular basis of hematopoietic differentiation is therefore important for understanding and treating disease. Retinoic acid induces expression of ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 (ASB2) in acute promyelocytic leukemia cells, and ASB2 expression inhibits growth and promotes commitment, recapitulating an early step critical for differentiation. ASB2 is the specificity subunit of an E3 ubiquitin ligase complex and is proposed to exert its effects by regulating the turnover of specific proteins; however, no ASB2 substrates had been identified. Here, we report that ASB2 targets the actin-binding proteins filamin A and B for proteasomal degradation. Knockdown of endogenous ASB2 in leukemia cells delays retinoic acid-induced differentiation and filamin degradation; conversely, ASB2 expression in leukemia cells induces filamin degradation. ASB2 expression inhibits cell spreading, and this effect is recapitulated by knocking down both filamin A and filamin B. Thus, we suggest that ASB2 may regulate hematopoietic cell differentiation by modulating cell spreading and actin remodeling through targeting of filamins for degradation. PMID:18799729

  20. HIV-1 Envelope Resistance to Proteasomal Cleavage: Implications for Vaccine Induced Immune Responses

    PubMed Central

    Steers, Nicholas J.; Ratto-Kim, Silvia; de Souza, Mark S.; Currier, Jeffrey R.; Kim, Jerome H.; Michael, Nelson L.; Alving, Carl R.; Rao, Mangala

    2012-01-01

    Background Antigen processing involves many proteolytic enzymes such as proteasomes and cathepsins. The processed antigen is then presented on the cell surface bound to either MHC class I or class II molecules and induces/interacts with antigen-specific CD8+ and CD4+ T-cells, respectively. Preliminary immunological data from the RV144 phase III trial indicated that the immune responses were biased towards the Env antigen with a dominant CD4+ T-cell response. Methods In this study, we examined the susceptibility of HIV-1 Env-A244 gp120 protein, one of the protein boost subunits of the RV144 Phase III vaccine trial, to proteasomes and cathepsins and identified the generated peptide epitope repertoire by mass spectrometry. The peptide fragments were tested for cytokine production in CD4+ T-cell lines derived from RV144 volunteers. Results Env-A244 was resistant to proteasomes, thus diminishing the possibility of the generation of class I epitopes by the classical MHC class I pathway. However, Env-A244 was efficiently cleaved by cathepsins generating peptide arrays identified by mass spectrometry that contained both MHC class I and class II epitopes as reported in the Los Alamos database. Each of the cathepsins generated distinct degradation patterns containing regions of light and dense epitope clusters. The sequence DKKQKVHALF that is part of the V2 loop of gp120 produced by cathepsins induced a polyfunctional cytokine response including the generation of IFN-γ from CD4+ T-cell lines-derived from RV144 vaccinees. This sequence is significant since antibodies to the V1/V2-loop region correlated inversely with HIV-1 infection in the RV144 trial. Conclusions Based on our results, the susceptibility of Env-A244 to cathepsins and not to proteasomes suggests a possible mechanism for the generation of Env-specific CD4+T cell and antibody responses in the RV144 vaccinees. PMID:22880042

  1. The Ubiquitin-Proteasome System: Potential Therapeutic Targets for Alzheimer's Disease and Spinal Cord Injury.

    PubMed

    Gong, Bing; Radulovic, Miroslav; Figueiredo-Pereira, Maria E; Cardozo, Christopher

    2016-01-01

    The ubiquitin-proteasome system (UPS) is a crucial protein degradation system in eukaryotes. Herein, we will review advances in the understanding of the role of several proteins of the UPS in Alzheimer's disease (AD) and functional recovery after spinal cord injury (SCI). The UPS consists of many factors that include E3 ubiquitin ligases, ubiquitin hydrolases, ubiquitin and ubiquitin-like molecules, and the proteasome itself. An extensive body of work links UPS dysfunction with AD pathogenesis and progression. More recently, the UPS has been shown to have vital roles in recovery of function after SCI. The ubiquitin hydrolase (Uch-L1) has been proposed to increase cellular levels of mono-ubiquitin and hence to increase rates of protein turnover by the UPS. A low Uch-L1 level has been linked with Aβ accumulation in AD and reduced neuroregeneration after SCI. One likely mechanism for these beneficial effects of Uch-L1 is reduced turnover of the PKA regulatory subunit and consequently, reduced signaling via CREB. The neuron-specific F-box protein Fbx2 ubiquitinates β-secretase thus targeting it for proteasomal degradation and reducing generation of Aβ. Both Uch-L1 and Fbx2 improve synaptic plasticity and cognitive function in mouse AD models. The role of Fbx2 after SCI has not been examined, but abolishing ß-secretase reduces neuronal recovery after SCI, associated with reduced myelination. UBB+1, which arises through a frame-shift mutation in the ubiquitin gene that adds 19 amino acids to the C-terminus of ubiquitin, inhibits proteasomal function and is associated with increased neurofibrillary tangles in patients with AD, Pick's disease and Down's syndrome. These advances in understanding of the roles of the UPS in AD and SCI raise new questions but, also, identify attractive and exciting targets for potential, future therapeutic interventions. PMID:26858599

  2. Link between Organ-specific Antigen Processing by 20S Proteasomes and CD8+ T Cell–mediated Autoimmunity

    PubMed Central

    Kuckelkorn, Ulrike; Ruppert, Thomas; Strehl, Britta; Jungblut, Peter R.; Zimny-Arndt, Ursula; Lamer, Stephanie; Prinz, Immo; Drung, Ilse; Kloetzel, Peter-M.; Kaufmann, Stefan H.E.; Steinhoff, Ulrich

    2002-01-01

    Adoptive transfer of cross-reactive HSP60-specific CD8+ T cells into immunodeficient mice causes autoimmune intestinal pathology restricted to the small intestine. We wondered whether local immunopathology induced by CD8+ T cells can be explained by tissue-specific differences in proteasome-mediated processing of major histocompatibility complex class I T cell epitopes. Our experiments demonstrate that 20S proteasomes of different organs display a characteristic composition of α and β chain subunits and produce distinct peptide fragments with respect to both quality and quantity. Digests of HSP60 polypeptides by 20S proteasomes show most efficient generation of the pathology related CD8+ T cell epitope in the small intestine. Further, we demonstrate that the organ-specific potential to produce defined T cell epitopes reflects quantities that are relevant for cytotoxic T lymphocyte recognition. We propose tissue-specific antigen processing by 20S proteasomes as a potential mechanism to control organ-specific immune responses. PMID:11956289

  3. Identification of substrates of the Mycobacterium tuberculosis proteasome

    PubMed Central

    Pearce, Michael J; Arora, Pooja; Festa, Richard A; Butler-Wu, Susan M; Gokhale, Rajesh S; Darwin, K Heran

    2006-01-01

    The putative proteasome-associated proteins Mpa (Mycobaterium proteasomal ATPase) and PafA (proteasome accessory factor A) of the human pathogen Mycobacterium tuberculosis (Mtb) are essential for virulence and resistance to nitric oxide. However, a direct link between the proteasome protease and Mpa or PafA has never been demonstrated. Furthermore, protein degradation by bacterial proteasomes in vitro has not been accomplished, possibly due to the failure to find natural degradation substrates or other necessary proteasome co-factors. In this work, we identify the first bacterial proteasome substrates, malonyl Co-A acyl carrier protein transacylase and ketopantoate hydroxymethyltransferase, enzymes that are required for the biosynthesis of fatty acids and polyketides that are essential for the pathogenesis of Mtb. Maintenance of the physiological levels of these enzymes required Mpa and PafA in addition to proteasome protease activity. Mpa levels were also regulated in a proteasome-dependent manner. Finally, we found that a conserved tyrosine of Mpa was essential for function. Thus, these results suggest that Mpa, PafA, and the Mtb proteasome degrade bacterial proteins that are important for virulence in mice. PMID:17082771

  4. Pupylation-dependent and -independent proteasomal degradation in mycobacteria.

    PubMed

    Imkamp, Frank; Ziemski, Michal; Weber-Ban, Eilika

    2015-08-01

    Bacteria make use of compartmentalizing protease complexes, similar in architecture but not homologous to the eukaryotic proteasome, for the selective and processive removal of proteins. Mycobacteria as members of the actinobacteria harbor proteasomes in addition to the canonical bacterial degradation complexes. Mycobacterial proteasomal degradation, although not essential during normal growth, becomes critical for survival under particular environmental conditions, like, for example, during persistence of the pathogenic Mycobacterium tuberculosis in host macrophages or of environmental mycobacteria under starvation. Recruitment of protein substrates for proteasomal degradation is usually mediated by pupylation, the post-translational modification of lysine side chains with the prokaryotic ubiquitin-like protein Pup. This substrate recruitment strategy is functionally reminiscent of ubiquitination in eukaryotes, but is the result of convergent evolution, relying on chemically and structurally distinct enzymes. Pupylated substrates are recognized by the ATP-dependent proteasomal regulator Mpa that associates with the 20S proteasome core. A pupylation-independent proteasome degradation pathway has recently been discovered that is mediated by the ATP-independent bacterial proteasome activator Bpa (also referred to as PafE), and that appears to play a role under stress conditions. In this review, mechanistic principles of bacterial proteasomal degradation are discussed and compared with functionally related elements of the eukaryotic ubiquitin-proteasome system. Special attention is given to an understanding on the molecular level based on structural and biochemical analysis. Wherever available, discussion of in vivo studies is included to highlight the biological significance of this unusual bacterial degradation pathway. PMID:26352358

  5. The Homeodomain Transcription Factor Hoxa2 Interacts with and Promotes the Proteasomal Degradation of the E3 Ubiquitin Protein Ligase RCHY1

    PubMed Central

    Bergiers, Isabelle; Bridoux, Laure; Nguyen, Nathan; Twizere, Jean-Claude; Rezsöhazy, René

    2013-01-01

    Hox proteins are conserved homeodomain transcription factors known to be crucial regulators of animal development. As transcription factors, the functions and modes of action (co-factors, target genes) of Hox proteins have been very well studied in a multitude of animal models. However, a handful of reports established that Hox proteins may display molecular activities distinct from gene transcription regulation. Here, we reveal that Hoxa2 interacts with 20S proteasome subunits and RCHY1 (also known as PIRH2), an E3 ubiquitin ligase that targets p53 for degradation. We further show that Hoxa2 promotes proteasome-dependent degradation of RCHY1 in an ubiquitin-independent manner. Correlatively, Hoxa2 alters the RCHY1-mediated ubiquitination of p53 and promotes p53 stabilization. Together, our data establish that Hoxa2 can regulate the proteasomal degradation of RCHY1 and stabilization of p53. PMID:24244684

  6. Cupriphilic compounds to aid in proteasome inhibition.

    PubMed

    Mukherjee, Sreya; Sparks, Robert; Metcalf, Rainer; Brooks, Wesley; Daniel, Kenyon; Guida, Wayne C

    2016-08-01

    It has been found that tumor cells and tissues, compared to normal cells, have higher levels of copper and possibly other metal ions. This presents a potential vulnerability of tumor cells that can serve as a physiological difference between cancer cells and normal cells and allows design of compounds that selectively target tumor cells while sparing normal cells. Recently we have identified compounds that have potential to inhibit the proteasome in tumor cells and induce cell death by mobilizing endogenous tumor copper resulting in in cellulo activation of the compound. These compounds hence act as pro-drugs, becoming active drugs in tumor cells with high copper content but remaining essentially inactive in normal cells, thereby greatly reducing adverse effects in patients. Such use would be of significant benefit in early detection and treatment of cancers, in particular, aggressive cancers such as pancreatic cancer which is usually not detected until it has reached an advanced stage. Six compounds were identified following virtual screening of the NCI Diversity Set with our proteasome computer model followed by confirmation with a biochemical assay that showed significant inhibition of the proteasome by the compounds in the presence of copper ions. In a dose response assay, NSC 37408 (6,7-dihydroxy-1-benzofuran-3-one), our best compound, exhibited an IC50 of 3μM in the presence of 100nM copper. PMID:27311892

  7. Combination Treatment with Sublethal Ionizing Radiation and the Proteasome Inhibitor, Bortezomib, Enhances Death-Receptor Mediated Apoptosis and Anti-Tumor Immune Attack

    PubMed Central

    Cacan, Ercan; Spring, Alexander M.; Kumari, Anita; Greer, Susanna F.; Garnett-Benson, Charlie

    2015-01-01

    Sub-lethal doses of radiation can modulate gene expression, making tumor cells more susceptible to T-cell-mediated immune attack. Proteasome inhibitors demonstrate broad anti-tumor activity in clinical and pre-clinical cancer models. Here, we use a combination treatment of proteasome inhibition and irradiation to further induce immunomodulation of tumor cells that could enhance tumor-specific immune responses. We investigate the effects of the 26S proteasome inhibitor, bortezomib, alone or in combination with radiotherapy, on the expression of immunogenic genes in normal colon and colorectal cancer cell lines. We examined cells for changes in the expression of several death receptors (DR4, DR5 and Fas) commonly used by T cells for killing of target cells. Our results indicate that the combination treatment resulted in increased cell surface expression of death receptors by increasing their transcript levels. The combination treatment further increases the sensitivity of carcinoma cells to apoptosis through FAS and TRAIL receptors but does not change the sensitivity of normal non-malignant epithelial cells. Furthermore, the combination treatment significantly enhances tumor cell killing by tumor specific CD8+ T cells. This study suggests that combining radiotherapy and proteasome inhibition may simultaneously enhance tumor immunogenicity and the induction of antitumor immunity by enhancing tumor-specific T-cell activity. PMID:26703577

  8. Substrate Ubiquitination Controls the Unfolding Ability of the Proteasome.

    PubMed

    Reichard, Eden L; Chirico, Giavanna G; Dewey, William J; Nassif, Nicholas D; Bard, Katelyn E; Millas, Nickolas E; Kraut, Daniel A

    2016-08-26

    In eukaryotic cells, proteins are targeted to the proteasome for degradation by polyubiquitination. These proteins bind to ubiquitin receptors, are engaged and unfolded by proteasomal ATPases, and are processively degraded. The factors determining to what extent the proteasome can successfully unfold and degrade a substrate are still poorly understood. We find that the architecture of polyubiquitin chains attached to a substrate affects the ability of the proteasome to unfold and degrade the substrate, with K48- or mixed-linkage chains leading to greater processivity than K63-linked chains. Ubiquitin-independent targeting of substrates to the proteasome gave substantially lower processivity of degradation than ubiquitin-dependent targeting. Thus, even though ubiquitin chains are removed early in degradation, during substrate engagement, remarkably they dramatically affect the later unfolding of a protein domain. Our work supports a model in which a polyubiquitin chain associated with a substrate switches the proteasome into an activated state that persists throughout the degradation process. PMID:27405762

  9. Could inhibition of the proteasome cause mad cow disease?

    PubMed

    Hooper, Nigel M

    2003-04-01

    The proteasome is the cellular machinery responsible for the degradation of normal and misfolded proteins. Inhibitors of the proteasome are being evaluated as therapeutic agents and recent work suggests that such inhibition might promote the neurotoxic properties of the prion protein (the causative agent of mad cow disease) and its conformational conversion to the infectious form, thus raising the question as to whether proteasome inhibitors might facilitate the development of prion diseases. PMID:12679058

  10. Tobacco RING E3 Ligase NtRFP1 Mediates Ubiquitination and Proteasomal Degradation of a Geminivirus-Encoded βC1.

    PubMed

    Shen, Qingtang; Hu, Tao; Bao, Min; Cao, Linge; Zhang, Huawei; Song, Fengmin; Xie, Qi; Zhou, Xueping

    2016-06-01

    The βC1 protein encoded by the Tomato yellow leaf curl China virus-associated betasatellite functions as a pathogenicity determinant. To better understand the molecular basis whereby βC1 functions in pathogenicity, a yeast two-hybrid screen of a tobacco cDNA library was carried out using βC1 as the bait. The screen revealed that βC1 interacts with a tobacco RING-finger protein designated NtRFP1, which was further confirmed by the bimolecular fluorescence complementation and co-immunoprecipitation assays in Nicotiana benthamiana cells. Expression of NtRFP1 was induced by βC1, and in vitro ubiquitination assays showed that NtRFP1 is a functional E3 ubiquitin ligase that mediates βC1 ubiquitination. In addition, βC1 was shown to be ubiquitinated in vivo and degraded by the plant 26S proteasome. After viral infection, plants overexpressing NtRFP1 developed attenuated symptoms, whereas plants with silenced expression of NtRFP1 showed severe symptoms. Other lines of evidence showed that NtRFP1 attenuates βC1-induced symptoms through promoting its degradation by the 26S proteasome. Taken together, our results suggest that tobacco RING E3 ligase NtRFP1 attenuates disease symptoms by interacting with βC1 to mediate its ubiquitination and degradation via the ubiquitin/26S proteasome system. PMID:27018391

  11. Proteasome-Mediated Processing of Def1, a Critical Step in the Cellular Response to Transcription Stress

    PubMed Central

    Wilson, Marcus D.; Harreman, Michelle; Taschner, Michael; Reid, James; Walker, Jane; Erdjument-Bromage, Hediye; Tempst, Paul; Svejstrup, Jesper Q.

    2013-01-01

    Summary DNA damage triggers polyubiquitylation and degradation of the largest subunit of RNA polymerase II (RNAPII), a “mechanism of last resort” employed during transcription stress. In yeast, this process is dependent on Def1 through a previously unresolved mechanism. Here, we report that Def1 becomes activated through ubiquitylation- and proteasome-dependent processing. Def1 processing results in the removal of a domain promoting cytoplasmic localization, resulting in nuclear accumulation of the clipped protein. Nuclear Def1 then binds RNAPII, utilizing a ubiquitin-binding domain to recruit the Elongin-Cullin E3 ligase complex via a ubiquitin-homology domain in the Ela1 protein. This facilitates polyubiquitylation of Rpb1, triggering its proteasome-mediated degradation. Together, these results outline the multistep mechanism of Rpb1 polyubiquitylation triggered by transcription stress and uncover the key role played by Def1 as a facilitator of Elongin-Cullin ubiquitin ligase function. PMID:23993092

  12. Proteasomes and protein conjugation across domains of life

    PubMed Central

    Maupin-Furlow, Julie

    2012-01-01

    Like other energy-dependent proteases, proteasomes, which are found across the three domains of life, are self-compartmentalized and important in the early steps of proteolysis. Proteasomes degrade improperly synthesized, damaged or misfolded proteins and hydrolyse regulatory proteins that must be specifically removed or cleaved for cell signalling. In eukaryotes, proteins are typically targeted for proteasome-mediated destruction through polyubiquitylation, although ubiquitin-independent pathways also exist. Interestingly, actinobacteria and archaea also covalently attach small proteins (prokaryotic ubiquitin-like protein (Pup) and small archaeal modifier proteins (Samps), respectively) to certain proteins, and this may serve to target the modified proteins for degradation by proteasomes. PMID:22183254

  13. 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

  14. 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

    2013-01-01

    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

  15. Relationship between the proteasomal system and autophagy

    PubMed Central

    Lilienbaum, Alain

    2013-01-01

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

  16. Ubiquitin proteasome system research in gastrointestinal cancer

    PubMed Central

    Zhong, Jia-Ling; Huang, Chang-Zhi

    2016-01-01

    The ubiquitin proteasome system (UPS) is important for the degradation of proteins in eukaryotic cells. It is involved in nearly every cellular process and plays an important role in maintaining body homeostasis. An increasing body of evidence has linked alterations in the UPS to gastrointestinal malignancies, including esophageal, gastric and colorectal cancers. Here, we summarize the current literature detailing the involvement of the UPS in gastrointestinal cancer, highlighting its role in tumor occurrence and development, providing information for therapeutic targets research and anti-gastrointestinal tumor drug design. PMID:26909134

  17. Cytosolic Hsp60 Can Modulate Proteasome Activity in Yeast*

    PubMed Central

    Kalderon, Bella; Kogan, Gleb; Bubis, Ettel; Pines, Ophry

    2015-01-01

    Hsp60, an essential oligomeric molecular mitochondrial chaperone, has been subject to rigorous basic and clinical research. With yeast as a model system, we provide evidence for the ability of cytosolic yHsp60 to inhibit the yeast proteasome. (i) Following biological turnover of murine Bax (a proteasome substrate), we show that co-expression of cytosolic yHsp60 stabilizes Bax, enhances its association with mitochondria, and enhances its killing capacity. (ii) Expression of yHsp60 in the yeast cytosol (yHsp60c) inhibits degradation of a cytosolic protein ΔMTS-Aco1 tagged with the degron SL17 (a ubiquitin-proteasome substrate). (iii) Conditions under which Hsp60 accumulates in the cytosol (elevated Hsp60c or growth at 37 °C) correlate with reduced 20 S peptidase activity in proteasomes purified from cell extracts. (iv) Elevated yHsp60 in the cytosol correlate with accumulation of polyubiquitinated proteins. (v) According to 20 S proteasome pulldown experiments, Hsp60 is physically associated with proteasomes in extracts of cells expressing Hsp60c or grown at 37 °C. Even mutant Hsp60 proteins, lacking chaperone activity, were still capable of proteasome inhibition. The results support the hypothesis that localization of Hsp60 to the cytosol may modulate proteasome activity according to cell need. PMID:25525272

  18. Identification of Novel Proteasome Inhibitors from an Enaminone Library.

    PubMed

    Elliott, Megan L; Thomas, Kevin; Kennedy, Steven; Koduri, Naga D; Hussaini, R Syed; Sheaff, Robert J

    2015-09-01

    A library of structurally distinct enaminones was synthesized using sonication or Ru(II) catalysis to couple primary, secondary, and tertiary thioamides with α-halocarbonyls or α-diazocarbonyls. Screening the library for proteasome inhibition using a luciferase-based assay identified seven structurally diverse compounds. Two of these molecules targeted luciferase, while the remaining five exhibited varying potency and specificity for the trypsin-like, chymotrypsin-like, or caspase-like protease activities of the proteasome. Physiological relevance was confirmed by showing these molecules inhibited proteasomal degradation of the full-length protein substrate p21cip1 expressed in tissue culture cells. A cell viability analysis revealed that the proteasome inhibitors differentially affected cell survival. Results indicate a subset of enaminones and precursor molecules identified in this study are good candidates for further development into novel proteasome inhibitors with potential therapeutic value. PMID:25494709

  19. Proteasome modulators: essential chemical genetic tools for understanding human diseases.

    PubMed

    Wehenkel, Marie; Hong, Jin Tae; Kim, Kyung Bo

    2008-04-01

    Primarily used for medicinal purposes in the past, biologically active small molecules have been increasingly employed to explore complex biological processes in the era of "chemical genetics". Since the contributions of this small molecule approach to biology have been extensive, we limit the focus of our review to the use of small-molecule modulators in the exciting field of proteasomal biology, one that has benefited significantly from a chemical genetics approach. Specifically, as the contributions of general inhibitors of proteasomal activity to the fields of cell biology and clinical oncology have been extensively discussed in several excellent reviews, we instead outline recent progress towards the development of novel, specific classes of proteasome modulators for studies of proteasomal biology and the types of proteasome inhibitors emerging as important new treatment options for cancer therapeutics. PMID:18354780

  20. Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells

    PubMed Central

    Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

    2016-01-01

    Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K

  1. Harnessing Proteasome Dynamics and Allostery in Drug Design

    PubMed Central

    Osmulski, Pawel A.

    2014-01-01

    Abstract Significance: The proteasome is the essential protease that is responsible for regulated cleavage of the bulk of intracellular proteins. Its central role in cellular physiology has been exploited in therapies against aggressive cancers where proteasome-specific competitive inhibitors that block proteasome active centers are very effectively used. However, drugs regulating this essential protease are likely to have broader clinical usefulness. The non-catalytic sites of the proteasome emerge as an attractive alternative target in search of highly specific and diverse proteasome regulators. Recent Advances: Crystallographic models of the proteasome leave the false impression of fixed structures with minimal molecular dynamics lacking long-distance allosteric signaling. However, accumulating biochemical and structural observations strongly support the notion that the proteasome is regulated by precise allosteric interactions arising from protein dynamics, encouraging the active search for allosteric regulators. Here, we discuss properties of several promising compounds that affect substrate gating and processing in antechambers, and interactions of the catalytic core with regulatory proteins. Critical Issues: Given the structural complexity of proteasome assemblies, it is a painstaking process to better understand their allosteric regulation and molecular dynamics. Here, we discuss the challenges and achievements in this field. We place special emphasis on the role of atomic force microscopy imaging in probing the allostery and dynamics of the proteasome, and in dissecting the mechanisms involving small-molecule allosteric regulators. Future Directions: New small-molecule allosteric regulators may become a next generation of drugs targeting the proteasome, which is critical to the development of new therapies in cancers and other diseases. Antioxid. Redox Signal. 21, 2286–2301. PMID:24410482

  2. A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases

    PubMed Central

    Liu, Ningning; Liu, Chunjiao; Li, Xiaofen; Liao, Siyan; Song, Wenbin; Yang, Changshan; Zhao, Chong; Huang, Hongbiao; Guan, Lixia; Zhang, Peiquan; Liu, Shouting; Hua, Xianliang; Chen, Xin; Zhou, Ping; Lan, Xiaoying; Yi, Songgang; Wang, Shunqing; Wang, Xuejun; Dou, Q. Ping; Liu, Jinbao

    2014-01-01

    The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy. PMID:24912524

  3. A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases.

    PubMed

    Liu, Ningning; Liu, Chunjiao; Li, Xiaofen; Liao, Siyan; Song, Wenbin; Yang, Changshan; Zhao, Chong; Huang, Hongbiao; Guan, Lixia; Zhang, Peiquan; Liu, Shouting; Hua, Xianliang; Chen, Xin; Zhou, Ping; Lan, Xiaoying; Yi, Songgang; Wang, Shunqing; Wang, Xuejun; Dou, Q Ping; Liu, Jinbao

    2014-01-01

    The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy. PMID:24912524

  4. Overview of proteasome inhibitor-based anti-cancer therapies: perspective on bortezomib and second generation proteasome inhibitors versus future generation inhibitors of ubiquitin-proteasome system.

    PubMed

    Dou, Q Ping; Zonder, Jeffrey A

    2014-01-01

    Over the past ten years, proteasome inhibition has emerged as an effective therapeutic strategy for treating multiple myeloma (MM) and some lymphomas. In 2003, Bortezomib (BTZ) became the first proteasome inhibitor approved by the U.S. Food and Drug Administration (FDA). BTZ-based therapies have become a staple for the treatment of MM at all stages of the disease. The survival rate of MM patients has improved significantly since clinical introduction of BTZ and other immunomodulatory drugs. However, BTZ has several limitations. Not all patients respond to BTZ based therapies and relapse occurs in many patients who initially responded. Solid tumors, in particular, are often resistant to BTZ. Furthermore, BTZ can induce dose-limiting peripheral neuropathy (PN). The second generation proteasome inhibitor Carfizomib (CFZ; U.S. FDA approved in August 2012) induces responses in a minority of MM patients relapsed from or refractory to BTZ. There is less PN compared to BTZ. Four other second-generation proteasome inhibitors (Ixazomib, Delanzomib, Oprozomib and Marizomib) with different pharmacologic properties and broader anticancer activities, have also shown some clinical activity in bortezomib-resistant cancers. While the mechanism of resistance to bortezomib in human cancers still remains to be fully understood, targeting the immunoproteasome, ubiquitin E3 ligases, the 19S proteasome and deubiquitinases in pre-clinical studies represents possible directions for future generation inhibitors of ubiquitin-proteasome system in the treatment of MM and other cancers. PMID:25092212

  5. Overview of Proteasome Inhibitor-Based Anti-cancer Therapies: Perspective on Bortezomib and Second Generation Proteasome Inhibitors versus Future Generation Inhibitors of Ubiquitin-Proteasome System

    PubMed Central

    Dou, Q. Ping; Zonder, Jeffrey A.

    2014-01-01

    Over the past ten years, proteasome inhibition has emerged as an effective therapeutic strategy for treating multiple myeloma (MM) and some lymphomas. In 2003, Bortezomib (BTZ) became the first proteasome inhibitor approved by the U.S. Food and Drug Administration (FDA). BTZ-based therapies have become a staple for the treatment of MM at all stages of the disease. The survival rate of MM patients has improved significantly since clinical introduction of BTZ and other immunomodulatory drugs. However, BTZ has several limitations. Not all patients respond to BTZ-based therapies and relapse occurs in many patients who initially responded. Solid tumors, in particular, are often resistant to BTZ. Furthermore, BTZ can induce dose-limiting peripheral neuropathy (PN). The second generation proteasome inhibitor Carfizomib (CFZ; U.S. FDA approved in August 2012) induces responses in a minority of MM patients relapsed from or refractory to BTZ. There is less PN compared to BTZ. Four other second-generation proteasome inhibitors (Ixazomib, Delanzomib, Oprozomib and Marizomib) with different pharmacologic properties and broader anticancer activities, have also shown some clinical activity in bortezomib-resistant cancers. While the mechanism of resistance to bortezomib in human cancers still remains to be fully understood, targeting the immunoproteasome, ubiquitin E3 ligases, the 19S proteasome and deubiquitinases in pre-clinical studies represents possible directions for future generation inhibitors of ubiquitin-proteasome system in the treatment of MM and other cancers. PMID:25092212

  6. Proteasome mutants, pre4-2 and ump1-2, suppress the essential function but not the mitochondrial RNase P function of the Saccharomyces cerevisiae gene RPM2.

    PubMed Central

    Lutz, M S; Ellis, S R; Martin, N C

    2000-01-01

    The Saccharomyces cerevisiae nuclear gene RPM2 encodes a component of the mitochondrial tRNA-processing enzyme RNase P. Cells grown on fermentable carbon sources do not require mitochondrial tRNA processing activity, but still require RPM2, indicating an additional function for the Rpm2 protein. RPM2-null cells arrest after 25 generations on fermentable media. Spontaneous mutations that suppress arrest occur with a frequency of approximately 9 x 10(-6). The resultant mutants do not grow on nonfermentable carbon sources. We identified two loci responsible for this suppression, which encode proteins that influence proteasome function or assembly. PRE4 is an essential gene encoding the beta-7 subunit of the 20S proteasome core. A Val-to-Phe substitution within a highly conserved region of Pre4p that disrupts proteasome function suppresses the growth arrest of RPM2-null cells on fermentable media. The other locus, UMP1, encodes a chaperone involved in 20S proteasome assembly. A nonsense mutation in UMP1 also disrupts proteasome function and suppresses Deltarpm2 growth arrest. In an RPM2 wild-type background, pre4-2 and ump1-2 strains fail to grow at restrictive temperatures on nonfermentable carbon sources. These data link proteasome activity with Rpm2p and mitochondrial function. PMID:10757750

  7. Mutant glucocerebrosidase in Gaucher disease recruits Hsp27 to the Hsp90 chaperone complex for proteasomal degradation

    PubMed Central

    Yang, Chunzhang; Wang, Herui; Zhu, Dongwang; Hong, Christopher S.; Dmitriev, Pauline; Zhang, Chao; Li, Yan; Ikejiri, Barbara; Brady, Roscoe O.; Zhuang, Zhengping

    2015-01-01

    Gaucher disease is caused by mutations of the GBA1 gene, which encodes the lysosomal anchored gluococerebrosidase (GCase). GBA1 mutations commonly result in protein misfolding, abnormal chaperone recognition, and premature degradation, but are less likely to affect catalytic activity. In the present study, we demonstrate that the Hsp90/HOP/Cdc37 complex recruits Hsp27 after recognition of GCase mutants with subsequent targeting of GCase mutant peptides to degradation mechanisms such as VCP and the 26S proteasome. Inhibition of Hsp27 not only increased the quantity of enzyme but also enhanced GCase activity in fibroblasts derived from patients with Gaucher disease. These findings provide insight into a possible therapeutic strategy for protein misfolding diseases by correcting chaperone binding and altering subsequent downstream patterns of protein degradation. PMID:25583479

  8. Graded Proteasome Dysfunction in Caenorhabditis elegans Activates an Adaptive Response Involving the Conserved SKN-1 and ELT-2 Transcription Factors and the Autophagy-Lysosome Pathway

    PubMed Central

    Chinchankar, Meghna N.; Ferguson, Annabel A.; Ghazi, Arjumand; Fisher, Alfred L.

    2016-01-01

    The maintenance of cellular proteins in a biologically active and structurally stable state is a vital endeavor involving multiple cellular pathways. One such pathway is the ubiquitin-proteasome system that represents a major route for protein degradation, and reductions in this pathway usually have adverse effects on the health of cells and tissues. Here, we demonstrate that loss-of-function mutants of the Caenorhabditis elegans proteasome subunit, RPN-10, exhibit moderate proteasome dysfunction and unexpectedly develop both increased longevity and enhanced resistance to multiple threats to the proteome, including heat, oxidative stress, and the presence of aggregation prone proteins. The rpn-10 mutant animals survive through the activation of compensatory mechanisms regulated by the conserved SKN-1/Nrf2 and ELT-2/GATA transcription factors that mediate the increased expression of genes encoding proteasome subunits as well as those mediating oxidative- and heat-stress responses. Additionally, we find that the rpn-10 mutant also shows enhanced activity of the autophagy-lysosome pathway as evidenced by increased expression of the multiple autophagy genes including atg-16.2, lgg-1, and bec-1, and also by an increase in GFP::LGG-1 puncta. Consistent with a critical role for this pathway, the enhanced resistance of the rpn-10 mutant to aggregation prone proteins depends on autophagy genes atg-13, atg-16.2, and prmt-1. Furthermore, the rpn-10 mutant is particularly sensitive to the inhibition of lysosome activity via either RNAi or chemical means. We also find that the rpn-10 mutant shows a reduction in the numbers of intestinal lysosomes, and that the elt-2 gene also plays a novel and vital role in controlling the production of functional lysosomes by the intestine. Overall, these experiments suggest that moderate proteasome dysfunction could be leveraged to improve protein homeostasis and organismal health and longevity, and that the rpn-10 mutant provides a unique

  9. Graded Proteasome Dysfunction in Caenorhabditis elegans Activates an Adaptive Response Involving the Conserved SKN-1 and ELT-2 Transcription Factors and the Autophagy-Lysosome Pathway.

    PubMed

    Keith, Scott A; Maddux, Sarah K; Zhong, Yayu; Chinchankar, Meghna N; Ferguson, Annabel A; Ghazi, Arjumand; Fisher, Alfred L

    2016-02-01

    The maintenance of cellular proteins in a biologically active and structurally stable state is a vital endeavor involving multiple cellular pathways. One such pathway is the ubiquitin-proteasome system that represents a major route for protein degradation, and reductions in this pathway usually have adverse effects on the health of cells and tissues. Here, we demonstrate that loss-of-function mutants of the Caenorhabditis elegans proteasome subunit, RPN-10, exhibit moderate proteasome dysfunction and unexpectedly develop both increased longevity and enhanced resistance to multiple threats to the proteome, including heat, oxidative stress, and the presence of aggregation prone proteins. The rpn-10 mutant animals survive through the activation of compensatory mechanisms regulated by the conserved SKN-1/Nrf2 and ELT-2/GATA transcription factors that mediate the increased expression of genes encoding proteasome subunits as well as those mediating oxidative- and heat-stress responses. Additionally, we find that the rpn-10 mutant also shows enhanced activity of the autophagy-lysosome pathway as evidenced by increased expression of the multiple autophagy genes including atg-16.2, lgg-1, and bec-1, and also by an increase in GFP::LGG-1 puncta. Consistent with a critical role for this pathway, the enhanced resistance of the rpn-10 mutant to aggregation prone proteins depends on autophagy genes atg-13, atg-16.2, and prmt-1. Furthermore, the rpn-10 mutant is particularly sensitive to the inhibition of lysosome activity via either RNAi or chemical means. We also find that the rpn-10 mutant shows a reduction in the numbers of intestinal lysosomes, and that the elt-2 gene also plays a novel and vital role in controlling the production of functional lysosomes by the intestine. Overall, these experiments suggest that moderate proteasome dysfunction could be leveraged to improve protein homeostasis and organismal health and longevity, and that the rpn-10 mutant provides a unique

  10. The amazing ubiquitin-proteasome system: structural components and implication in aging.

    PubMed

    Tsakiri, Eleni N; Trougakos, Ioannis P

    2015-01-01

    Proteome quality control (PQC) is critical for the maintenance of cellular functionality and it is assured by the curating activity of the proteostasis network (PN). PN is constituted of several complex protein machines that under conditions of proteome instability aim to, firstly identify, and then, either rescue or degrade nonnative polypeptides. Central to the PN functionality is the ubiquitin-proteasome system (UPS) which is composed from the ubiquitin-conjugating enzymes and the proteasome; the latter is a sophisticated multi-subunit molecular machine that functions in a bimodal way as it degrades both short-lived ubiquitinated normal proteins and nonfunctional polypeptides. UPS is also involved in PQC of the nucleus, the endoplasmic reticulum and the mitochondria and it also interacts with the other main cellular degradation axis, namely the autophagy-lysosome system. UPS functionality is optimum in the young organism but it is gradually compromised during aging resulting in increasing proteotoxic stress; these effects correlate not only with aging but also with most age-related diseases. Herein, we present a synopsis of the UPS components and of their functional alterations during cellular senescence or in vivo aging. We propose that mild UPS activation in the young organism will, likely, promote antiaging effects and/or suppress age-related diseases. PMID:25619718

  11. Proteasome-associated autoinflammatory syndromes: advances in pathogeneses, clinical presentations, diagnosis, and management.

    PubMed

    McDermott, Amelia; Jacks, Jennifer; Kessler, Marcus; Emanuel, Peter D; Gao, Ling

    2015-02-01

    The disease spectrum currently known as the proteasome-associated autoinflammatory syndromes (PRAAS) was first described in 1939 in patients who presented with recurrent fevers beginning in infancy or early childhood, which were accompanied by nodular erythema, a pernio-like rash, and joint contractures. Since then, several syndromes, such as chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) syndrome, Nakajo-Nishimura syndrome (NNS), joint contractures, muscle atrophy, microcytic anemia and panniculitis-induced lipodystrophy (JMP) syndrome, and Japanese autoinflammatory syndrome with lipodystrophy (JASL), have been used to categorize patients with diseases within the same spectrum. Recently, independent studies have identified mutations in the human proteasome subunit β type 8 (PSMB8) gene, which result in a sustained inflammatory response in all syndromes. Further functional studies not only suggest a causative role of PSMB8 mutations but also imply that they represent one disease spectrum, referred to as PRAAS. In this paper, we review the clinical presentations and laboratory findings of PRAAS, as well as the most recent advances in pathogeneses, diagnosis, and treatment options for patients with diseases in this spectrum. PMID:25521013

  12. Salinosporamide Natural Products: Potent 20S Proteasome Inhibitors as Promising Cancer Chemotherapeutics

    PubMed Central

    Gulder, Tobias A. M.

    2010-01-01

    Proteasome inhibitors are rapidly evolving as potent treatment options in cancer therapy. One of the most promising drug candidates of this type is salinosporamide A from the bacterium Salinispora tropica. This marine natural product possesses a complex, densely functionalized γ-lactam-β-lactone pharmacophore, which is responsible for its irreversible binding to its target, the β subunit of the 20S proteasome. Salinosporamide A entered phase I clinical trials for the treatment of multiple myeloma only three years after its discovery. The strong biological activity and the challenging structure of this compound have fueled intense academic and industrial research in recent years, which has led to the development of more than ten syntheses, the elucidation of its biosynthetic pathway, and the generation of promising structure–activity relationships and oncological data. Salinosporamide A thus serves as an intriguing example of the successful interplay of modern drug discovery and biomedical research, medicinal chemistry and pharmacology, natural product synthesis and analysis, as well as biosynthesis and bioengineering. PMID:20927786

  13. 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

  14. Ribosomal proteins produced in excess are degraded by the ubiquitin-proteasome system.

    PubMed

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

    2016-09-01

    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

  15. 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. PMID:27448207

  16. Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa

    SciTech Connect

    Wang, T.; Li, H; Lin, G; Tang, C; Li, D; Nathan, C; Heran Darwin, K

    2009-01-01

    Proteasome-mediated protein turnover in all domains of life is an energy-dependent process that requires ATPase activity. Mycobacterium tuberculosis (Mtb) was recently shown to possess a ubiquitin-like proteasome pathway that plays an essential role in Mtb resistance to killing by products of host macrophages. Here we report our structural and biochemical investigation of Mpa, the presumptive Mtb proteasomal ATPase. We demonstrate that Mpa binds to the Mtb proteasome in the presence of ATPS, providing the physical evidence that Mpa is the proteasomal ATPase. X-ray crystallographic determination of the conserved interdomain showed a five stranded double {beta} barrel structure containing a Greek key motif. Structure and mutational analysis indicate a major role of the interdomain for Mpa hexamerization. Our mutational and functional studies further suggest that the central channel in the Mpa hexamer is involved in protein substrate translocation and degradation. These studies provide insights into how a bacterial proteasomal ATPase interacts with and facilitates protein degradation by the proteasome.

  17. Abnormally high expression of proteasomes in human leukemic cells.

    PubMed Central

    Kumatori, A; Tanaka, K; Inamura, N; Sone, S; Ogura, T; Matsumoto, T; Tachikawa, T; Shin, S; Ichihara, A

    1990-01-01

    Proteasomes are eukaryotic ring-shaped or cylindrical particles with multicatalytic protease activities. To clarify the involvement of proteasomes in tumorigenesis of human blood cells, we compared their expression in human hematopoietic malignant tumor cells with that in normal peripheral blood mononuclear cells. Immunohistochemical staining showed considerably increased concentrations of proteasomes in leukemic cells from the bone marrow of patients with various types of leukemia and the predominant localization of these proteasomes in the nuclei. Moreover, enzyme immunoassay and Northern blot analysis indicated that the concentrations of proteasomes and their mRNA levels were consistently much higher in a variety of malignant human hematopoietic cell lines than in resting peripheral lymphocytes and monocytes from healthy adults. Proteasome expression was also greatly increased in normal blood mononuclear cells during blastogenic transformation induced by phytohemagglutinin; their expression increased in parallel with induction of DNA synthesis and returned to the basal level with progress of the cell cycle. Thus, abnormally high expression of proteasomes may play an important role in transformation and proliferation of blood cells and in specific functions of hematopoietic tumor cells. Images PMID:2205851

  18. Deciphering preferential interactions within supramolecular protein complexes: the proteasome case

    PubMed Central

    Fabre, Bertrand; Lambour, Thomas; Garrigues, Luc; Amalric, François; Vigneron, Nathalie; Menneteau, Thomas; Stella, Alexandre; Monsarrat, Bernard; Van den Eynde, Benoît; Burlet-Schiltz, Odile; Bousquet-Dubouch, Marie-Pierre

    2015-01-01

    In eukaryotic cells, intracellular protein breakdown is mainly performed by the ubiquitin–proteasome system. Proteasomes are supramolecular protein complexes formed by the association of multiple sub-complexes and interacting proteins. Therefore, they exhibit a very high heterogeneity whose function is still not well understood. Here, using a newly developed method based on the combination of affinity purification and protein correlation profiling associated with high-resolution mass spectrometry, we comprehensively characterized proteasome heterogeneity and identified previously unknown preferential associations within proteasome sub-complexes. In particular, we showed for the first time that the two main proteasome subtypes, standard proteasome and immunoproteasome, interact with a different subset of important regulators. This trend was observed in very diverse human cell types and was confirmed by changing the relative proportions of both 20S proteasome forms using interferon-γ. The new method developed here constitutes an innovative and powerful strategy that could be broadly applied for unraveling the dynamic and heterogeneous nature of other biologically relevant supramolecular protein complexes. PMID:25561571

  19. Activities of proteasome and m-calpain are essential for Chikungunya virus replication.

    PubMed

    Karpe, Yogesh A; Pingale, Kunal D; Kanade, Gayatri D

    2016-10-01

    Replication of many viruses is dependent on the ubiquitin proteasome system. The present study demonstrates that Chikungunya virus replication increases proteasome activity and induces unfolded protein response (UPR) in cultured cells. Further, it was seen that the virus replication was dependent on the activities of proteasomes and m-calpain. Proteasome inhibition induced accumulation of polyubiquitinated proteins and earlier visualization of UPR. PMID:27206501

  20. Modulation of autophagy and ubiquitin-proteasome pathways during ultra-endurance running.

    PubMed

    Jamart, Cécile; Francaux, Marc; Millet, Guillaume Y; Deldicque, Louise; Frère, Delphine; Féasson, Léonard

    2012-05-01

    In this study, the coordinated activation of ubiquitin-proteasome pathway (UPP), autophagy-lysosomal pathway (ALP), and mitochondrial remodeling including mitophagy was assessed by measuring protein markers during ultra-endurance running exercise in human skeletal muscle. Eleven male, experienced ultra-endurance athletes ran for 24 h on a treadmill. Muscle biopsy samples were taken from the vastus lateralis muscle 2 h before starting and immediately after finishing exercise. Athletes ran 149.8 ± 16.3 km with an effective running time of 18 h 42 min ( ± 41 min). The phosphorylation state of Akt (-74 ± 5%; P < 0.001), FOXO3a (-49 ± 9%; P < 0.001), mTOR Ser2448 (-32 ± 14%; P = 0.028), and 4E-BP1 (-34 ± 7%; P < 0.001) was decreased, whereas AMPK phosphorylation state increased by 247 ± 170% (P = 0.042). Proteasome β2 subunit activity increased by 95 ± 44% (P = 0.028), whereas the activities associated with the β1 and β5 subunits remained unchanged. MuRF1 protein level increased by 55 ± 26% (P = 0.034), whereas MAFbx protein and ubiquitin-conjugated protein levels did not change. LC3bII increased by 554 ± 256% (P = 0.005), and the form of ATG12 conjugated to ATG5 increased by 36 ± 17% (P = 0.042). The mitochondrial fission marker phospho-DRP1 increased by 110 ± 47% (P = 0.003), whereas the fusion marker Mfn1 and the mitophagy markers Parkin and PINK1 remained unchanged. These results fit well with a coordinated regulation of ALP and UPP triggered by FOXO3 and AMPK during ultra-endurance exercise. PMID:22345427

  1. The ubiquitin-proteasome system meets angiogenesis.

    PubMed

    Rahimi, Nader

    2012-03-01

    A strict physiological balance between endogenous proangiogenic and antiangiogenic factors controls endothelial cell functions, such that endothelial cell growth is normally restrained. However, in pathologic angiogenesis, a shift occurs in the balance of regulators, favoring endothelial growth. Much of the control of angiogenic events is instigated through hypoxia-induced VEGF expression. The ubiquitin-proteasome system (UPS) plays a central role in fine-tuning the functions of core proangiogenic proteins, including VEGF, VEGFR-2, angiogenic signaling proteins (e.g., the PLCγ1 and PI3 kinase/AKT pathways), and other non-VEGF angiogenic pathways. The emerging mechanisms by which ubiquitin modification of angiogenic proteins control angiogenesis involve both proteolytic and nonproteolytic functions. Here, I review recent advances that link the UPS to regulation of angiogenesis and highlight the potential therapeutic value of the UPS in angiogenesis-associated diseases. PMID:22357635

  2. Development of novel proteasome inhibitors based on phthalazinone scaffold.

    PubMed

    Yang, Lingfei; Wang, Wei; Sun, Qi; Xu, Fengrong; Niu, Yan; Wang, Chao; Liang, Lei; Xu, Ping

    2016-06-15

    In this study we designed a series of proteasome inhibitors using pyridazinone as initial scaffold, and extended the structure with rational design by computer aided drug design (CADD). Two different synthetic routes were explored and the biological evaluation of the phthalazinone derivatives was investigated. Most importantly, electron positive triphenylphosphine group was first introduced in the structure of proteasome inhibitors and potent inhibition was achieved. As 6c was the most potent inhibitor of proteasome, we examined the structure-activity relationship (SAR) of 6c analogs. PMID:27158142

  3. Zinc ionophores pyrithione inhibits herpes simplex virus replication through interfering with proteasome function and NF-κB activation.

    PubMed

    Qiu, Min; Chen, Yu; Chu, Ying; Song, Siwei; Yang, Na; Gao, Jie; Wu, Zhiwei

    2013-10-01

    Pyrithione (PT), known as a zinc ionophore, is effective against several pathogens from the Streptococcus and Staphylococcus genera. The antiviral activity of PT was also reported against a number of RNA viruses. In this paper, we showed that PT could effectively inhibit herpes simplex virus types 1 and 2 (HSV-1 and HSV-2). PT inhibited HSV late gene (Glycoprotein D, gD) expression and the production of viral progeny, and this action was dependent on Zn(2+). Further studies showed that PT suppressed the expression of HSV immediate early (IE) gene, the infected cell polypeptide 4 (ICP4), but had less effect on another regulatory IE protein, ICP0. It was found that PT treatment could interfere with cellular ubiquitin-proteasome system (UPS), leading to the inhibition of HSV-2-induced IκB-α degradation to inhibit NF-κB activation and enhanced promyelocytic leukemia protein (PML) stability in nucleus. However, PT did not show direct inhibition of 26S proteasome activity. Instead, it induced Zn(2+) influx, which facilitated the dysregulation of UPS and the accumulation of intracellular ubiquitin-conjugates. UPS inhibition by PT caused disruption of IκB-α degradation and NF-κB activation thus leading to marked reduction of viral titer. PMID:23867132

  4. The GST-BHMT assay reveals a distinct mechanism underlying proteasome inhibition-induced macroautophagy in mammalian cells

    PubMed Central

    Rui, Yan-Ning; Xu, Zhen; Chen, Zhihua; Zhang, Sheng

    2015-01-01

    By monitoring the fragmentation of a GST-BHMT (a protein fusion of glutathionine S-transferase N-terminal to betaine-homocysteine S-methyltransferase) reporter in lysosomes, the GST-BHMT assay has previously been established as an endpoint, cargo-based assay for starvation-induced autophagy that is largely nonselective. Here, we demonstrate that under nutrient-rich conditions, proteasome inhibition by either pharmaceutical or genetic manipulations induces similar autophagy-dependent GST-BHMT processing. However, mechanistically this proteasome inhibition-induced autophagy is different from that induced by starvation as it does not rely on regulation by MTOR (mechanistic target of rapamycin [serine/threonine kinase]) and PRKAA/AMPK (protein kinase, AMP-activated, α catalytic subunit), the upstream central sensors of cellular nutrition and energy status, but requires the presence of the cargo receptors SQSTM1/p62 (sequestosome 1) and NBR1 (neighbor of BRCA1 gene 1) that are normally involved in the selective autophagy pathway. Further, it depends on ER (endoplasmic reticulum) stress signaling, in particular ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) and its main downstream effector MAPK8/JNK1 (mitogen-activated protein kinase 8), but not XBP1 (X-box binding protein 1), by regulating the phosphorylation-dependent disassociation of BCL2 (B-cell CLL/lymphoma 2) from BECN1 (Beclin 1, autophagy related). Moreover, the multimerization domain of GST-BHMT is required for its processing in response to proteasome inhibition, in contrast to its dispensable role in starvation-induced processing. Together, these findings support a model in which under nutrient-rich conditions, proteasome inactivation induces autophagy-dependent processing of the GST-BHMT reporter through a distinct mechanism that bears notable similarity with the yeast Cvt (cytoplasm-to-vacuole targeting) pathway, and suggest the GST-BHMT reporter might be employed as a convenient assay to study

  5. The Ubiquitin-Proteasome System: Potential Therapeutic Targets for Alzheimer’s Disease and Spinal Cord Injury

    PubMed Central

    Gong, Bing; Radulovic, Miroslav; Figueiredo-Pereira, Maria E.; Cardozo, Christopher

    2016-01-01

    The ubiquitin-proteasome system (UPS) is a crucial protein degradation system in eukaryotes. Herein, we will review advances in the understanding of the role of several proteins of the UPS in Alzheimer’s disease (AD) and functional recovery after spinal cord injury (SCI). The UPS consists of many factors that include E3 ubiquitin ligases, ubiquitin hydrolases, ubiquitin and ubiquitin-like molecules, and the proteasome itself. An extensive body of work links UPS dysfunction with AD pathogenesis and progression. More recently, the UPS has been shown to have vital roles in recovery of function after SCI. The ubiquitin hydrolase (Uch-L1) has been proposed to increase cellular levels of mono-ubiquitin and hence to increase rates of protein turnover by the UPS. A low Uch-L1 level has been linked with Aβ accumulation in AD and reduced neuroregeneration after SCI. One likely mechanism for these beneficial effects of Uch-L1 is reduced turnover of the PKA regulatory subunit and consequently, reduced signaling via CREB. The neuron-specific F-box protein Fbx2 ubiquitinates β-secretase thus targeting it for proteasomal degradation and reducing generation of Aβ. Both Uch-L1 and Fbx2 improve synaptic plasticity and cognitive function in mouse AD models. The role of Fbx2 after SCI has not been examined, but abolishing ß-secretase reduces neuronal recovery after SCI, associated with reduced myelination. UBB+1, which arises through a frame-shift mutation in the ubiquitin gene that adds 19 amino acids to the C-terminus of ubiquitin, inhibits proteasomal function and is associated with increased neurofibrillary tangles in patients with AD, Pick’s disease and Down’s syndrome. These advances in understanding of the roles of the UPS in AD and SCI raise new questions but, also, identify attractive and exciting targets for potential, future therapeutic interventions. PMID:26858599

  6. Conserved Sequence Preferences Contribute to Substrate Recognition by the Proteasome*

    PubMed Central

    Yu, Houqing; Singh Gautam, Amit K.; Wilmington, Shameika R.; Wylie, Dennis; Martinez-Fonts, Kirby; Kago, Grace; Warburton, Marie; Chavali, Sreenivas; Inobe, Tomonao; Finkelstein, Ilya J.; Babu, M. Madan

    2016-01-01

    The proteasome has pronounced preferences for the amino acid sequence of its substrates at the site where it initiates degradation. Here, we report that modulating these sequences can tune the steady-state abundance of proteins over 2 orders of magnitude in cells. This is the same dynamic range as seen for inducing ubiquitination through a classic N-end rule degron. The stability and abundance of His3 constructs dictated by the initiation site affect survival of yeast cells and show that variation in proteasomal initiation can affect fitness. The proteasome's sequence preferences are linked directly to the affinity of the initiation sites to their receptor on the proteasome and are conserved between Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human cells. These findings establish that the sequence composition of unstructured initiation sites influences protein abundance in vivo in an evolutionarily conserved manner and can affect phenotype and fitness. PMID:27226608

  7. The drosophila Bcl-2 family protein Debcl is targeted to the proteasome by the β-TrCP homologue slimb.

    PubMed

    Colin, Jessie; Garibal, Julie; Clavier, Amandine; Rincheval-Arnold, Aurore; Gaumer, Sébastien; Mignotte, Bernard; Guénal, Isabelle

    2014-10-01

    The ubiquitin-proteasome system is one of the main proteolytic pathways. It inhibits apoptosis by degrading pro-apoptotic regulators, such as caspases or the tumor suppressor p53. However, it also stimulates cell death by degrading pro-survival regulators, including IAPs. In Drosophila, the control of apoptosis by Bcl-2 family members is poorly documented. Using a genetic modifier screen designed to identify regulators of mammalian bax-induced apoptosis in Drosophila, we identified the ubiquitin activating enzyme Uba1 as a suppressor of bax-induced cell death. We then demonstrated that Uba1 also regulates apoptosis induced by Debcl, the only counterpart of Bax in Drosophila. Furthermore, we show that these apoptotic processes involve the same multimeric E3 ligase-an SCF complex consisting of three common subunits and a substrate-recognition variable subunit identified in these processes as the Slimb F-box protein. Thus, Drosophila Slimb, the homologue of β-TrCP targets Bax and Debcl to the proteasome. These new results shed light on a new aspect of the regulation of apoptosis in fruitfly that identifies the first regulation of a Drosophila member of the Bcl-2 family. PMID:25208640

  8. A single-nucleotide deletion in the POMP 5' UTR causes a transcriptional switch and altered epidermal proteasome distribution in KLICK genodermatosis.

    PubMed

    Dahlqvist, Johanna; Klar, Joakim; Tiwari, Neha; Schuster, Jens; Törmä, Hans; Badhai, Jitendra; Pujol, Ramon; van Steensel, Maurice A M; Brinkhuizen, Tjinta; Brinkhuijzen, Tjinta; Gijezen, Lieke; Chaves, Antonio; Tadini, Gianluca; Vahlquist, Anders; Dahl, Niklas

    2010-04-01

    KLICK syndrome is a rare autosomal-recessive skin disorder characterized by palmoplantar keratoderma, linear hyperkeratotic papules, and ichthyosiform scaling. In order to establish the genetic cause of this disorder, we collected DNA samples from eight European probands. Using high-density genome-wide SNP analysis, we identified a 1.5 Mb homozygous candidate region on chromosome 13q. Sequence analysis of the ten annotated genes in the candidate region revealed homozygosity for a single-nucleotide deletion at position c.-95 in the proteasome maturation protein (POMP) gene, in all probands. The deletion is included in POMP transcript variants with long 5' untranslated regions (UTRs) and was associated with a marked increase of these transcript variants in keratinocytes from KLICK patients. POMP is a ubiquitously expressed protein and functions as a chaperone for proteasome maturation. Immunohistochemical analysis of skin biopsies from KLICK patients revealed an altered epidermal distribution of POMP, the proteasome subunit proteins alpha 7 and beta 5, and the ER stress marker CHOP. Our results suggest that KLICK syndrome is caused by a single-nucleotide deletion in the 5' UTR of POMP resulting in altered distribution of POMP in epidermis and a perturbed formation of the outermost layers of the skin. These findings imply that the proteasome has a prominent role in the terminal differentiation of human epidermis. PMID:20226437

  9. Quantitative Proteomic Analysis Revealed 4-(methylnitrosamino)-1-(3-pyridinyl)-1-butanone-induced Up-regulation of 20S Proteasome in Cultured Human Fibroblast Cells

    PubMed Central

    Prins, John M.; Wang, Yinsheng

    2012-01-01

    The tobacco-specific N-nitrosamine, 4-(methylnitrosamino)-1-(3-pyridinyl)-1-butanone (NNK), is a well-known carcinogen. Although the ability of the metabolically activated form of NNK to generate DNA adducts is well established, little is known about the cellular pathways perturbed by NNK in its native state. In this study, we utilized stable isotope labeling by amino acid in cell culture (SILAC), together with mass spectrometry, to assess the perturbation of protein expression in GM00637 human skin fibroblast cells upon NNK exposure. With this approach, we were able to quantify 1412 proteins and 137 of them were with significantly altered expression following NNK exposure, including the up-regulation of all subunits of the 20S proteasome core complex. The up-regulation of the 20S core complex was also reflected by a significant increase in 20S proteasome activities in GM00637, IMR90 and MCF-7 cells upon NNK treatment. Furthermore, the β-adrenergic receptor (β-AR) antagonist propranolol could attenuate significantly the NNK-induced increase in proteasome activity in all the three cell lines, suggesting that up-regulation of the 20S proteasome may be mediated through the β-AR. Additionally, we found that NNK treatment altered the expression levels of other important proteins including mitochondrial proteins, cytoskeleton-associated proteins, and proteins involved in glycolysis and gluconeogenesis. Results from the present study provided novel insights into the cellular mechanisms targeted by NNK. PMID:22369695

  10. A spliced antigenic peptide comprising a single spliced amino acid is produced in the proteasome by reverse splicing of a longer peptide fragment followed by trimming.

    PubMed

    Michaux, Alexandre; Larrieu, Pierre; Stroobant, Vincent; Fonteneau, Jean-François; Jotereau, Francine; Van den Eynde, Benoît J; Moreau-Aubry, Agnès; Vigneron, Nathalie

    2014-02-15

    Peptide splicing is a novel mechanism of production of peptides relying on the proteasome and involving the linkage of fragments originally distant in the parental protein. Peptides produced by splicing can be presented on class I molecules of the MHC and recognized by CTLs. In this study, we describe a new antigenic peptide, which is presented by HLA-A3 and comprises two noncontiguous fragments of the melanoma differentiation Ag gp100(PMEL17) spliced together in the reverse order to that in which they appear in the parental protein. Contrary to the previously described spliced peptides, which are produced by the association of fragments of 3-6 aa, the peptide described in this work results from the ultimate association of an 8-aa fragment with a single arginine residue. As described before, peptide splicing takes place in the proteasome by transpeptidation involving an acyl-enzyme intermediate linking one of the peptide fragment to a catalytic subunit of the proteasome. Interestingly, we observe that the peptide causing the nucleophilic attack on the acyl-enzyme intermediate must be at least 3 aa long to give rise to a spliced peptide. The spliced peptide produced from this reaction therefore bears an extended C terminus that needs to be further trimmed to produce the final antigenic peptide. We show that the proteasome is able to perform the final trimming step required to produce the antigenic peptide described in this work. PMID:24453253

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

    NASA Technical Reports Server (NTRS)

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

    1995-01-01

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

  12. Rational Design of Proteasome Inhibitors as Antimalarial Drugs.

    PubMed

    Le Chapelain, Camille; Groll, Michael

    2016-05-23

    One life, two strategies: Crucial structural differences between the human and the Plasmodium falciparum proteasomes were recently identified. A combination of cryo-EM and functional characterization enabled the design of a selective antimalarial proteasome inhibitor that shows low toxicity in the host. When used with artemisinin, this ligand offers a new approach for the efficient treatment of malaria at all stages of the parasite lifecycle. PMID:27079849

  13. Dynamic Association of Proteasomal Machinery with the Centrosome

    PubMed Central

    Christian Wigley, W.; Fabunmi, Rosalind P.; Lee, Min Goo; Marino, Christopher R.; Muallem, Shmuel; DeMartino, George N.; Thomas, Philip J.

    1999-01-01

    Although the number of pathologies known to arise from the inappropriate folding of proteins continues to grow, mechanisms underlying the recognition and ultimate disposition of misfolded polypeptides remain obscure. For example, how and where such substrates are identified and processed is unknown. We report here the identification of a specific subcellular structure in which, under basal conditions, the 20S proteasome, the PA700 and PA28 (700- and 180-kD proteasome activator complexes, respectively), ubiquitin, Hsp70 and Hsp90 (70- and 90-kD heat shock protein, respectively) concentrate in HEK 293 and HeLa cells. The structure is perinuclear, surrounded by endoplasmic reticulum, adjacent to the Golgi, and colocalizes with γ-tubulin, an established centrosomal marker. Density gradient fractions containing purified centrosomes are enriched in proteasomal components and cell stress chaperones. The centrosome-associated structure enlarges in response to inhibition of proteasome activity and the level of misfolded proteins. For example, folding mutants of CFTR form large inclusions which arise from the centrosome upon inhibition of proteasome activity. At high levels of misfolded protein, the structure not only expands but also extensively recruits the cytosolic pools of ubiquitin, Hsp70, PA700, PA28, and the 20S proteasome. Thus, the centrosome may act as a scaffold, which concentrates and recruits the systems which act as censors and modulators of the balance between folding, aggregation, and degradation. PMID:10225950

  14. Dynamic association of proteasomal machinery with the centrosome.

    PubMed

    Wigley, W C; Fabunmi, R P; Lee, M G; Marino, C R; Muallem, S; DeMartino, G N; Thomas, P J

    1999-05-01

    Although the number of pathologies known to arise from the inappropriate folding of proteins continues to grow, mechanisms underlying the recognition and ultimate disposition of misfolded polypeptides remain obscure. For example, how and where such substrates are identified and processed is unknown. We report here the identification of a specific subcellular structure in which, under basal conditions, the 20S proteasome, the PA700 and PA28 (700- and 180-kD proteasome activator complexes, respectively), ubiquitin, Hsp70 and Hsp90 (70- and 90-kD heat shock protein, respectively) concentrate in HEK 293 and HeLa cells. The structure is perinuclear, surrounded by endoplasmic reticulum, adjacent to the Golgi, and colocalizes with gamma-tubulin, an established centrosomal marker. Density gradient fractions containing purified centrosomes are enriched in proteasomal components and cell stress chaperones. The centrosome-associated structure enlarges in response to inhibition of proteasome activity and the level of misfolded proteins. For example, folding mutants of CFTR form large inclusions which arise from the centrosome upon inhibition of proteasome activity. At high levels of misfolded protein, the structure not only expands but also extensively recruits the cytosolic pools of ubiquitin, Hsp70, PA700, PA28, and the 20S proteasome. Thus, the centrosome may act as a scaffold, which concentrates and recruits the systems which act as censors and modulators of the balance between folding, aggregation, and degradation. PMID:10225950

  15. Proteasome dysfunction induces muscle growth defects and protein aggregation

    PubMed Central

    Kitajima, Yasuo; Tashiro, Yoshitaka; Suzuki, Naoki; Warita, Hitoshi; Kato, Masaaki; Tateyama, Maki; Ando, Risa; Izumi, Rumiko; Yamazaki, Maya; Abe, Manabu; Sakimura, Kenji; Ito, Hidefumi; Urushitani, Makoto; Nagatomi, Ryoichi; Takahashi, Ryosuke; Aoki, Masashi

    2014-01-01

    ABSTRACT The ubiquitin–proteasome and autophagy–lysosome pathways are the two major routes of protein and organelle clearance. The role of the proteasome pathway in mammalian muscle has not been examined in vivo. In this study, we report that the muscle-specific deletion of a crucial proteasomal gene, Rpt3 (also known as Psmc4), resulted in profound muscle growth defects and a decrease in force production in mice. Specifically, developing muscles in conditional Rpt3-knockout animals showed dysregulated proteasomal activity. The autophagy pathway was upregulated, but the process of autophagosome formation was impaired. A microscopic analysis revealed the accumulation of basophilic inclusions and disorganization of the sarcomeres in young adult mice. Our results suggest that appropriate proteasomal activity is important for muscle growth and for maintaining myofiber integrity in collaboration with autophagy pathways. The deletion of a component of the proteasome complex contributed to myofiber degeneration and weakness in muscle disorders that are characterized by the accumulation of abnormal inclusions. PMID:25380823

  16. Ziram Causes Dopaminergic Cell Damage by Inhibiting E1 Ligase of the Proteasome*

    PubMed Central

    Chou, Arthur P.; Maidment, Nigel; Klintenberg, Rebecka; Casida, John E.; Li, Sharon; Fitzmaurice, Arthur G.; Fernagut, Pierre-Olivier; Mortazavi, Farzad; Chesselet, Marie-Francoise; Bronstein, Jeff M.

    2008-01-01

    The etiology of Parkinson disease (PD) is unclear but may involve environmental toxins such as pesticides leading to dysfunction of the ubiquitin proteasome system (UPS). Here, we measured the relative toxicity of ziram (a UPS inhibitor) and analogs to dopaminergic neurons and examined the mechanism of cell death. UPS (26 S) activity was measured in cell lines after exposure to ziram and related compounds. Dimethyl- and diethyldithiocarbamates including ziram were potent UPS inhibitors. Primary ventral mesencephalic cultures were exposed to ziram, and cell toxicity was assessed by staining for tyrosine hydroxylase (TH) and NeuN antigen. Ziram caused a preferential damage to TH+ neurons and elevated α-synuclein levels but did not increase aggregate formation. Mechanistically, ziram altered UPS function through interfering with the targeting of substrates by inhibiting ubiquitin E1 ligase. Sodium dimethyldithiocarbamate administered to mice for 2 weeks resulted in persistent motor deficits and a mild reduction in striatal TH staining but no nigral cell loss. These results demonstrate that ziram causes selective dopaminergic cell damage in vitro by inhibiting an important degradative pathway implicated in the etiology of PD. Chronic exposure to widely used dithiocarbamate fungicides may contribute to the development of PD, and elucidation of its mechanism would identify a new potential therapeutic target. PMID:18818210

  17. Ubiquitin–Proteasome-dependent Degradation of a Mitofusin, a Critical Regulator of Mitochondrial Fusion

    PubMed Central

    Cohen, Mickael M.J.; Leboucher, Guillaume P.; Livnat-Levanon, Nurit

    2008-01-01

    The mitochondrion is a dynamic membranous network whose morphology is conditioned by the equilibrium between ongoing fusion and fission of mitochondrial membranes. In the budding yeast, Saccharomyces cerevisiae, the transmembrane GTPase Fzo1p controls fusion of mitochondrial outer membranes. Deletion or overexpression of Fzo1p have both been shown to alter the mitochondrial fusion process indicating that maintenance of steady-state levels of Fzo1p are required for efficient mitochondrial fusion. Cellular levels of Fzo1p are regulated through degradation of Fzo1p by the F-box protein Mdm30p. How Mdm30p promotes degradation of Fzo1p is currently unknown. We have now determined that during vegetative growth Mdm30p mediates ubiquitylation of Fzo1p and that degradation of Fzo1p is an ubiquitin-proteasome–dependent process. In vivo, Mdm30p associates through its F-box motif with other core components of Skp1-Cullin-F-box (SCF) ubiquitin ligases. We show that the resulting SCFMdm30p ligase promotes ubiquitylation of Fzo1p at mitochondria and its subsequent degradation by the 26S proteasome. These results provide the first demonstration that a cytosolic ubiquitin ligase targets a critical regulatory molecule at the mitochondrial outer membrane. This study provides a framework for developing an understanding of the function of Mdm30p-mediated Fzo1p degradation in the multistep process of mitochondrial fusion. PMID:18353967

  18. Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome

    PubMed Central

    Stadelmann, Britta; Birkestedt, Sandra; Hellman, Ulf; Svärd, Staffan G.

    2012-01-01

    In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes. PMID:22611020

  19. A selective inhibitor of the immunoproteasome subunit LMP2 induces apoptosis in PC-3 cells and suppresses tumour growth in nude mice

    PubMed Central

    Wehenkel, M; Ban, J-O; Ho, Y-K; Carmony, K C; Hong, J T; Kim, K B

    2012-01-01

    Background: Although the proteasome is a validated anticancer target, the clinical application of its inhibitors has been limited because of inherent systemic toxicity. To broaden clinical utility of proteasome inhibitors as anticancer agents, it is critical to develop strategies to selectively target proteasomes in cancer cells. The immunoproteasome is an alternative form of the constitutive proteasome that is expressed at high levels in cancer tissues, but not in most normal cells in the body. Methods: To validate the immunoproteasome as a chemotherapeutic target, an immunoproteasome catalytic subunit LMP2-targeting inhibitor and siRNA were used. The sensitivity of PC-3 prostate cancer cells to these reagents was investigated using viability assays. Further, a xenograft model of prostate cancer was studied to test the in vivo effects of LMP2 inhibition. Results: A small molecule inhibitor of the immunoproteasome subunit LMP2, UK-101, induced apoptosis of PC-3 cells and resulted in significant inhibition (∼50–60%) of tumour growth in vivo. Interestingly, UK-101 did not block degradation of IκBα in PC-3 cells treated with TNF-α, suggesting that its mode of action may be different from that of general proteasome inhibitors, such as bortezomib, which block IκBα degradation. Conclusion: These results strongly suggest that the immunoproteasome has important roles in cancer cell growth and thus provide a rationale for targeting the immunoproteasome in the treatment of prostate cancer. PMID:22677907

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26215701

  1. From Bortezomib to other Inhibitors of the Proteasome and Beyond

    PubMed Central

    Buac, Daniela; Shen, Min; Schmitt, Sara; Kona, Fathima Rani; Deshmukh, Rahul; Zhang, Zhen; Neslund-Dudas, Christine; Mitra, Bharati; Dou, Q. Ping

    2013-01-01

    The cancer drug discovery field has placed much emphasis on the identification of novel and cancer-specific molecular targets. A rich source of such targets for the design of novel anti-tumor agents is the ubiqutin-proteasome system (UP-S), a tightly regulated, highly specific pathway responsible for the vast majority of protein turnover within the cell. Because of its critical role in almost all cell processes that ensure normal cellular function, its inhibition at one point in time was deemed non-specific and therefore not worth further investigation as a molecular drug target. However, today the proteasome is one of the most promising anti-cancer drug targets of the century. The discovery that tumor cells are in fact more sensitive to proteasome inhibitors than normal cells indeed paved the way for the design of its inhibitors. Such efforts have led to bortezomib, the first FDA approved proteasome inhibitor now used as a frontline treatment for newly diagnosed multiple myeloma (MM), relapsed/refractory MM and mantle cell lymphoma. Though successful in improving clinical outcomes for patients with hematological malignancies, relapse often occurs in those who initially responded to bortezomib. Therefore, the acquisition of bortezomib resistance is a major issue with its therapy. Furthermore, some neuro-toxicities have been associated with bortezomib treatment and its efficacy in solid tumors is lacking. These observations have encouraged researchers to pursue the next generation of proteasome inhibitors, which would ideally overcome bortezomib resistance, have reduced toxicities and a broader range of anti-cancer activity. This review summarizes the success and limitations of bortezomib, and describes recent advances in the field, including, and most notably, the most recent FDA approval of carfilzomib in July, 2012, a second generation proteasome inhibitor. Other proteasome inhibitors currently in clinical trials and those that are currently experimental grade

  2. Oxidative Stress-Mediated Regulation of Proteasome Complexes*

    PubMed Central

    Aiken, Charity T.; Kaake, Robyn M.; Wang, Xiaorong; Huang, Lan

    2011-01-01

    Oxidative stress has been implicated in aging and many human diseases, notably neurodegenerative disorders and various cancers. The reactive oxygen species that are generated by aerobic metabolism and environmental stressors can chemically modify proteins and alter their biological functions. Cells possess protein repair pathways to rescue oxidized proteins and restore their functions. If these repair processes fail, oxidized proteins may become cytotoxic. Cell homeostasis and viability are therefore dependent on the removal of oxidatively damaged proteins. Numerous studies have demonstrated that the proteasome plays a pivotal role in the selective recognition and degradation of oxidized proteins. Despite extensive research, oxidative stress-triggered regulation of proteasome complexes remains poorly defined. Better understanding of molecular mechanisms underlying proteasome function in response to oxidative stress will provide a basis for developing new strategies aimed at improving cell viability and recovery as well as attenuating oxidation-induced cytotoxicity associated with aging and disease. Here we highlight recent advances in the understanding of proteasome structure and function during oxidative stress and describe how cells cope with oxidative stress through proteasome-dependent degradation pathways. PMID:21543789

  3. Proteasome Inhibitors in the Treatment of Multiple Myeloma

    PubMed Central

    Shah, Jatin J.; Orlowski, Robert Z.

    2016-01-01

    Targeting intracellular protein turnover by inhibiting the ubiquitin-proteasome pathway as a strategy for cancer therapy is a new addition to our chemotherapeutic armamentarium, and has seen its greatest successes against multiple myeloma. The first-in-class proteasome inhibitor bortezomib was initially approved for treatment of patients in the relapsed/refractory setting as a single agent, and was recently shown to induce even greater benefits as part of rationally-designed combinations that overcome chemoresistance. Modulation of proteasome function is also a rational approach to achieve chemosensitization to other anti-myeloma agents, and bortezomib has now been incorporated into the front-line setting. Bortezomib-based induction regimens are able to achieve higher overall response rates and response qualities than was the case with prior standards of care, and unlike these older approaches, maintain efficacy in patients with clinically- and molecularly-defined high-risk disease. Second-generation proteasome inhibitors with novel properties, such as NPI-0052 and carfilzomib, are entering the clinical arena, and showing evidence of anti-myeloma activity. In this spotlight review, we provide an overview of the current state of the art use of bortezomib and other proteasome inhibitors against multiple myeloma, and highlight areas for future study that will further optimize our ability to benefit patients with this disease. PMID:19741722

  4. Proteasome inhibition: a new anti-inflammatory strategy.

    PubMed

    Elliott, Peter J; Zollner, Thomas Matthias; Boehncke, Wolf-Henning

    2003-04-01

    The ubiquitin-proteasome pathway has a central role in the selective degradation of intracellular proteins. Among the key proteins modulated by the proteasome are those involved in the control of inflammatory processes, cell cycle regulation, and gene expression. Consequently proteasome inhibition is a potential treatment option for cancer and inflammatory conditions. Thus far, proof of principle has been obtained from studies in numerous animal models for a variety of human diseases including cancer, reperfusion injury, and inflammatory conditions such as rheumatoid arthritis, asthma, multiple sclerosis, and psoriasis. Two proteasome inhibitors, each representing a unique chemical class, are currently under clinical evaluation. Velcade (PS-341) is currently being evaluated in multiple phase II clinical trials for several solid tumor indications and has just entered a phase III trial for multiple myeloma. PS-519, representing another class of inhibitors, focuses on the inflammatory events following ischemia and reperfusion injury. Since proteasome inhibitors exhibit anti-inflammatory and antiproliferative effects, diseases characterized by both of these processes simultaneously, as is the case in rheumatoid arthritis or psoriasis, might also represent clinical opportunities for such drugs. PMID:12700891

  5. Proteasome Inhibition Suppresses Dengue Virus Egress in Antibody Dependent Infection

    PubMed Central

    Costa, Vivian V.; Tan, Hwee Cheng; Horrevorts, Sophie; Ooi, Eng Eong

    2015-01-01

    The mosquito-borne dengue virus (DENV) is a cause of significant global health burden, with an estimated 390 million infections occurring annually. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP) to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined. We show here that proteasome inhibition decouples infectious virus production from viral RNA replication in antibody-dependent infection of THP-1 cells. Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress. Intriguingly, the licensed proteasome inhibitor, bortezomib, is able to inhibit DENV titers at low nanomolar drug concentrations for different strains of all four serotypes of DENV in primary monocytes. Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes. Our findings suggest that preventing DENV egress through proteasome inhibition could be a suitable therapeutic strategy against dengue. PMID:26565697

  6. Proteasome Inhibition Suppresses Dengue Virus Egress in Antibody Dependent Infection.

    PubMed

    Choy, Milly M; Zhang, Summer L; Costa, Vivian V; Tan, Hwee Cheng; Horrevorts, Sophie; Ooi, Eng Eong

    2015-11-01

    The mosquito-borne dengue virus (DENV) is a cause of significant global health burden, with an estimated 390 million infections occurring annually. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP) to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined. We show here that proteasome inhibition decouples infectious virus production from viral RNA replication in antibody-dependent infection of THP-1 cells. Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress. Intriguingly, the licensed proteasome inhibitor, bortezomib, is able to inhibit DENV titers at low nanomolar drug concentrations for different strains of all four serotypes of DENV in primary monocytes. Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes. Our findings suggest that preventing DENV egress through proteasome inhibition could be a suitable therapeutic strategy against dengue. PMID:26565697

  7. New orally active proteasome inhibitors in multiple myeloma.

    PubMed

    Allegra, Alessandro; Alonci, Andrea; Gerace, Demetrio; Russo, Sabina; Innao, Vanessa; Calabrò, Laura; Musolino, Caterina

    2014-01-01

    Bortezomib is the first proteasome inhibitor approved for the therapy of multiple myeloma (MM). Although Bortezomib has renovated the treatment of MM, a considerable proportion of subjects fail to respond to Bortezomib treatment and almost all patients relapse from this drug either alone or when used in combination therapies. However, the good clinical outcome of Bortezomib treatment in MM patients gave impulsion for the development of second generation proteasome inhibitors with the ambition of improving efficacy of proteasome inhibition, enhancing antitumor activity, and decreasing toxicity, as well as providing flexible dosing schedules and patient convenience. This review provides an overview of the role of oral proteasome inhibitors including Marizomib, Oprozomib, Delanzomib, chemical proteasome inhibitors, and cinnabaramides, in the therapy of MM, focusing on developments over the past five years. These emerging drugs with different mechanisms of action have exhibited promising antitumor activity in patients with relapsed/refractory MM, and they are creating chances to target multiple pathways, overcome resistance, and improve clinical outcomes, mainly for those subjects who are refractory to approved agents. Future steps in the clinical development of oral inhibitors include the optimization of the schedule and the definition of their antitumor activity in MM. PMID:24239172

  8. Quiescent fibroblasts are protected from proteasome inhibition–mediated toxicity

    PubMed Central

    Legesse-Miller, Aster; Raitman, Irene; Haley, Erin M.; Liao, Albert; Sun, Lova L.; Wang, David J.; Krishnan, Nithya; Lemons, Johanna M. S.; Suh, Eric J.; Johnson, Elizabeth L.; Lund, Benjamin A.; Coller, Hilary A.

    2012-01-01

    Proteasome inhibition is used as a treatment strategy for multiple types of cancers. Although proteasome inhibition can induce apoptotic cell death in actively proliferating cells, it is less effective in quiescent cells. In this study, we used primary human fibroblasts as a model system to explore the link between the proliferative state of a cell and proteasome inhibition–mediated cell death. We found that proliferating and quiescent fibroblasts have strikingly different responses to MG132, a proteasome inhibitor; proliferating cells rapidly apoptosed, whereas quiescent cells maintained viability. Moreover, MG132 treatment of proliferating fibroblasts led to increased superoxide anion levels, juxtanuclear accumulation of ubiquitin- and p62/SQSTM1-positive protein aggregates, and apoptotic cell death, whereas MG132-treated quiescent cells displayed fewer juxtanuclear protein aggregates, less apoptosis, and higher levels of mitochondrial superoxide dismutase. In both cell states, reducing reactive oxygen species with N-acetylcysteine lessened protein aggregation and decreased apoptosis, suggesting that protein aggregation promotes apoptosis. In contrast, increasing cellular superoxide levels with 2-methoxyestradiol treatment or inhibition of autophagy/lysosomal pathways with bafilomycin A1 sensitized serum-starved quiescent cells to MG132-induced apoptosis. Thus, antioxidant defenses and the autophagy/lysosomal pathway protect serum-starved quiescent fibroblasts from proteasome inhibition–induced cytotoxicity. PMID:22875985

  9. Targeting Tumor Ubiquitin-Proteasome Pathway with Polyphenols for Chemosensitization

    PubMed Central

    Shen, Min; Chan, Tak Hang; Dou, Q. Ping

    2012-01-01

    The development of tumor drug resistance is one of the biggest obstacles on the way to achieve a favorable outcome of chemotherapy. Among various strategies that have been explored to overcome drug resistance, the combination of current chemotherapy with plant polyphenols as a chemosensitizer has emerged as a promising one. Plant polyphenols are a group of phytochemicals characterized by the presence of more than one phenolic group. Mechanistic studies suggest that polyphenols have multiple intracellular targets, one of which is the proteasome complex. The proteasome is a proteolytic enzyme complex responsible for intracellular protein degradation and has been shown to play an important role in tumor growth and the development of drug resistance. Therefore, proteasome inhibition by plant polyphenols could be one of the mechanisms contributing to their chemosensitizing effect. Plant polyphenols that have been identified to possess proteasome-inhibitory activity include (−)-epigallocatechins-3-gallate (EGCG), genistein, luteolin, apigenin, chrysin, quercetin, curcumin and tannic acid. These polyphenols have exhibited an appreciable effect on overcoming resistance to various chemotherapeutic drugs as well as multidrug resistance in a broad spectrum of tumors ranging from carcinoma and sarcoma to hematological malignances. The in vitro and in vivo studies on polyphenols with proteasome-inhibitory activity have built a solid foundation to support the idea that they could serve as a chemosensitizer for the treatment of cancer. In-depth mechanistic studies and identification of optimal regimen are needed in order to eventually translate this laboratory concept into clinical trials to actually benefit current chemotherapy. PMID:22292765

  10. The Cellular Level of PR500, a Protein Complex Related to the 19S Regulatory Particle of the Proteasome, Is Regulated in Response to Stresses in Plants

    PubMed Central

    Peng, Zhaohua; Staub, Jeffrey M.; Serino, Giovanna; Kwok, Shing F.; Kurepa, Jasmina; Bruce, Barry D.; Vierstra, Richard D.; Wei, Ning; Deng, Xing-Wang

    2001-01-01

    In Arabidopsis seedlings and cauliflower florets, Rpn6 (a proteasome non-ATPase regulatory subunit) was found in two distinct protein complexes of ∼800 and 500 kDa, respectively. The large complex likely represents the proteasome 19S regulator particle (RP) because it displays the expected subunit composition and all characteristics. The small complex, designated PR500, shares at least three subunits with the “lid” subcomplex of 19S RP and is loosely associated with an hsp70 protein. In Arabidopsis COP9 signalosome mutants, PR500 was specifically absent or reduced to an extent that correlates with the severity of the mutations. Furthermore, PR500 was also diminished in response to potential protein-misfolding stresses caused by the heat shock and canavanine treatment. Immunofluorescence studies suggest that PR500 has a distinct localization pattern and is enriched in specific nuclear foci. We propose that PR500 may be evolved in higher plants to cope with the frequently encountered environmental stresses. PMID:11179422

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

    PubMed Central

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

    2014-01-01

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

  12. The initiator caspase Dronc is subject of enhanced autophagy upon proteasome impairment in Drosophila.

    PubMed

    Lee, T V; Kamber Kaya, H E; Simin, R; Baehrecke, E H; Bergmann, A

    2016-09-01

    A major function of ubiquitylation is to deliver target proteins to the proteasome for degradation. In the apoptotic pathway in Drosophila, the inhibitor of apoptosis protein 1 (Diap1) regulates the activity of the initiator caspase Dronc (death regulator Nedd2-like caspase; caspase-9 ortholog) by ubiquitylation, supposedly targeting Dronc for degradation by the proteasome. Using a genetic approach, we show that Dronc protein fails to accumulate in epithelial cells with impaired proteasome function suggesting that it is not degraded by the proteasome, contrary to the expectation. Similarly, decreased autophagy, an alternative catabolic pathway, does not result in increased Dronc protein levels. However, combined impairment of the proteasome and autophagy triggers accumulation of Dronc protein levels suggesting that autophagy compensates for the loss of the proteasome with respect to Dronc turnover. Consistently, we show that loss of the proteasome enhances endogenous autophagy in epithelial cells. We propose that enhanced autophagy degrades Dronc if proteasome function is impaired. PMID:27104928

  13. Transcriptional upregulation of BAG3 upon proteasome inhibition

    SciTech Connect

    Wang Huaqin Liu Haimei; Zhang Haiyan; Guan Yifu; Du Zhenxian

    2008-01-11

    Proteasome inhibitors exhibit antitumoral activity against malignancies of different histology. Emerging evidence indicates that antiapoptotic factors may also accumulate as a consequence of exposure to these drugs, thus it seems plausible that activation of survival signaling cascades might compromise their antitumoral effects. Bcl-2-associated athanogene (BAG) family proteins are characterized by their property of interaction with a variety of partners involved in modulating the proliferation/death balance, including heat shock proteins (HSP), Bcl-2, Raf-1. In this report, we demonstrated that BAG3 is a novel antiapoptotic molecule induced by proteasome inhibitors in various cancer cells at the transcriptional level. Moreover, we demonstrated that BAG3 knockdown by siRNA sensitized cancer cells to MG132-induced apoptosis. Taken together, our results suggest that BAG3 induction might represents as an unwanted molecular consequence of utilizing proteasome inhibitors to combat tumors.

  14. A novel proteasome inhibitor NPI-0052 as an anticancer therapy

    PubMed Central

    Chauhan, D; Hideshima, T; Anderson, K C

    2006-01-01

    Proteasome inhibitor Bortezomib/Velcade has emerged as an effective anticancer therapy for the treatment of relapsed and/or refractory multiple myeloma (MM), but prolonged treatment can be associated with toxicity and development of drug resistance. In this review, we discuss the recent discovery of a novel proteasome inhibitor, NPI-0052, that is distinct from Bortezomib in its chemical structure, mechanisms of action, and effects on proteasomal activities; most importantly, it overcomes resistance to conventional and Bortezomib therapies. In vivo studies using human MM xenografts shows that NPI-0052 is well tolerated, prolongs survival, and reduces tumour recurrence. These preclinical studies provided the basis for Phase-I clinical trial of NPI-0052 in relapsed/refractory MM patients. PMID:17047643

  15. Proteasome inhibitors - molecular basis and current perspectives in multiple myeloma.

    PubMed

    Kubiczkova, Lenka; Pour, Ludek; Sedlarikova, Lenka; Hajek, Roman; Sevcikova, Sabina

    2014-06-01

    Inhibition of proteasome, a proteolytic complex responsible for the degradation of ubiquitinated proteins, has emerged as a powerful strategy for treatment of multiple myeloma (MM), a plasma cell malignancy. First-in-class agent, bortezomib, has demonstrated great positive therapeutic efficacy in MM, both in pre-clinical and in clinical studies. However, despite its high efficiency, a large proportion of patients do not achieve sufficient clinical response. Therefore, the development of a second-generation of proteasome inhibitors (PIs) with improved pharmacological properties was needed. Recently, several of these new agents have been introduced into clinics including carfilzomib, marizomib and ixazomib. Further, new orally administered second-generation PI oprozomib is being investigated. This review provides an overview of main mechanisms of action of PIs in MM, focusing on the ongoing development and progress of novel anti-proteasome therapeutics. PMID:24712303

  16. Marchantin M: a novel inhibitor of proteasome induces autophagic cell death in prostate cancer cells

    PubMed Central

    Jiang, H; Sun, J; Xu, Q; Liu, Y; Wei, J; Young, C Y F; Yuan, H; Lou, H

    2013-01-01

    We previously reported that marchantin M (Mar) is an active agent to induce apoptosis in human prostate cancer (PCa), but the molecular mechanisms of action remain largely unknown. Here, we demonstrate that Mar potently inhibited chymotrypsin-like and peptidyl-glutamyl peptide-hydrolyzing activities of 20S proteasome both in in vitro and intracellular systems and significantly induced the accumulation of polyubiquitinated proteins in PCa cells. The computational modeling analysis suggested that Mar non-covalently bound to active sites of proteasome β5 and β1 subunits, resulting in a non-competitive inhibition. Proteasome inhibition by Mar subsequently resulted in endoplasmic reticulum (ER) stress, as evidenced by elevated glucose-regulated protein 78 and CHOP, increased phospho-eukaryotic translation initiation factor 2α (eIF2α), splicing of X-box-binding protein-1 and dilation of the ER. However, Mar-mediated cell death was not completely impaired by a pan inhibitor of caspases. Further studies revealed that the Mar-induced cell death was greatly associated with the activation of autophagy, as indicated by the significant induction of microtubule-associated protein-1 light chain-3 beta (LC3B) expression and conversion. Electron microscopic and green fluorescent protein-tagged LC3B analyses further demonstrated the ability of autophagy induction by Mar. Time kinetic studies revealed that Mar induced a rapid and highly sustained processing of LC3B in treated cells and simultaneously decreased the expression of p62/SQSTM1. Pharmacological blockade or knockdown of LC3B and Atg5 attenuated Mar-mediated cell death. The autophagic response triggered by Mar required the activation of RNA-dependent protein kinase-like ER kinase/eIF2α and suppression of the phosphatidylinositol-3 kinase/Akt/mammalian target of rapamycin axis via preventing activation and expression of Akt. Our results identified a novel mechanism for the cytotoxic effect of Mar, which strengthens it as

  17. Modifications in endopeptidase and 20S proteasome expression and activities in cadmium treated tomato (Solanum lycopersicum L.) plants.

    PubMed

    Djebali, Wahbi; Gallusci, Philippe; Polge, Cécile; Boulila, Latifa; Galtier, Nathalie; Raymond, Philippe; Chaibi, Wided; Brouquisse, Renaud

    2008-02-01

    The effects of cadmium (Cd) on cellular proteolytic responses were investigated in the roots and leaves of tomato (Solanum lycopersicum L., var Ibiza) plants. Three-week-old plants were grown for 3 and 10 days in the presence of 0.3-300 microM Cd and compared to control plants grown in the absence of Cd. Roots of Cd treated plants accumulated four to fivefold Cd as much as mature leaves. Although 10 days of culture at high Cd concentrations inhibited plant growth, tomato plants recovered and were still able to grow again after Cd removal. Tomato roots and leaves are not modified in their proteolytic response with low Cd concentrations (< or =3 microM) in the incubation medium. At higher Cd concentration, protein oxidation state and protease activities are modified in roots and leaves although in different ways. The soluble protein content of leaves decreased and protein carbonylation level increased indicative of an oxidative stress. Conversely, protein content of roots increased from 30 to 50%, but the amount of oxidized proteins decreased by two to threefold. Proteolysis responded earlier in leaves than in root to Cd stress. Additionally, whereas cysteine- and metallo-endopeptidase activities, as well as proteasome chymotrypsin activity and subunit expression level, increased in roots and leaves, serine-endopeptidase activities increased only in leaves. This contrasted response between roots and leaves may reflect differences in Cd compartmentation and/or complexation, antioxidant responses and metabolic sensitivity to Cd between plant tissues. The up-regulation of the 20S proteasome gene expression and proteolytic activity argues in favor of the involvement of the 20S proteasome in the degradation of oxidized proteins in plants. PMID:17952456

  18. The role of hypercholesterolemic diet and vitamin E on Nrf2 pathway, endoplasmic reticulum stress and proteasome activity.

    PubMed

    Bozaykut, Perinur; Sozen, Erdi; Yazgan, Burak; Karademir, Betul; Kartal-Ozer, Nesrin

    2014-10-01

    Hypercholesterolemia is the major risk factor for the development of atherosclerosis and vitamin E is suggested to have a preventive role in this process (1), although the mechanism of action still remains unclear.The ubiquitin-proteasome system (UPS) may in?uence atherosclerosis by affecting disease-relevant cellular processes such as apoptosis, proliferation, and differentiation, or by affecting cellular stress responses and/or adaptive phenomena, such as ER stress, in?ammation, and redox homeostasis (2). NF-E2-related factor 2 (Nrf2) is a transcription factor that controls the expression of phase II detoxi?cation and antioxidant genes. Nrf2 signaling has additionally been shown to upregulate the expression of the proteasome catalytic subunits (3). In the present study, we investigated the role of Nrf2 pathway on oxidative and ER stress conditions induced by cholesterol diet and the effects of vitamin E on related signaling pathways in in vivo model of atherosclerosis. All experimental procedures were approved by the Marmara University Ethics Committee. Twenty-one male albino rabbits (23 months old) were assigned randomly to four groups fed for 8 weeks: (i) vitamin E deficient diet, (ii) vitamin E deficient diet containing 2% cholesterol, and (iii) vitamin E deficient diet containing 2% cholesterol with daily intramuscular injections of vitamin E (50mg/kg), (iv) vitamin E deficient diet with daily intramuscular injections of vitamin E (50mg/kg). In order to elucidate in vivo role of oxidative stress and ER stress in cardiovascular system of hypercholesterolemic rabbits, we investigated serum levels of cholesterol, MDA and vitamin E and Nrf2, GST-1, GRP78, GRP94, PERK, IRE1 protein levels and the proteasomal activity in aortic tissues will be discussed. PMID:26461313

  19. Proteasome function shapes innate and adaptive immune responses.

    PubMed

    Kammerl, Ilona E; Meiners, Silke

    2016-08-01

    The proteasome system degrades more than 80% of intracellular proteins into small peptides. Accordingly, the proteasome is involved in many essential cellular functions, such as protein quality control, transcription, immune responses, cell signaling, and apoptosis. Moreover, degradation products are loaded onto major histocompatibility class I molecules to communicate the intracellular protein composition to the immune system. The standard 20S proteasome core complex contains three distinct catalytic active sites that are exchanged upon stimulation with inflammatory cytokines to form the so-called immunoproteasome. Immunoproteasomes are constitutively expressed in immune cells and have different proteolytic activities compared with standard proteasomes. They are rapidly induced in parenchymal cells upon intracellular pathogen infection and are crucial for priming effective CD8(+) T-cell-mediated immune responses against infected cells. Beyond shaping these adaptive immune reactions, immunoproteasomes also regulate the function of immune cells by degradation of inflammatory and immune mediators. Accordingly, they emerge as novel regulators of innate immune responses. The recently unraveled impairment of immunoproteasome function by environmental challenges and by genetic variations of immunoproteasome genes might represent a currently underestimated risk factor for the development and progression of lung diseases. In particular, immunoproteasome dysfunction will dampen resolution of infections, thereby promoting exacerbations, may foster autoimmunity in chronic lung diseases, and possibly contributes to immune evasion of tumor cells. Novel pharmacological tools, such as site-specific inhibitors of the immunoproteasome, as well as activity-based probes, however, hold promises as innovative therapeutic drugs for respiratory diseases and biomarker profiling, respectively. PMID:27343191

  20. The Ubiquitin-Proteasome Pathway and Synaptic Plasticity

    ERIC Educational Resources Information Center

    Hegde, Ashok N.

    2010-01-01

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

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

    PubMed Central

    Huang, Qian; Figueiredo-Pereira, Maria E.

    2010-01-01

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

  2. 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…

  3. PKA turnover by the REGγ-proteasome modulates FoxO1 cellular activity and VEGF-induced angiogenesis

    PubMed Central

    Liu, Shuang; Lai, Li; Zuo, Qiuhong; Dai, Fujun; Wu, Lin; Wang, Yan; Zhou, Qingxia; Liu, Jian; Liu, Jiang; Li, Lei; Lin, Qingxiang; Creighton, Chad J.; Costello, Myra Grace; Huang, Shixia; Jia, Caifeng; Liao, Lujian; Luo, Honglin; Fu, Junjiang; Liu, Mingyao; Yi, Zhengfang; Xiao, Jianru; Li, Xiaotao

    2014-01-01

    The REGγ-proteasome serves as a short-cut for the destruction of certain intact mammalian proteins in the absence of ubiquitin-and ATP. The biological roles of the proteasome activator REGγ are not completely understood. Here we demonstrate that REGγ controls degradation of protein kinase A catalytic subunit-α (PKAca) both in primary human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblast cells (MEFs). Accumulation of PKAca in REGγ-deficient HUVECs or MEFs results in phosphorylation and nuclear exclusion of the transcription factor FoxO1, indicating that REGγ is involved in preserving FoxO1 transcriptional activity. Consequently, VEGF-induced expression of the FoxO1 responsive genes, VCAM-1 and E-Selectin, was tightly controlled by REGγ in a PKA dependent manner. Functionally, REGγ is crucial for the migration of HUVECs. REGγ−/− mice display compromised VEGF-instigated neovascularization in cornea and aortic ring models. Implanted matrigel plugs containing VEGF in REGγ−/− mice induced fewer capillaries than in REGγ+/+ littermates. Taken together, our study identifies REGγ as a novel angiogenic factor that plays an important role in VEGF-induced expression of VCAM-1 and E-Selectin by antagonizing PKA signaling. Identification of the REGγ–PKA–FoxO1 pathway in endothelial cells (ECs) provides another potential target for therapeutic intervention in vascular diseases. PMID:24560667

  4. [Antiatherogenic characteristics of korvitin: effect on proteasome activity of the aorta, heart, and blood cells].

    PubMed

    Pashevin, D O; Dosenko, B Ie; Byts', Iu V; Moĭbenko, O O

    2009-01-01

    We studied the changes in proteasomal proteolisis during modelling of rabbit cholesterol-induced atherosclerosis. It was determined that in aorta the TL activity of proteasome increased 2.4-fold (P < 0.05), CTL activity increased by 43%, and PGPG--by 10%. In heart tissue it was observed the increase of CTL proteasome activity by 14%. The application of "Korvitin" (water-soluble form of quercetine) followed by considerable decrease of proteasomal activity both in tissues (aorta and heart) and leucocytes. The intensity ofatherosclerotic changes in aorta was significantly smaller. Obtained data suggest that "Korvitin" reveales angioprotective properties mediated by it effect on proteasomal proteolisis. PMID:19827630

  5. Molecular sequelae of proteasome inhibition in human multiple myeloma cells

    PubMed Central

    Mitsiades, Nicholas; Mitsiades, Constantine S.; Poulaki, Vassiliki; Chauhan, Dharminder; Fanourakis, Galinos; Gu, Xuesong; Bailey, Charles; Joseph, Marie; Libermann, Towia A.; Treon, Steven P.; Munshi, Nikhil C.; Richardson, Paul G.; Hideshima, Teru; Anderson, Kenneth C.

    2002-01-01

    The proteasome inhibitor PS-341 inhibits IκB degradation, prevents NF-κB activation, and induces apoptosis in several types of cancer cells, including chemoresistant multiple myeloma (MM) cells. PS-341 has marked clinical activity even in the setting of relapsed refractory MM. However, PS-341-induced apoptotic cascade(s) are not yet fully defined. By using gene expression profiling, we characterized the molecular sequelae of PS-341 treatment in MM cells and further focused on molecular pathways responsible for the anticancer actions of this promising agent. The transcriptional profile of PS-341-treated cells involved down-regulation of growth/survival signaling pathways, and up-regulation of molecules implicated in proapoptotic cascades (which are both consistent with the proapoptotic effect of proteasome inhibition), as well as up-regulation of heat-shock proteins and ubiquitin/proteasome pathway members (which can correspond to stress responses against proteasome inhibition). Further studies on these pathways showed that PS-341 decreases the levels of several antiapoptotic proteins and triggers a dual apoptotic pathway of mitochondrial cytochrome c release and caspase-9 activation, as well as activation of Jun kinase and a Fas/caspase-8-dependent apoptotic pathway [which is inhibited by a dominant negative (decoy) Fas construct]. Stimulation with IGF-1, as well as overexpression of Bcl-2 or constitutively active Akt in MM cells also modestly attenuates PS-341-induced cell death, whereas inhibitors of the BH3 domain of Bcl-2 family members or the heat-shock protein 90 enhance tumor cell sensitivity to proteasome inhibition. These data provide both insight into the molecular mechanisms of antitumor activity of PS-341 and the rationale for future clinical trials of PS-341, in combination with conventional and novel therapies, to improve patient outcome in MM. PMID:12391322

  6. Resistance Gene-Guided Genome Mining: Serial Promoter Exchanges in Aspergillus nidulans Reveal the Biosynthetic Pathway for Fellutamide B, a Proteasome Inhibitor.

    PubMed

    Yeh, Hsu-Hua; Ahuja, Manmeet; Chiang, Yi-Ming; Oakley, C Elizabeth; Moore, Shauna; Yoon, Olivia; Hajovsky, Heather; Bok, Jin-Woo; Keller, Nancy P; Wang, Clay C C; Oakley, Berl R

    2016-08-19

    Fungal genome projects are revealing thousands of cryptic secondary metabolism (SM) biosynthetic gene clusters that encode pathways that potentially produce valuable compounds. Heterologous expression systems should allow these clusters to be expressed and their products obtained, but approaches are needed to identify the most valuable target clusters. The inp cluster of Aspergillus nidulans contains a gene, inpE, that encodes a proteasome subunit, leading us to hypothesize that the inp cluster produces a proteasome inhibitor and inpE confers resistance to this compound. Previous efforts to express this cluster have failed, but by sequentially replacing the promoters of the genes of the cluster with a regulatable promotor, we have expressed them successfully. Expression reveals that the product of the inp cluster is the proteasome inhibitor fellutamide B, and our data allow us to propose a biosynthetic pathway for the compound. By deleting inpE and activating expression of the inp cluster, we demonstrate that inpE is required for resistance to internally produced fellutamide B. These data provide experimental validation for the hypothesis that some fungal SM clusters contain genes that encode resistant forms of the enzymes targeted by the compound produced by the cluster. PMID:27294372

  7. LDL suppresses angiogenesis through disruption of the HIF pathway via NF-κB inhibition which is reversed by the proteasome inhibitor BSc2118

    PubMed Central

    Doeppner, Thorsten R.; Niu, Feng; Li, Qiaochuan; Yang, Yanping; Kuckelkorn, Ulrike; Hagemann, Nina; Li, Wei; Hermann, Dirk M.; Dai, Yun; Zhou, Wen; Jin, Fengyan

    2015-01-01

    Since disturbance of angiogenesis predisposes to ischemic injuries, attempts to promote angiogenesis have been made to improve clinical outcomes of patients with many ischemic disorders. While hypoxia inducible factors (HIFs) stimulate vascular remodeling and angiogenesis, hyperlipidemia impairs angiogenesis in response to various pro-angiogenic factors. However, it remains uncertain how HIFs regulate angiogenesis under hyperlipidemia. Here, we report that exposure to low-density lipoprotein (LDL) suppressed in vitro angiogenesis of human brain microvascular endothelial cells. Whereas LDL exposure diminished expression of HIF-1α and HIF-2α induced by hypoxia, it inhibited DMOG- and TNFα-induced HIF-1α and HIF-2α expression in normoxia. Notably, in both hypoxia and normoxia, LDL markedly reduced expression of HIF-1β, a constitutively stable HIF subunit, an event associated with NF-κB inactivation. Moreover, knockdown of HIF-1β down-regulated HIF-1α and HIF-2α expression, in association with increased HIF-1α hydroxylation and 20S proteasome activity after LDL exposure. Significantly, the proteasome inhibitor BSc2118 prevented angiogenesis attenuation by LDL through restoring expression of HIFs. Together, these findings argue that HIF-1β might act as a novel cross-link between the HIF and NF-κB pathways in suppression of angiogenesis by LDL, while proteasome inhibitors might promote angiogenesis by reactivating this signaling cascade under hyperlipidemia. PMID:26388611

  8. Analysis of the Protein Kinase A-Regulated Proteome of Cryptococcus neoformans Identifies a Role for the Ubiquitin-Proteasome Pathway in Capsule Formation

    PubMed Central

    Geddes, J. M. H.; Caza, M.; Croll, D.; Stoynov, N.; Foster, L. J.

    2016-01-01

    ABSTRACT The opportunistic fungal pathogen Cryptococcus neoformans causes life-threatening meningitis in immunocompromised individuals. The expression of virulence factors, including capsule and melanin, is in part regulated by the cyclic-AMP/protein kinase A (cAMP/PKA) signal transduction pathway. In this study, we investigated the influence of PKA on the composition of the intracellular proteome to obtain a comprehensive understanding of the regulation that underpins virulence. Through quantitative proteomics, enrichment and bioinformatic analyses, and an interactome study, we uncovered a pattern of PKA regulation for proteins associated with translation, the proteasome, metabolism, amino acid biosynthesis, and virulence-related functions. PKA regulation of the ubiquitin-proteasome pathway in C. neoformans showed a striking parallel with connections between PKA and protein degradation in chronic neurodegenerative disorders and other human diseases. Further investigation of proteasome function with the inhibitor bortezomib revealed an impact on capsule production as well as hypersusceptibility for strains with altered expression or activity of PKA. Parallel studies with tunicamycin also linked endoplasmic reticulum stress with capsule production and PKA. Taken together, the data suggest a model whereby expression of PKA regulatory and catalytic subunits and the activation of PKA influence proteostasis and the function of the endoplasmic reticulum to control the elaboration of the polysaccharide capsule. Overall, this study revealed both broad and conserved influences of the cAMP/PKA pathway on the proteome and identified proteostasis as a potential therapeutic target for the treatment of cryptococcosis. PMID:26758180

  9. Depletion of guanine nucleotides leads to the Mdm2-dependent proteasomal degradation of nucleostemin.

    PubMed

    Huang, Min; Itahana, Koji; Zhang, Yanping; Mitchell, Beverly S

    2009-04-01

    Nucleostemin is a positive regulator of cell proliferation and is highly expressed in a variety of stem cells, tumors, and tumor cell lines. The protein shuttles between the nucleolus and the nucleus in a GTP-dependent fashion. Selective depletion of intracellular guanine nucleotides by AVN-944, an inhibitor of the de novo purine synthetic enzyme, IMP dehydrogenase, leads to the rapid disappearance of nucleostemin protein in tumor cell lines, an effect that does not occur with two other nucleolar proteins, nucleophosmin or nucleolin. Endogenous nucleostemin protein is completely stabilized by MG132, an inhibitor of the 26S proteasome, as are the levels of expressed enhanced green fluorescent protein-tagged nucleostemin, both wild-type protein and protein containing mutations at the G(1) GTP binding site. Nutlin-3a, a small molecule that disrupts the binding of the E3 ubiquitin ligase, Mdm2, to p53, stabilizes nucleostemin protein in the face of guanine nucleotide depletion, as does siRNA-mediated knockdown of Mdm2 expression and overexpression of a dominant-negative form of Mdm2. Neither Doxorubicin nor Actinomycin D, which cause the release of nucleostemin from the nucleolus, results in nucleostemin degradation. We conclude that nucleostemin is a target for Mdm2-mediated ubiquitination and degradation when not bound to GTP. Because this effect does not occur with other chemotherapeutic agents, the induction of nucleostemin protein degradation in tumor cells by IMP dehydrogenase inhibition or by other small molecules that disrupt GTP binding may offer a new approach to the treatment of certain neoplastic diseases. PMID:19318567

  10. Chemical and biological evaluation of dipeptidyl boronic acid proteasome inhibitors for use in prodrugs and pro-soft drugs targeting solid tumors.

    PubMed

    Milo, Lawrence J; Lai, Jack H; Wu, Wengen; Liu, Yuxin; Maw, Hlaing; Li, Youhua; Jin, Zhiping; Shu, Ying; Poplawski, Sarah E; Wu, Yong; Sanford, David G; Sudmeier, James L; Bachovchin, William W

    2011-07-14

    Bortezomib, a dipeptidyl boronic acid and potent inhibitor of the 26S proteasome, is remarkably effective against multiple myeloma (MM) but not against solid tumors. Dose-limiting adverse effects from "on target" inhibition of the proteasome in normal cells and tissues appear to be a key obstacle. Achieving efficacy against solid tumors therefore is likely to require making the inhibitor more selective for tumor tissue over normal tissues. The simplest strategy that might provide such tissue specificity would be to employ a tumor specific protease to release an inhibitor from a larger, noninhibitory structure. However, such release would necessarily generate an inhibitor with a free N-terminal amino group, raising a key question: Can short peptide boronic acids with N-terminal amino groups have the requisite properties to serve as warheads in prodrugs? Here we show that dipeptides of boroLeu, the smallest plausible candidates for the task, can indeed be sufficiently potent, cell-penetrating, cytotoxic, and stable to degradation by cellular peptidases to serve in this capacity. PMID:21634429

  11. Dopamine or biopterin deficiency potentiates phosphorylation at (40)Ser and ubiquitination of tyrosine hydroxylase to be degraded by the ubiquitin proteasome system.

    PubMed

    Kawahata, Ichiro; Ohtaku, Shiori; Tomioka, Yoshihisa; Ichinose, Hiroshi; Yamakuni, Tohru

    2015-09-11

    The protein amount of tyrosine hydroxylase (TH), that is the rate-limiting enzyme for the biosynthesis of dopamine (DA), should be tightly regulated, whereas its degradation pathway is largely unknown. In this study, we analyzed how the TH protein is chemically modified and subsequently degraded under deficiencies of DA and tetrahydrobiopterin (BH4), a cofactor for TH, by using pharmacological agents in PC12D cells and cultured mesencephalic neurons. When inhibition of DA- or BH4-synthesizing enzymes greatly reduced the DA contents in PC12D cells, a marked and persistent increase in phosphorylated TH at (40)Ser (p40-TH) was concomitantly observed. This phosphorylation was mediated by D2 dopamine auto-receptor and cAMP-dependent protein kinase (PKA). Our immunoprecipitation experiments showed that the increase in the p40-TH level was accompanied with its poly-ubiquitination. Treatment of PC12D cells with cycloheximide showed that total-TH protein level was reduced by the DA- or BH4-depletion. Notably, this reduction in the total-TH protein level was sensitive not only to a 26S proteasomal inhibitor, MG-132, but also to a PKA inhibitor, H-89. These data demonstrated that DA deficiency should induce compensatory activation of TH via phosphorylation at (40)Ser through D2-autoreceptor and PKA-mediated pathways, which in turn give a rise to its degradation through an ubiquitin-proteasome pathway, resulting in a negative spiral of DA production when DA deficiency persists. PMID:26225746

  12. Gene therapy by proteasome activator, PA28γ, improves motor coordination and proteasome function in Huntington's disease YAC128 mice.

    PubMed

    Jeon, J; Kim, W; Jang, J; Isacson, O; Seo, H

    2016-06-01

    Huntington's disease (HD) is neurologically characterized by involuntary movements, associated with degeneration of the medium-sized spiny neurons (MSNs) and ubiquitin-positive neuronal intranuclear inclusions (NIIs). It has been reported that the proteolytic activities of the ubiquitin-proteasome system (UPS) are generally inhibited in HD patient's brain. We previously discovered that a proteasome activator (PA), PA28γ enhances proteasome activities and cell survival in in vitro HD model. In this study, we aimed to find whether PA28γ gene transfer improves the proteasome activities and pathological symptoms in in vivo HD model. We stereotaxically injected lenti-PA28γ virus into the striatum of mutant (MT) YAC128 HD mice and littermate (LM) controls at 14-18months of age, and validated their behavioral and biochemical changes at 12weeks after the injection. YAC128 mice showed a significant increase in their peptidyl-glutamyl preferring hydrolytic (PGPH) proteasome activity and the mRNA or protein levels of brain-derived neurotrophic factor (BDNF) and pro-BDNF after lenti-PA28γ injection. The number of ubiquitin-positive inclusion bodies was reduced in the striatum of YAC128 mice after lenti-PA28γ injection. YAC128 mice showed significant improvement of latency to fall on the rota-rod test after lenti-PA28γ injection. These data demonstrate that the gene therapy with PA, PA28γ can improve UPS function as well as behavioral abnormalities in HD model mice. PMID:26944602

  13. Genome-wide RNAi Screening Identifies Protein Modules Required for 40S Subunit Synthesis in Human Cells.

    PubMed

    Badertscher, Lukas; Wild, Thomas; Montellese, Christian; Alexander, Leila T; Bammert, Lukas; Sarazova, Marie; Stebler, Michael; Csucs, Gabor; Mayer, Thomas U; Zamboni, Nicola; Zemp, Ivo; Horvath, Peter; Kutay, Ulrike

    2015-12-29

    Ribosome biogenesis is a highly complex process requiring many assisting factors. Studies in yeast have yielded comprehensive knowledge of the cellular machinery involved in this process. However, many aspects of ribosome synthesis are different in higher eukaryotes, and the global set of mammalian ribosome biogenesis factors remains unexplored. We used an imaging-based, genome-wide RNAi screen to find human proteins involved in 40S ribosomal subunit biogenesis. Our analysis identified ∼ 300 factors, many part of essential protein modules such as the small subunit (SSU) processome, the eIF3 and chaperonin complexes, and the ubiquitin-proteasome system. We demonstrate a role for the vertebrate-specific factor RBIS in ribosome synthesis, uncover a requirement for the CRL4 E3 ubiquitin ligase in nucleolar ribosome biogenesis, and reveal that intracellular glutamine synthesis supports 40S subunit production. PMID:26711351

  14. Degradation of the Retinoblastoma Tumor Suppressor by the Human Papillomavirus Type 16 E7 Oncoprotein Is Important for Functional Inactivation and Is Separable from Proteasomal Degradation of E7

    PubMed Central

    Gonzalez, Sonia L.; Stremlau, Matt; He, Xi; Basile, John R.; Münger, Karl

    2001-01-01

    The steady-state level and metabolic half-life of retinoblastoma tumor suppressor protein pRB are decreased in cells that express high-risk human papillomavirus (HPV) E7 proteins. Here we show that pRB degradation is a direct activity of E7 and does not reflect a property of cell lines acquired during the selection process for E7 expression. An amino-terminal domain of E7 that does not directly contribute to pRB binding but is required for transformation is also necessary for E7-mediated pRB degradation. Treatment with inhibitors of the 26S proteasome not only blocks E7-mediated pRB degradation but also causes the stabilization of E7. Mutagenic analyses, however, reveal that the processes of proteasomal degradation of E7 and pRB are not linked processes. HPV type 16 E7 also targets the pRB-related proteins p107 and p130 for destabilization by a proteasome-dependent mechanism. Using the SAOS2 flat-cell assay as a biological indicator for pRB function, we demonstrate that pRB degradation, not solely binding, is important for the E7-induced inactivation of pRB. PMID:11462030

  15. KLHL20 links the ubiquitin-proteasome system to autophagy termination.

    PubMed

    Liu, Chin-Chih; Chen, Ruey-Hwa

    2016-05-01

    Autophagy is a dynamic and self-limiting process. The amplitude and duration of this process need to be properly controlled to maintain cell homeostasis, and excessive or insufficient autophagy activity could each lead to disease states. Compared to our understanding of the molecular mechanisms of autophagy induction, little is known about how the autophagy process is turned off after its activation. We recently identified KLHL20 as a key regulator of autophagy termination. By functioning as a substrate-binding subunit of CUL3 ubiquitin ligase, KLHL20 targets the activated ULK1 and phagophore-residing PIK3C3/VPS34 and BECN1 for ubiquitination and proteasomal degradation, which in turn triggers a destabilization of their complex components ATG13 and ATG14. These hierarchical degradation events cause the exhaustion of the autophagic pool of ULK1 and PIK3C3/VPS34 complexes, thereby preventing persistent and excessive autophagy activity. Impairment of KLHL20-dependent feedback regulation of autophagy enhances cell death under prolonged starvation and aggravates muscle atrophy in diabetic mice, which highlights the pathophysiological significance of this autophagy termination mechanism in cell survival and tissue homeostasis. Modulation of this autophagy termination pathway may be effective for treating diseases associated with deregulation of autophagy activity. PMID:26985984

  16. Proteasome immunosubunits protect against the development of CD8 T-cell-mediated autoimmune diseases

    PubMed Central

    Zaiss, Dietmar M.W.; Bekker, Cornelis P.J.; Gröne, Andrea; Lie, Benedicte A.; Sijts, Alice J.A.M.

    2011-01-01

    Exposure of cells to inflammatory cytokines induces the expression of three proteasome immunosubunits, two of which are encoded in the MHC-II region. The induced subunits replace their constitutive homologues in newly formed, so called immunoproteasomes. Immunosubunit incorporation enhances the proteasome’ proteolytic activity and modifies the proteasome’ cleavage site preferences, which improves the generation of many MHC-I presented peptides and shapes the fine-specificity of pathogen-specific CD8 T cell responses. We here report on a second effect of immunoproteasome formation on CD8 T cell responses. We show that mice deficient for the immunosubunits β5i/LMP7 and β2i/MECL-1 develop early-stage multi-organ autoimmunity following irradiation and BM transplantation. Disease symptoms are caused by CD8 T cells and transferrable into immunosubunit-deficient, RAG1-deficient mice. Moreover, using the human Type 1 Diabetes Genetics Consortium (T1DGC) MHC dataset, we identified two SNPs within the β5i/LMP7-encoding gene sequences, that were in strong linkage disequilibrium (LD), as independent genetic risk factors for T1D development in humans. Strikingly, these SNPs significantly enhanced the risk conferred by HLA haplotypes that were formerly shown to predispose for T1D. These data suggest that inflammation-induced immunosubunit expression in peripheral tissues constitutes a mechanism that prevents the development of CD8 T cell mediated autoimmune diseases. PMID:21804012

  17. The proteasome immunosubunits, PA28 and ER-aminopeptidase 1 protect melanoma cells from efficient MART-126-35 -specific T-cell recognition.

    PubMed

    Keller, Martin; Ebstein, Frédéric; Bürger, Elke; Textoris-Taube, Kathrin; Gorny, Xenia; Urban, Sabrina; Zhao, Fang; Dannenberg, Tanja; Sucker, Antje; Keller, Christin; Saveanu, Loredana; Krüger, Elke; Rothkötter, Hermann-Josef; Dahlmann, Burkhardt; Henklein, Petra; Voigt, Antje; Kuckelkorn, Ulrike; Paschen, Annette; Kloetzel, Peter-Michael; Seifert, Ulrike

    2015-12-01

    The immunodominant MART-1(26(27)-35) epitope, liberated from the differentiation antigen melanoma antigen recognized by T cells/melanoma antigen A (MART-1/Melan-A), has been frequently targeted in melanoma immunotherapy, but with limited clinical success. Previous studies suggested that this is in part due to an insufficient peptide supply and epitope presentation, since proteasomes containing the immunosubunits β5i/LMP7 (LMP, low molecular weight protein) or β1i/LMP2 and β5i/LMP7 interfere with MART-1(26-35) epitope generation in tumor cells. Here, we demonstrate that in addition the IFN-γ-inducible proteasome subunit β2i/MECL-1 (multicatalytic endopeptidase complex-like 1), proteasome activator 28 (PA28), and ER-resident aminopeptidase 1 (ERAP1) impair MART-1(26-35) epitope generation. β2i/MECL-1 and PA28 negatively affect C- and N-terminal cleavage and therefore epitope liberation from the proteasome, whereas ERAP1 destroys the MART-1(26-35) epitope by overtrimming activity. Constitutive expression of PA28 and ERAP1 in melanoma cells indicate that both interfere with MART-1(26-35) epitope generation even in the absence of IFN-γ. In summary, our results provide first evidence that activities of different antigen-processing components contribute to an inefficient MART-1(26-35) epitope presentation, suggesting the tumor cell's proteolytic machinery might have an important impact on the outcome of epitope-specific immunotherapies. PMID:26399368

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

    DOE PAGESBeta

    Jastrab, Jordan B.; Wang, Tong; Murphy, J. Patrick; Bai, Lin; Hu, Kuan; Merkx, Remco; Huang, Jessica; Chatterjee, Champak; Ovaa, Huib; Gygi, Steven P.; et al

    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

  19. 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.

  20. Fellutamide B is a Potent Inhibitor of the Mycobacterium tuberculosis Proteasome

    SciTech Connect

    Lin, G.; Li, D; Chidawanyika, T; Nathan, C; Li, H

    2010-01-01

    Via high-throughput screening of a natural compound library, we have identified a lipopeptide aldehyde, fellutamide B (1), as the most potent inhibitor of the Mycobacterium tuberculosis (Mtb) proteasome tested to date. Kinetic studies reveal that 1 inhibits both Mtb and human proteasomes in a time-dependent manner under steady-state condition. Remarkably, 1 inhibits the Mtb proteasome in a single-step binding mechanism with K{sub i} = 6.8 nM, whereas it inhibits the human proteasome {beta}5 active site following a two-step mechanism with K{sub i} = 11.5 nM and K*{sub i} = 0.93 nM. Co-crystallization of 1 bound to the Mtb proteasome revealed a structural basis for the tight binding of 1 to the active sites of the Mtb proteasome. The hemiacetal group of 1 in the Mtb proteasome takes the (R)-configuration, whereas in the yeast proteasome it takes the (S)-configuration, indicating that the pre-chiral CHO group of 1 binds to the active site Thr1 in a different orientation. Re-examination of the structure of the yeast proteasome in complex with 1 showed significant conformational changes at the substrate-binding cleft along the active site. These structural differences are consistent with the different kinetic mechanisms of 1 against Mtb and human proteasomes.

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

    PubMed Central

    Chen, Di; Dou, Q. Ping

    2012-01-01

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

  2. Distinct specificities of Mycobacterium tuberculosis and mammalian proteasomes for N-acetyl tripeptide substrates.

    PubMed

    Lin, Gang; Tsu, Christopher; Dick, Lawrence; Zhou, Xi K; Nathan, Carl

    2008-12-01

    The proteasome of Mycobacterium tuberculosis (Mtb) is a validated and drug-treatable target for therapeutics. To lay ground-work for developing peptide-based inhibitors with a useful degree of selectivity for the Mtb proteasome over those of the host, we used a library of 5,920 N-acetyl tripeptide-aminomethylcoumarins to contrast the substrate preferences of the recombinant Mtb proteasome wild type and open gate mutant, the Rhodococcus erythropolis proteasome, and the bovine proteasome with activator PA28. The Mtb proteasome was distinctive in strictly preferring P1 = tryptophan, particularly in combination with P3 = glycine, proline, lysine or arginine. Screening results were validated with Michalis-Menten kinetic analyses of 21 oligopeptide aminomethyl-coumarin substrates. Bortezomib, a proteasome inhibitor in clinical use, and 17 analogs varying only at P1 were used to examine the differential impact of inhibitors on human and Mtb proteasomes. The results with the inhibitor panel confirmed those with the substrate panel in demonstrating differential preferences of Mtb and mammalian proteasomes at the P1 amino acid. Changing P1 in bortezomib from Leu to m-CF(3)-Phe led to a 220-fold increase in IC(50) against the human proteasome, whereas changing a P1 Ala to m-F-Phe decreased the IC(50) 400-fold against the Mtb proteasome. The change of a P1 Ala to m-Cl-Phe led to an 8000-fold shift in inhibitory potency in favor of the Mtb proteasome, resulting in 8-fold selectivity. Combinations of preferred amino acids at different sites may thus improve the species selectivity of peptide-based inhibitors that target the Mtb proteasome. PMID:18829465

  3. Multiple proteolytic systems, including the proteasome, contribute to CFTR processing.

    PubMed

    Jensen, T J; Loo, M A; Pind, S; Williams, D B; Goldberg, A L; Riordan, J R

    1995-10-01

    The molecular components of the quality control system that rapidly degrades abnormal membrane and secretory proteins have not been identified. The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane protein to which this quality control is stringently applied; approximately 75% of the wild-type precursor and 100% of the delta F508 CFTR variant found in most CF patients are rapidly degraded before exiting from the ER. We now show that this ER degradation is sensitive to inhibitors of the cytosolic proteasome, including lactacystin and certain peptide aldehydes. One of the latter compounds, MG-132, also completely blocks the ATP-dependent conversion of the wild-type precursor to the native folded form that enables escape from degradation. Hence, CFTR and presumably other intrinsic membrane proteins are substrates for proteasomal degradation during their maturation within the ER. PMID:7553864

  4. Colorectal Carcinogenesis, Radiation Quality, and the Ubiquitin-Proteasome Pathway

    PubMed Central

    Datta, Kamal; Suman, Shubhankar; Kumar, Santosh; Fornace, Albert J

    2016-01-01

    Adult colorectal epithelium undergoes continuous renewal and maintains homeostatic balance through regulated cellular proliferation, differentiation, and migration. The canonical Wnt signaling pathway involving the transcriptional co-activator β-catenin is important for colorectal development and normal epithelial maintenance, and deregulated Wnt/β-catenin signaling has been implicated in colorectal carcinogenesis. Colorectal carcinogenesis has been linked to radiation exposure, and radiation has been demonstrated to alter Wnt/β-catenin signaling, as well as the proteasomal pathway involved in the degradation of the signaling components and thus regulation of β-catenin. The current review discusses recent progresses in our understanding of colorectal carcinogenesis in relation to different types of radiation and roles that radiation quality plays in deregulating β-catenin and ubiquitin-proteasome pathway (UPP) for colorectal cancer initiation and progression. PMID:26819641

  5. TRIB1 Is Regulated Post-Transcriptionally by Proteasomal and Non-Proteasomal Pathways

    PubMed Central

    Soubeyrand, Sébastien; Martinuk, Amy; Lau, Paulina; McPherson, Ruth

    2016-01-01

    The TRIB1 gene has been associated with multiple malignancies, plasma triglycerides and coronary artery disease (CAD). Despite the clinical significance of this pseudo-kinase, there is little information on the regulation of TRIB1. Previous studies reported TRIB1 mRNA to be unstable, hinting that TRIB1 might be subject to post-transcriptional regulation. This work explores TRIB1 regulation, focusing on its post-transcriptional aspects. In 3 distinct model systems (HEK293T, HeLa and arterial smooth muscle cells) TRIB1 was undetectable as assessed by western blot. Using recombinant TRIB1 as a proxy, we demonstrate TRIB1 to be highly unstable at the protein and RNA levels. By contrast, recombinant TRIB1 was stable in cellular extracts. Blocking proteasome function led to increased protein steady state levels but failed to rescue protein instability, demonstrating that the 2 processes are uncoupled. Unlike as shown for TRIB2, CUL1 and TRCPβ did not play a role in mediating TRIB1 instability although TRCPβ suppression increased TRIB1 expression. Lastly, we demonstrate that protein instability is independent of TRIB1 subcellular localization. Following the identification of TRIB1 nuclear localization signal, a cytosolic form was engineered. Despite being confined to the cytosol, TRIB1 remained unstable, suggesting that instability occurs at a stage that precedes its nuclear translocation and downstream nuclear function. These results uncover possible avenues of intervention to regulate TRIB1 function by identifying two distinct regulatory axes that control TRIB1 at the post-transcriptional level. PMID:27019349

  6. MG132, a proteasome inhibitor, induces apoptosis in tumor cells.

    PubMed

    Guo, Na; Peng, Zhilan

    2013-03-01

    The balance between cell proliferation and apoptosis is critical for normal development and for the maintenance of homeostasis in adult organisms. Disruption of this balance has been implicated in a large number of disease processes, ranging from autoimmunity and neurodegenerative disorders to cancer. The ubiquitin-proteasome pathway, responsible for mediating the majority of intracellular proteolysis, plays a crucial role in the regulation of many normal cellular processes, including the cell cycle, differentiation and apoptosis. Apoptosis in cancer cells is closely connected with the activity of ubiquitin-proteasome pathway. The peptide-aldehyde proteasome inhibitor MG132 (carbobenzoxyl-L-leucyl-L-leucyl-L-leucine) induces the apoptosis of cells by a different intermediary pathway. Although the pathway of induction of apoptosis is different, it plays a crucial role in anti-tumor treatment. There are many cancer-related molecules in which the protein levels present in cells are regulated by a proteasomal pathway; for example, tumor inhibitors (P53, E2A, c-Myc, c-Jun, c-Fos), transcription factors (transcription factor nuclear factor-kappa B, IκBα, HIFI, YYI, ICER), cell cycle proteins (cyclin A and B, P27, P21, IAP1/3), MG132 induces cell apoptosis through formation of reactive oxygen species or the upregulation and downregulation of these factors, which is ultimately dependent upon the activation of the caspase family of cysteine proteases. In this article we review the mechanism of the induction of apoptosis in order to provide information required for research. PMID:22897979

  7. Formation of Tankyrase Inhibitor-Induced Degradasomes Requires Proteasome Activity

    PubMed Central

    Pedersen, Nina Marie; Thorvaldsen, Tor Espen; Schultz, Sebastian Wolfgang; Wenzel, Eva Maria; Stenmark, Harald

    2016-01-01

    In canonical Wnt signaling, the protein levels of the key signaling mediator β-catenin are under tight regulation by the multimeric destruction complex that mediates proteasomal degradation of β-catenin. In colorectal cancer, destruction complex activity is often compromised due to mutations in the multifunctional scaffolding protein Adenomatous Polyposis Coli (APC), leading to a stabilization of β-catenin. Recently, tankyrase inhibitors (TNKSi), a novel class of small molecule inhibitors, were shown to re-establish a functional destruction complex in APC-mutant cancer cell lines by stabilizing AXIN1/2, whose protein levels are usually kept low via poly(ADP-ribosyl)ation by the tankyrase enzymes (TNKS1/2). Surprisingly, we found that for the formation of the morphological correlates of destruction complexes, called degradasomes, functional proteasomes are required. In addition we found that AXIN2 is strongly upregulated after 6 h of TNKS inhibition. The proteasome inhibitor MG132 counteracted TNKSi-induced degradasome formation and AXIN2 stabilization, and this was accompanied by reduced transcription of AXIN2. Mechanistically we could implicate the transcription factor FoxM1 in this process, which was recently shown to be a transcriptional activator of AXIN2. We observed a substantial reduction in TNKSi-induced stabilization of AXIN2 after siRNA-mediated depletion of FoxM1 and found that proteasome inhibition reduced the active (phosphorylated) fraction of FoxM1. This can explain the decreased protein levels of AXIN2 after MG132 treatment. Our findings have implications for the design of in vitro studies on the destruction complex and for clinical applications of TNKSi. PMID:27482906

  8. Interaction of factor XIII subunits.

    PubMed

    Katona, Eva; Pénzes, Krisztina; Csapó, Andrea; Fazakas, Ferenc; Udvardy, Miklós L; Bagoly, Zsuzsa; Orosz, Zsuzsanna Z; Muszbek, László

    2014-03-13

    Coagulation factor XIII (FXIII) is a heterotetramer consisting of 2 catalytic A subunits (FXIII-A2) and 2 protective/inhibitory B subunits (FXIII-B2). FXIII-B, a mosaic protein consisting of 10 sushi domains, significantly prolongs the lifespan of catalytic subunits in the circulation and prevents their slow progressive activation in plasmatic conditions. In this study, the biochemistry of the interaction between the 2 FXIII subunits was investigated. Using a surface plasmon resonance technique and an enzyme-linked immunosorbent assay-type binding assay, the equilibrium dissociation constant (Kd) for the interaction was established in the range of 10(-10) M. Based on the measured Kd, it was calculated that in plasma approximately 1% of FXIII-A2 should be in free form. This value was confirmed experimentally by measuring FXIII-A2 in plasma samples immunodepleted of FXIII-A2B2. Free plasma FXIII-A2 is functionally active, and when activated by thrombin and Ca(2+), it can cross-link fibrin. In cerebrospinal fluid and tears with much lower FXIII subunit concentrations, >80% of FXIII-A2 existed in free form. A monoclonal anti-FXIII-B antibody that prevented the interaction between the 2 subunits reacted with the recombinant combined first and second sushi domains of FXIII-B, and its epitope was localized to the peptide spanning positions 96 to 103 in the second sushi domain. PMID:24408323

  9. CD147 is a regulatory subunit of the gamma-secretase complex inAlzheimer's disease amyloid beta-peptide production

    SciTech Connect

    Zhou, Shuxia; Zhou, Hua; Walian, Peter J.; Jap, Bing K.

    2005-04-06

    , CD44, DCC, ErbB4, E-cadherin, LRP, N-cadherin, Nectin-1, and Notch, within their transmembranous regions (2-11); therefore, in addition to its role in AD, {gamma}-secretase has been found to participate in other important biological functions, such as intracellular signaling. {gamma}-secretase processing of APP requires prior removal of a major fragment of the APP extracellular domain (sAPP{sub {beta}}) by {beta}-secretase to yield a membrane bound fragment (APP CTF{sub {beta}}). Subsequent cleavage of this membrane bound fragment by {gamma}-secretase results in the release of the Alzheimer's disease (AD) associated amyloid {beta}-peptides (12). The proteolytic activity of {gamma}-secretase is found not to be critically dependent on the specific sequence, but instead on the size of the extracellular domain (13); such sequence independent characteristics of the substrate are reminiscent of those of the 26S proteasome complex that cleaves substrates in a non-sequence specific manner. {gamma}-secretase is present in almost all animal species, vertebrates and invertebrates; it is expressed in many human organs and tissues.

  10. The role of allostery in the ubiquitin-proteasome system

    PubMed Central

    Liu, Jin; Nussinov, Ruth

    2012-01-01

    The Ubiquitin-Proteasome System is involved in many cellular processes including protein degradation. Degradation of a protein via this system involves two successive steps: ubiquitination and degradation. Ubiquitination tags the target protein with ubiquitin-like proteins, such as ubiquitin, SUMO and NEDD8, via a cascade involving three enzymes: activating enzyme E1, conjugating enzyme E2, and E3 ubiquitin ligases. The proteasomes recognize the ubiquitin-like protein tagged substrate proteins and degrade them. Accumulating evidence indicates that allostery is a central player in the regulation of ubiquitination, as well as deubiquitination and degradation. Here, we provide an overview of the key mechanistic roles played by allostery in all steps of these processes, and highlight allosteric drugs targeting them. Throughout the review, we emphasize the crucial mechanistic role played by linkers in allosterically controlling the Ubiquitin-Proteasome System action by biasing the sampling of the conformational space, which facilitate the catalytic reactions of the ubiquitination and degradation. Finally, we propose that allostery may similarly play key roles in the regulation of molecular machines in the cell, and as such allosteric drugs can be expected to be increasingly exploited in therapeutic regimes. PMID:23234564

  11. Marizomib, a potent second generation proteasome inhibitor from natural origin.

    PubMed

    Ma, Long; Diao, Aipo

    2015-01-01

    The malignance of cancers reinforces the need to find potent antineoplastic agents. In the past decades, proteasome has been witnessed as a potential target to fulfil this purpose, as evidenced by the fact that the first-in-class proteasome inhibitor Bortezomib was marketed in 2003. Marizomib (Salinosporamide A, NPI-0052), as a marine natural product, promises to be of high efficacy against multiple myeloma (MM), relapsed/refractory MM and other types of solid tumours. Compared with Bortezomib, it arguably has fewer severe side effects. Marizomib has been termed as orphan drug against multiple myeloma by US Food and Drug Administration (FDA) in 2013 and by European Medicines Agency (EMA) in 2014. As one of the second generation proteasome inhibitors (PIs), Marizomib is expected to bring about a sustained and complete therapeutic to extend cancer patients' life span. In this article, we intended to briefly review the historical developments, mechanisms, pharmacology, biosynthesis and side effects of this agent, aiming to provide concise coverage for a broad readership. In the end, we proposed our perspective for its futuristic applications. PMID:25403165

  12. Proteasomal Degradation of TRIM5α during Retrovirus Restriction

    PubMed Central

    Rold, Christopher James; Aiken, Christopher

    2008-01-01

    The host protein TRIM5α inhibits retroviral infection at an early post-penetration stage by targeting the incoming viral capsid. While the detailed mechanism of restriction remains unclear, recent studies have implicated the activity of cellular proteasomes in the restriction of retroviral reverse transcription imposed by TRIM5α. Here, we show that TRIM5α is rapidly degraded upon encounter of a restriction-susceptible retroviral core. Inoculation of TRIM5α-expressing human 293T cells with a saturating level of HIV-1 particles resulted in accelerated degradation of the HIV-1-restrictive rhesus macaque TRIM5α protein but not the nonrestrictive human TRIM5α protein. Exposure of cells to HIV-1 also destabilized the owl monkey restriction factor TRIMCyp; this was prevented by addition of the inhibitor cyclosporin A and was not observed with an HIV-1 virus containing a mutation in the capsid protein that relieves restriction by TRIMCyp IVHIV. Likewise, human TRIM5α was rapidly degraded upon encounter of the restriction-sensitive N-tropic murine leukemia virus (N-MLV) but not the unrestricted B-MLV. Pretreatment of cells with proteasome inhibitors prevented the HIV-1-induced loss of both rhesus macaque TRIM5α and TRIMCyp proteins. We also detected degradation of endogenous TRIM5α in rhesus macaque cells following HIV-1 infection. We conclude that engagement of a restriction-sensitive retrovirus core results in TRIM5α degradation by a proteasome-dependent mechanism. PMID:18497858

  13. Computational Approaches for the Discovery of Human Proteasome Inhibitors: An Overview.

    PubMed

    Guedes, Romina A; Serra, Patrícia; Salvador, Jorge A R; Guedes, Rita C

    2016-01-01

    Proteasome emerged as an important target in recent pharmacological research due to its pivotal role in degrading proteins in the cytoplasm and nucleus of eukaryotic cells, regulating a wide variety of cellular pathways, including cell growth and proliferation, apoptosis, DNA repair, transcription, immune response, and signaling processes. The last two decades witnessed intensive efforts to discover 20S proteasome inhibitors with significant chemical diversity and efficacy. To date, the US FDA approved to market three proteasome inhibitors: bortezomib, carfilzomib, and ixazomib. However new, safer and more efficient drugs are still required. Computer-aided drug discovery has long being used in drug discovery campaigns targeting the human proteasome. The aim of this review is to illustrate selected in silico methods like homology modeling, molecular docking, pharmacophore modeling, virtual screening, and combined methods that have been used in proteasome inhibitors discovery. Applications of these methods to proteasome inhibitors discovery will also be presented and discussed to raise improvements in this particular field. PMID:27438821

  14. Calcium-dependent proteasome activation is required for axonal neurofilament degradation.

    PubMed

    Park, Joo Youn; Jang, So Young; Shin, Yoon Kyung; Suh, Duk Joon; Park, Hwan Tae

    2013-12-25

    Even though many studies have identified roles of proteasomes in axonal degeneration, the molecular mechanisms by which axonal injury regulates proteasome activity are still unclear. In the present study, we found evidence indicating that extracellular calcium influx is an upstream regulator of proteasome activity during axonal degeneration in injured peripheral nerves. In degenerating axons, the increase in proteasome activity and the degradation of ubiquitinated proteins were significantly suppressed by extracellular calcium chelation. In addition, electron microscopic findings revealed selective inhibition of neurofilament degradation, but not microtubule depolymerization or mitochondrial swelling, by the inhibition of calpain and proteasomes. Taken together, our findings suggest that calcium increase and subsequent proteasome activation are an essential initiator of neurofilament degradation in Wallerian degeneration. PMID:25206662

  15. Quantitative time-resolved analysis reveals intricate, differential regulation of standard- and immuno-proteasomes.

    PubMed

    Liepe, Juliane; Holzhütter, Hermann-Georg; Bellavista, Elena; Kloetzel, Peter M; Stumpf, Michael P H; Mishto, Michele

    2015-01-01

    Proteasomal protein degradation is a key determinant of protein half-life and hence of cellular processes ranging from basic metabolism to a host of immunological processes. Despite its importance the mechanisms regulating proteasome activity are only incompletely understood. Here we use an iterative and tightly integrated experimental and modelling approach to develop, explore and validate mechanistic models of proteasomal peptide-hydrolysis dynamics. The 20S proteasome is a dynamic enzyme and its activity varies over time because of interactions between substrates and products and the proteolytic and regulatory sites; the locations of these sites and the interactions between them are predicted by the model, and experimentally supported. The analysis suggests that the rate-limiting step of hydrolysis is the transport of the substrates into the proteasome. The transport efficiency varies between human standard- and immuno-proteasomes thereby impinging upon total degradation rate and substrate cleavage-site usage. PMID:26393687

  16. Proteasomal control of cytokinin synthesis protects Mycobacterium tuberculosis against nitric oxide

    PubMed Central

    Samanovic, Marie I.; Tu, Shengjiang; Novák, Ondřej; Iyer, Lakshminarayan M.; McAllister, Fiona E.; Aravind, L.; Gygi, Steven P.; Hubbard, Stevan R.; Strnad, Miroslav; Darwin, K. Heran

    2015-01-01

    Summary One of several roles of the Mycobacterium tuberculosis proteasome is to defend against host-produced nitric oxide (NO), a free radical that can damage numerous biological macromolecules. Mutations that inactivate proteasomal degradation in Mycobacterium tuberculosis result in bacteria that are hypersensitive to NO and attenuated for growth in vivo, but it was not known why. To elucidate the link between proteasome function, NO-resistance, and pathogenesis, we screened for suppressors of NO hypersensitivity in a mycobacterial proteasome ATPase mutant and identified mutations in Rv1205. We determined that Rv1205 encodes a pupylated proteasome substrate. Rv1205 is a homologue of the plant enzyme LONELY GUY, which catalyzes the production of hormones called cytokinins. Remarkably, we report for the first time that an obligate human pathogen secretes several cytokinins. Finally, we determined that the Rv1205-dependent accumulation of cytokinin breakdown products is likely responsible for the sensitization of Mycobacterium tuberculosis proteasome-associated mutants to NO. PMID:25728768

  17. Priming the proteasome by protein kinase G: a novel cardioprotective mechanism of sildenafil

    PubMed Central

    Zhang, Hanming; Wang, Xuejun

    2015-01-01

    The proteasome mediates the degradation of most cellular proteins including misfolded proteins, pivotal to intracellular protein hemostasis. Proteasome functional insufficiency is implicated in a large subset of human failing hearts. Experimental studies have established proteasome functional insufficiency as a major pathogenic factor, rationalizing proteasome enhancement as a potentially new therapeutic strategy for congestive heart failure. Protein kinase G activation known to be cardioprotective was recently found to facilitate proteasomal degradation of misfolded proteins in cardiomyocytes; sildenafil was shown to activate myocardial protein kinase G, improve cardiac protein quality control and slow down the progression of cardiac proteinopathy in mice. This identifies the first clinically used drug that is capable of benign proteasome enhancement and unveils a potentially novel cardioprotective mechanism for sildenafil. PMID:25760877

  18. Proteasome activity is required for the initiation of precancerous pancreatic lesions.

    PubMed

    Furuyama, Takaki; Tanaka, Shinji; Shimada, Shu; Akiyama, Yoshimitsu; Matsumura, Satoshi; Mitsunori, Yusuke; Aihara, Arihiro; Ban, Daisuke; Ochiai, Takanori; Kudo, Atsushi; Fukamachi, Hiroshi; Arii, Shigeki; Kawaguchi, Yoshiya; Tanabe, Minoru

    2016-01-01

    Proteasome activity is significantly increased in advanced cancers, but its role in cancer initiation is not clear, due to difficulties in monitoring this process in vivo. We established a line of transgenic mice that carried the ZsGreen-degron(ODC) (Gdeg) proteasome reporter to monitor the proteasome activity. In combination with Pdx-1-Cre;LSL-Kras(G12D) model, proteasome activity was investigated in the initiation of precancerous pancreatic lesions (PanINs). Normal pancreatic acini in Gdeg mice had low proteasome activity. By contrast, proteasome activity was increased in the PanIN lesions that developed in Gdeg;Pdx-1-Cre;LSL-Kras(G12D) mice. Caerulein administration to Gdeg;Pdx-1-Cre;LSL-Kras(G12D) mice induced constitutive elevation of proteasome activity in pancreatic tissues and accelerated PanIN formation. The proteasome inhibitor markedly reduced PanIN formation in Gdeg;Pdx-1-Cre;LSL-Kras(G12D) mice (P = 0.001), whereas it had no effect on PanIN lesions that had already formed. These observations indicated the significance of proteasome activity in the initiation of PanIN but not the maintenance per se. In addition, the expressions of pERK and its downstream factors including cyclin D1, NF-κB, and Cox2 were decreased after proteasome inhibition in PanINs. Our studies showed activation of proteasome is required specifically for the initiation of PanIN. The roles of proteasome in the early stages of pancreatic carcinogenesis warrant further investigation. PMID:27244456

  19. Proteasome activity is required for the initiation of precancerous pancreatic lesions

    PubMed Central

    Furuyama, Takaki; Tanaka, Shinji; Shimada, Shu; Akiyama, Yoshimitsu; Matsumura, Satoshi; Mitsunori, Yusuke; Aihara, Arihiro; Ban, Daisuke; Ochiai, Takanori; Kudo, Atsushi; Fukamachi, Hiroshi; Arii, Shigeki; Kawaguchi, Yoshiya; Tanabe, Minoru

    2016-01-01

    Proteasome activity is significantly increased in advanced cancers, but its role in cancer initiation is not clear, due to difficulties in monitoring this process in vivo. We established a line of transgenic mice that carried the ZsGreen-degronODC (Gdeg) proteasome reporter to monitor the proteasome activity. In combination with Pdx-1-Cre;LSL-KrasG12D model, proteasome activity was investigated in the initiation of precancerous pancreatic lesions (PanINs). Normal pancreatic acini in Gdeg mice had low proteasome activity. By contrast, proteasome activity was increased in the PanIN lesions that developed in Gdeg;Pdx-1-Cre;LSL-KrasG12D mice. Caerulein administration to Gdeg;Pdx-1-Cre;LSL-KrasG12D mice induced constitutive elevation of proteasome activity in pancreatic tissues and accelerated PanIN formation. The proteasome inhibitor markedly reduced PanIN formation in Gdeg;Pdx-1-Cre;LSL-KrasG12D mice (P = 0.001), whereas it had no effect on PanIN lesions that had already formed. These observations indicated the significance of proteasome activity in the initiation of PanIN but not the maintenance per se. In addition, the expressions of pERK and its downstream factors including cyclin D1, NF-κB, and Cox2 were decreased after proteasome inhibition in PanINs. Our studies showed activation of proteasome is required specifically for the initiation of PanIN. The roles of proteasome in the early stages of pancreatic carcinogenesis warrant further investigation. PMID:27244456

  20. Changes in expression of proteasome in rats at different stages of atherosclerosis

    PubMed Central

    Oenzil, Fadil; Yanwirasti; Yerizel, Eti

    2016-01-01

    It has been suggested that proteasome system has a role in initiation, progression, and complication stages of atherosclerosis. Although there is still controversy, there has been no research that compares the expression of proteasome in tissue and serum at each of these stages. This study aimed to investigated the expression of proteasome at different stages of atherosclerosis using rat model. We measured the expression of aortic proteasome by immunohistochemical analyses and were then analyzed using ImageJ software for percentage of area and integrated density. We used Photoshop version 3.0 to analyze aortic proteasome expression as a comparison. We measured serum proteasome expression by enzyme linked immunosorbents assays. Kruskal-Wallis test was used to compare mean value of percentage of area and serum proteasome. Analysis of variance test was used to compare mean value of integrated density. Correlation test between vascular proteasome expression and serum proteasome expression was made using Spearman test. A P-value of 0.05 was considered statistically significant. Compared with normal, percentage of area was higher in initiation, progression, and complication. Compared with normal, integrated density was higher in initiation and further higher in progression and complication. Data from Image J is similar with data from Photoshop. Serum proteasome expression was higher in initiation compared with normal, and further higher in progression and complication. It was concluded that there were different vascular proteasome expression and serum proteasome expression at the stages of atherosclerosis. These results may be used in research into new marker and therapeutic target in atherosclerosis. PMID:27382511

  1. Clinical Use of Proteasome Inhibitors in the Treatment of Multiple Myeloma

    PubMed Central

    Merin, Noah M.; Kelly, Kevin R.

    2014-01-01

    Multiple myeloma (MM) is an incurable hematological malignancy characterized by the clonal proliferation of neoplastic plasma cells. The use of proteasome inhibitors in the treatment of MM has led to significant improvements in outcomes. This article reviews data on the use of the two approved proteasome inhibitors (bortezomib and carlfilzomib), as well as newer agents under development. Emphasis is placed on the clinical use of proteasome inhibitors, including management of side effects and combination with other agents. PMID:25545164

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

    PubMed

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

    2004-06-01

    Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis (fALS). The present study demonstrated impaired proteasomal function in the lumbar spinal cord of transgenic mice expressing human SOD-1 with the ALS-causing mutation G93A (SOD-1(G93A)) compared to non-transgenic littermates (LM) and SOD-1(WT) transgenic mice. Chymotrypsin-like activity was decreased as early as 45 days of age. By 75 days, chymotrypsin-, trypsin-, and caspase-like activities of the proteasome were impaired, at about 50% of control activity in lumbar spinal cord, but unchanged in thoracic spinal cord and liver. Both total and specific activities of the proteasome were reduced to a similar extent, indicating that a change in proteasome function, rather than a decrease in proteasome levels, had occurred. Similar decreases of total and specific activities of the proteasome were observed in NIH 3T3 cell lines expressing fALS mutants SOD-1(G93A) and SOD-1(G41S), but not in SOD-1(WT) controls. Although overall levels of proteasome were maintained in spinal cord of SOD-1(G93A) transgenic mice, the level of 20S proteasome was substantially reduced in lumbar spinal motor neurons relative to the surrounding neuropil. It is concluded that impairment of the proteasome is an early event and contributes to ALS pathogenesis. PMID:15189335

  3. ACTIVATION OF PERK KINASE IN NEURAL CELLS BY PROTEASOME INHIBITOR TREATMENT

    PubMed Central

    Zhang, Le; Ebenezer, Philip J; Dasuri, Kalavathi; Bruce-Keller, Annadora J.; Fernandez-Kim, Sun Ok; Liu, Ying; Keller, Jeffrey N.

    2010-01-01

    Inhibition of the proteasome proteolytic pathway occurs as the result of normal aging, as well as in a variety of neurodegenerative conditions, and is believed to promote cellular toxicity in each of these conditions through diverse mechanisms. In the present study we examined whether proteasome inhibition alters the protein kinase (PKR)-like ER kinase (PERK). Our studies demonstrate that proteasome inhibitors induce the transient activation of PERK in both primary rat neurons as well as the N2a neural cell line. Experiments with siRNA to PERK demonstrated that the modulation of PERK was not significant involved in regulating toxicity, ubiquitinated protein levels, or ribosome perturbations in response to proteasome inhibitor treatment. Surprisingly, PERK was observed to be involved in the upregulation of p38 kinase following proteasome inhibitor treatment. Taken together, these data demonstrate the ability of proteasome inhibition to activate PERK and demonstrate evidence for novel cross talk between PERK and the activation of p38 kinase in neural cells following proteasome inhibition. Taken together, these data have implications for understanding the basis by which proteasome inhibition alters neural homeostasis, and the basis by which cell signaling cascades are regulated by proteasome inhibition. PMID:19860852

  4. Force Spectroscopy of Substrate Molecules En Route to the Proteasome's Active Sites

    PubMed Central

    Classen, Mirjam; Breuer, Sarah; Baumeister, Wolfgang; Guckenberger, Reinhard; Witt, Susanne

    2011-01-01

    We used an atomic force microscope to study the mechanism underlying the translocation of substrate molecules inside the proteasome. Our specific experimental setup allowed us to measure interaction forces between the 20S proteasome and its substrates. The substrate (β-casein) was covalently bound either via a thiol-Au bond or by a PEG-based binding procedure to the atomic force microscope cantilever tip and offered as bait to proteasomes from Methanosarcina mazei. The proteasomes were immobilized densely in an upright orientation on mica, which made their upper pores accessible for substrates to enter. Besides performing conventional single-molecule force spectroscopy experiments, we developed a three-step procedure that allows the detection of specific proteasome-substrate single-molecule events without tip-sample contact. Using the active 20S wild type and an inactive active-site mutant, as well as two casein mutants bound with opposite termini to the microscope tip, we detected no directional preference of the proteasome-substrate interactions. By comparing the distribution of the measured forces for the proteasome-substrate interactions, were observed that a significant proportion of interaction events occurred at higher forces for the active versus the inactive proteasome. These forces can be attributed to the translocation of substrate en route to the active sites that are harbored deep inside the proteasome. PMID:21244845

  5. Increased 4-hydroxy-2-nonenal-induced proteasome dysfunction is correlated with cardiac damage in streptozotocin-injected rats with isoproterenol infusion.

    PubMed

    Deshpande, Mandar; Mali, Vishal R; Pan, Guodong; Xu, Jiang; Yang, Xiao-Ping; Thandavarayan, Rajarajan A; Palaniyandi, Suresh Selvaraj

    2016-07-01

    Increase in 4-hydroxy-2-nonenal (4HNE) due to oxidative stress has been observed in a variety of cardiac diseases such as diabetic cardiomyopathy. 4HNE exerts a damaging effect in the myocardium by interfering with subcellular organelles like mitochondria by forming adducts. Therefore, we hypothesized that increased 4HNE adduct formation in the heart results in proteasome inactivation in isoproterenol (ISO)-infused type 1 diabetes mellitus (DM) rats. Eight-week-old male Sprague Dawley rats were injected with streptozotocin (STZ, 65 mg kg(-1) ). The rats were infused with ISO (5 mg kg(-1) ) for 2 weeks by mini pumps, after 8 weeks of STZ injection. We studied normal control (n = 8) and DM + ISO (n = 10) groups. Cardiac performance was assessed by echocardiography and Millar catheter at the end of the protocol at 20 weeks. Initially, we found an increase in 4HNE adducts in the hearts of the DM + ISO group. There was also a decrease in myocardial proteasomal peptidase (chymotrypsin and trypsin-like) activity. Increases in cardiomyocyte area (446 ± 32·7 vs 221 ± 10·83) (µm(2) ), per cent area of cardiac fibrosis (7·4 ± 0·7 vs 2·7 ± 0·5) and cardiac dysfunction were also found in DM + ISO (P < 0·05) relative to controls. We also found increased 4HNE adduct formation on proteasomal subunits. Furthermore, reduced aldehyde dehydrogenase 2 activity was observed in the myocardium of the DM + ISO group. Treatment with 4HNE (100 μM) for 4 h on cultured H9c2 cardiomyocytes attenuated proteasome activity. Therefore, we conclude that the 4HNE-induced decrease in proteasome activity may be involved in the cardiac pathology in STZ-injected rats infused with ISO. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27273517

  6. Decreased Proteasomal Activity Causes Photoreceptor Degeneration in Mice

    PubMed Central

    Ando, Ryo; Noda, Kousuke; Tomaru, Utano; Kamoshita, Mamoru; Ozawa, Yoko; Notomi, Shoji; Hisatomi, Toshio; Noda, Mika; Kanda, Atsuhiro; Ishibashi, Tatsuro; Kasahara, Masanori; Ishida, Susumu

    2014-01-01

    Purpose. To study the retinal degeneration caused by decreased proteasomal activity in β5t transgenic (β5t-Tg) mice, an animal model of senescence acceleration. Methods. β5t-Tg mice and age-matched littermate control (WT) mice were used. Proteasomal activities and protein level of poly-ubiquitinated protein in retinal extracts were quantified. Fundus images of β5t-Tg mice were taken and their features were assessed. For histologic evaluation, the thicknesses of inner nuclear layer (INL), outer nuclear layer (ONL), and photoreceptor outer segment (OS) were measured. For functional analysis, ERG was recorded under scotopic and photopic illumination conditions. Immunofluorescence (IF) staining and TUNEL were performed to investigate the mechanism of photoreceptor degeneration. Results. Chymotrypsin-like activity was partially suppressed in retinal tissues of β5t-Tg mice. Retinal degenerative changes with arterial attenuation were present in β5t-Tg, but not in WT mice. Inner nuclear layer thickness showed no significant change between β5t-Tg and WT mice at 1, 3, 6, and 9 months of age. By contrast, thicknesses of ONL and OS in β5t-Tg mice were significantly decreased at 3, 6, and 9 months compared with those in WT mice. Electroretinograms showed decrease of scotopic a-wave amplitude in β5t-Tg mice. The number of TUNEL-positive cells in ONL were significantly increased in β5t-Tg mice and colocalized with apoptosis-inducing factor, but not with cleaved caspase-3 and -9, indicating that the photoreceptor cell death was induced via a caspase-independent pathway. Conclusions. The current data showed that impaired proteasomal function causes photoreceptor degeneration. PMID:24994871

  7. The Ubiquitin–Proteasome System of Saccharomyces cerevisiae

    PubMed Central

    Finley, Daniel; Ulrich, Helle D.; Sommer, Thomas; Kaiser, Peter

    2012-01-01

    Protein modifications provide cells with exquisite temporal and spatial control of protein function. Ubiquitin is among the most important modifiers, serving both to target hundreds of proteins for rapid degradation by the proteasome, and as a dynamic signaling agent that regulates the function of covalently bound proteins. The diverse effects of ubiquitylation reflect the assembly of structurally distinct ubiquitin chains on target proteins. The resulting ubiquitin code is interpreted by an extensive family of ubiquitin receptors. Here we review the components of this regulatory network and its effects throughout the cell. PMID:23028185

  8. JMJ24 targets CHROMOMETHYLASE3 for proteasomal degradation in Arabidopsis

    PubMed Central

    Deng, Shulin; Jang, In-Cheol; Su, Linlin; Xu, Jun; Chua, Nam-Hai

    2016-01-01

    H3K9 methylation is usually associated with DNA methylation, and together they symbolize transcriptionally silenced heterochromatin. A number of proteins involved in epigenetic processes have been characterized. However, how the stability of these proteins is regulated at the post-translational level is largely unknown. Here, we show that an Arabidopsis JmjC domain protein, JMJ24, possesses ubiquitin E3 ligase activity. JMJ24 directly targets a DNA methyltransferase, CHROMOMETHYLASE 3 (CMT3), for proteasomal degradation to initiate destabilization of the heterochromatic state of endogenous silenced loci. Our results uncover an additional connection between two conserved epigenetic modifications: histone modification and DNA methylation. PMID:26798133

  9. Characterization of the proteasome ß2 subunit gene and its mutant allele in the tephritid fruit fly pest, Anastrepha suspensa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Conditional lethal release (CLR) is a proposed variation of the sterile insect technique (SIT) for the biological control of pest insects that would result from the release of transgenic insects carrying dominant conditional lethal genes. After mating with pest insects in the field, lethal gene exp...

  10. A novel combination treatment for breast cancer cells involving BAPTA-AM and proteasome inhibitor bortezomib

    PubMed Central

    YERLIKAYA, AZMI; ERDOĞAN, ELIF; OKUR, EMRAH; YERLIKAYA, ŞERIFE; SAVRAN, BIRCAN

    2016-01-01

    Glucose-regulated protein 78 kDa/binding immunoglobulin protein (GRP78/BIP) is a well-known endoplasmic reticulum (ER) chaperone protein regulating ER stress by facilitating protein folding, assembly and Ca2+ binding. GRP78 is also a member of the heat shock protein 70 gene family and induces tumor cell survival and resistance to chemotherapeutics. Bortezomib is a highly specific 26S proteasome inhibitor that has been approved as treatment for patients with multiple myeloma. The present study first examined the dose- and time-dependent effects of bortezomib on GRP78 expression levels in the highly metastatic mouse breast cancer 4T1 cell line using western blot analysis. The analysis results revealed that GRP78 levels were significantly increased by bortezomib at a dose as low as 10 nM. Time-dependent experiments indicated that the accumulation of GRP78 was initiated after a 24 h incubation period following the addition of 10 nM bortezomib. Subsequently, the present study determined the half maximal inhibitory concentration of intracellular calcium chelator BAPTA-AM (13.6 µM) on 4T1 cells. The combination effect of BAPTA-AM and bortezomib on the 4T1 cells was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and WST-1 assays and an iCELLigence system. The results revealed that the combination of 10 nM bortezomib + 5 µM BAPTA-AM is more cytotoxic compared with monotherapies, including 10 nM bortezomib, 1 µM BAPTA-AM and 5 µM BAPTA-AM. In addition, the present results revealed that bortezomib + BAPTA-AM combination causes cell death through the induction of apoptosis. The present results also revealed that bortezomib + BAPTA-AM combination-induced apoptosis is associated with a clear increase in the phosphorylation of stress-activated protein kinase/Jun amino-terminal kinase SAPK/JNK. Overall, the present results suggest that bortezomib and BAPTA-AM combination therapy may be a novel therapeutic strategy for breast cancer treatment

  11. The ribosomal subunit assembly line

    PubMed Central

    Dlakić, Mensur

    2005-01-01

    Recent proteomic studies in Saccharomyces cerevisiae have identified nearly 200 proteins, other than the structural ribosomal proteins, that participate in the assembly of ribosomal subunits and their transport from the nucleus. In a separate line of research, proteomic studies of mature plant ribosomes have revealed considerable variability in the protein composition of individual ribosomes. PMID:16207363

  12. Essential role of proteasomes in maintaining self-renewal in neural progenitor cells

    PubMed Central

    Zhao, Yunhe; Liu, Xueqin; He, Zebin; Niu, Xiaojie; Shi, Weijun; Ding, Jian M.; Zhang, Li; Yuan, Tifei; Li, Ang; Yang, Wulin; Lu, Li

    2016-01-01

    Protein turnover and homeostasis are regulated by the proteasomal system, which is critical for cell function and viability. Pluripotency of stem cells also relies on normal proteasomal activity that mitigates senescent phenotypes induced by intensive cell replications, as previously demonstrated in human bone marrow stromal cells. In this study, we investigated the role of proteasomes in self-renewal of neural progenitor cells (NPCs). Through both in vivo and in vitro analyses, we found that the expression of proteasomes was progressively decreased during aging. Likewise, proliferation and self-renewal of NPCs were also impaired in aged mice, suggesting that the down-regulation of proteasomes might be responsible for this senescent phenotype. Lowering proteasomal activity by loss-of-function manipulations mimicked the senescence of NPCs both in vitro and in vivo; conversely, enhancing proteasomal activity restored and improved self-renewal in aged NPCs. These results collectively indicate that proteasomes work as a key regulator in promoting self-renewal of NPCs. T