共查询到20条相似文献,搜索用时 15 毫秒
1.
Muthukumar Elangovan Lavanya Sukumaran Yung Joon Yoo 《Biochemical and biophysical research communications》2010,396(2):425-428
It is well known that S5a and hRpn13 are two major ubiquitin (Ub) receptors in the proteasome but little is known about their functional difference in recruiting ubiquitinated substrates. In this study using siRNA-mediated knockdown of S5a or hRpn13, we found that two Ub receptors had different substrate specificity although similar level of accumulation of high molecular weight Ub-conjugates was observed. Interesting enough, depletion of S5a, but not hRpn13, resulted in the Ub-containing aggregates and induced ER chaperones such as Grp78 and Grp94. ERAD substrates such as α-TCR and α1-antitrypsin were also stabilized by the depletion of S5a but not hRpn13. Our results suggest that there is different substrate specificity between S5a and hRpn13 at the level of delivery and S5a may be the major docking site for ERAD substrates. 相似文献
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Increased degradation of oxidized proteins in yeast defective in 26 S proteasome assembly 总被引:1,自引:0,他引:1
An Rpn9-disrupted yeast strain, Delta rpn9, whose growth is temperature sensitive with defective assembly of the 26 S proteasome complex, was studied. This mutant yeast was more resistant to hydrogen peroxide treatment and able to degrade carbonylated proteins more efficiently than wild type. Nondenaturing gel electrophoresis followed by activity staining revealed that Delta rpn9 yeast cells had a higher activity of 20 S proteasome than wild type and that in both Delta rpn9 and wild-type cells treated with hydrogen peroxide, 20 S proteasome activity was increased with a concomitant decrease in 26 S proteasome activity. Protein multiubiquitination was not observed in the hydrogen peroxide-treated cells. Taken together, these results suggest that the 20 S proteasome degrades oxidized proteins without ubiquitination of target proteins. 相似文献
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The ubiquitin–proteasome system (UPS) is the primary selective degradation system in the nuclei and cytoplasm of eukaryotic cells, required for the turnover of myriad soluble proteins. The hundreds of factors that comprise the UPS include an enzymatic cascade that tags proteins for degradation via the covalent attachment of a poly-ubiquitin chain, and a large multimeric enzyme that degrades ubiquitinated proteins, the proteasome. Protein degradation by the UPS regulates many pathways and is a crucial component of the cellular proteostasis network. Dysfunction of the ubiquitination machinery or the proteolytic activity of the proteasome is associated with numerous human diseases. In this review we discuss the contributions of the proteasome to human pathology, describe mechanisms that regulate the proteolytic capacity of the proteasome, and discuss strategies to modulate proteasome function as a therapeutic approach to ameliorate diseases associated with altered UPS function. This article is part of a Special Issue entitled: Ubiquitin–Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf. 相似文献
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Owen M. Wilkins Sondra L. Downey Emily S. Weyburne David A. Williams Anne C. Mirabella Herman S. Overkleeft Alexei F. Kisselev 《Analytical biochemistry》2014
Proteasome-Glo is a homogeneous cell-based assay of proteasomal chymotrypsin-like, trypsin-like, and caspase-like activities using luminogenic substrates, commercially available from Promega. Here we report that the background activity from cleavage of the substrate of the trypsin-like sites by nonproteasomal proteases in multiple breast and lung cancer cell lines exceeds the activity of the proteasome. We also observed substantial background chymotrypsin-like activity in some cell lines. Thus, Proteasome-Glo assay must be used with caution, and it is necessary to include a specific proteasome inhibitor to determine the background for each proteasome activity. 相似文献
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Eisele F Braun B Pfirrmann T Wolf DH 《Biochemical and biophysical research communications》2006,350(2):329-333
Selective proteolysis is an important regulatory mechanism in all cells. In eukaryotes, this process gains specificity by tagging proteins with the small protein ubiquitin. K48 linked polyubiquitin chains of four and more ubiquitin moieties target proteins for hydrolysis by the proteasome. Prior to degradation the polyubiquitin chain is removed from the protein, cleaved into single units, and recycled. The deubiquitinating enzyme Ubp14 is an important catalyst of this process. Mutants of Ubp14 had been shown to accumulate non-cleaved oligo- and polyubiquitin chains, which resulted in inhibition of overall ubiquitin-proteasome linked proteolysis as well as in inhibition of degradation of some known substrates. Here we show that accumulation of ubiquitin chains due to defective Ubp14 does not uniformly lead to inhibition of ubiquitin-proteasome linked protein degradation. Instead, inhibition of degradation depends on the substrate tested. The results indicate the existence of different paths through which proteins enter the proteasome. 相似文献
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Nomura M Nomura N Yamashita J 《Biochemical and biophysical research communications》2005,335(3):900-905
Checkpoint kinase 1 (Chk1) is a cell cycle regulator and a heat shock protein 90 (Hsp90) client. It is essential for cell proliferation and survival. In this report, we analyzed the mechanisms of Chk1 regulation in U87MG glioblastoma cells using Geldanamycin (GA), which interferes with the function of Hsp90. GA reduced Chk1 protein level but not its mRNA level in glioblastoma cells. Co-treatment with GA and cycloheximide (CHX), a protein synthesis inhibitor, induced a decrease of half-life of the Chk1 protein to 3h and resulted in Chk1 down-regulation. CHX alone induced only 32% reduction of Chk1 protein even after 24h. These findings indicated that reduction of Chk1 by GA was due to destabilization and degradation of the protein. In addition, GA-induced down-regulation of Chk1 was reversed by MG132, a specific proteasome inhibitor. And it was revealed that Chk1 was ubiquitinated by GA. These results have indicated that degradation of Chk1 by GA was mediated by the ubiquitin-proteasome pathway in U87MG glioblastoma cells. 相似文献
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Miguel A. Aguileta Jelena Korac Thomas M. Durcan Jean-Fran?ois Trempe Michael Haber Kalle Gehring Suzanne Elsasser Oliver Waidmann Edward A. Fon Koraljka Husnjak 《The Journal of biological chemistry》2015,290(12):7492-7505
Mutations in the Park2 gene, encoding the RING-HECT hybrid E3 ubiquitin ligase parkin, are responsible for a common familial form of Parkinson disease. By mono- and polyubiquitinating target proteins, parkin regulates various cellular processes, including degradation of proteins within the 26 S proteasome, a large multimeric degradation machine. In our attempt to further elucidate the function of parkin, we have identified the proteasomal ubiquitin receptor Rpn13/ADRM1 as a parkin-interacting protein. We show that the N-terminal ubiquitin-like (Ubl) domain of parkin binds directly to the pleckstrin-like receptor for ubiquitin (Pru) domain within Rpn13. Using mutational analysis and NMR, we find that Pru binding involves the hydrophobic patch surrounding Ile-44 in the parkin Ubl, a region that is highly conserved between ubiquitin and Ubl domains. However, compared with ubiquitin, the parkin Ubl exhibits greater than 10-fold higher affinity for the Pru domain. Moreover, knockdown of Rpn13 in cells increases parkin levels and abrogates parkin recruitment to the 26 S proteasome, establishing Rpn13 as the major proteasomal receptor for parkin. In contrast, silencing Rpn13 did not impair parkin recruitment to mitochondria or parkin-mediated mitophagy upon carbonyl cyanide m-chlorophenyl hydrazone-induced mitochondrial depolarization. However, it did delay the clearance of mitochondrial proteins (TIM23, TIM44, and TOM20) and enhance parkin autoubiquitination. Taken together, these findings implicate Rpn13 in linking parkin to the 26 S proteasome and regulating the clearance of mitochondrial proteins during mitophagy. 相似文献
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Dehvari N Mahmud T Persson J Bengtsson T Graff C Winblad B Rönnbäck A Behbahani H 《Neurochemistry international》2012,60(5):533-542
Aggresomes are cytoplasmic inclusions which are localized at the microtubule organizing center (MTOC) as a result of induced proteasome inhibition, stress or over-expression of certain proteins. Aggresomes are linked to the pathogenesis of many neurodegenerative diseases. Here we studied whether amyloid precursor protein (APP), a type-I transmembrane glycoprotein, is localized in aggresomes after exposure to stress condition. Using confocal microscopy we found that APP is located in aggresomes and co-localized with vimentin, γ-tubulin, 20S and ubiquitin at the MTOC in response to proteasome dysfunction. An interaction between vimentin and APP was found after proteasome inhibition suggesting that APP is an additional protein constituent of aggresomes. Suppression of the proteasome system in APP-HEK293 cells overexpressing APP or transfected with APP Swedish mutation caused an accumulation of stable, detergent-insoluble forms of APP containing poly-ubiquitinated proteins. In addition, brain homogenates from transgenic mice expressing human APP with the Arctic mutation demonstrated an interaction between APP and the aggresomal-marker vimentin. These data suggest that malfunctioning of the proteasome system caused by mutation or overexpression of pathological or non-pathological proteins may lead to the accumulation of stable aggresomes, perhaps contributing to the neurodegeneration. 相似文献
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Saeki Y Sone T Toh-e A Yokosawa H 《Biochemical and biophysical research communications》2002,291(4):813-819
The high resolution structure of hemalbumin was determined by single crystal X-ray diffraction to a resolution of 1.9 A. The structure revealed the protoporphyrin IX bound to a single site within a hydrophobic cavity in subdomain IB, one of the principal binding sites for long chain fatty acid. The iron is penta coordinated with the fifth ligand comprised of the hydroxyl oxygen of Tyr-161 (phenolic oxygen to heme plane distance: 2.73 A) in an otherwise completely hydrophobic pocket. The heme propionic acid residues form salt bridges with His-142 and Lys-190, which together with a series of hydrophobic interactions, enclose and secure the heme within the IB helical motif. A detailed discussion of the structure together with its implications for the development of potential blood substitutes is presented. 相似文献
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Screen M Britton M Downey SL Verdoes M Voges MJ Blom AE Geurink PP Risseeuw MD Florea BI van der Linden WA Pletnev AA Overkleeft HS Kisselev AF 《The Journal of biological chemistry》2010,285(51):40125-40134
Proteasomes degrade most proteins in mammalian cells and are established targets of anti-cancer drugs. The majority of proteasome inhibitors are composed of short peptides with an electrophilic functionality (pharmacophore) at the C terminus. All eukaryotic proteasomes have three types of active sites as follows: chymotrypsin-like, trypsin-like, and caspase-like. It is widely believed that active site specificity of inhibitors is determined primarily by the peptide sequence and not the pharmacophore. Here, we report that active site specificity of inhibitors can also be tuned by the chemical nature of the pharmacophore. Specifically, replacement of the epoxyketone by vinyl sulfone moieties further improves the selectivity of β5-specific inhibitors NC-005, YU-101, and PR-171 (carfilzomib). This increase in specificity is likely the basis of the decreased cytotoxicity of vinyl sulfone-based inhibitors to HeLa cells as compared with that of epoxyketone-based inhibitors. 相似文献
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Hosoi A Takeda Y Sakuta K Ueha S Kurachi M Kimura K Maekawa R Kakimi K 《Biochemical and biophysical research communications》2008,371(2):242-246
Dendritic cells (DCs) transfected with mRNA encoding tumor-associated antigens (TAAs) can induce tumor-specific T-cell responses. To potentiate this, we transfected mature DCs (mDCs) with mRNA encoding TAA targeted to the proteasome. DCs were generated from bone marrow cells by culture with 20 ng/ml GM-CSF and maturation with 1 μg/ml LPS. These mDCs were then electroporated with 10 μg of mRNA. Antigen presentation after electroporation with in vitro transcribed mRNA was compared with mRNA from a construct of the TAA preceded by ubiquitin. Proteasomal targeting of mRNA encoding cotranslationally ubiquitinated antigen was found to enhance intracellular degradation of target protein, and result in more efficient priming and expansion of TAA-specific CD8+ T-cells. We therefore suggest that RNA-transfected DC vaccine efficacy could be improved by the use of mRNA targeted to the proteasome. 相似文献
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Structural defects in the regulatory particle-core particle interface of the proteasome induce a novel proteasome stress response 总被引:1,自引:0,他引:1
Proteasomes consist of a 19-subunit regulatory particle (RP) and 28-subunit core particle (CP), an α(7)β(7)β(7)α(7) structure. The RP recognizes substrates and translocates them into the CP for degradation. At the RP-CP interface, a heterohexameric Rpt ring joins to a heteroheptameric CP α ring. Rpt C termini insert individually into the α ring pockets to form a salt bridge with a pocket lysine residue. We report that substitutions of α pocket lysine residues produce an unexpected block to CP assembly, arising from a late stage defect in β ring assembly. Substitutions α5(K66A) and α6(K62A) resulted in abundant incorporation of immature CP β subunits, associated with a complete β ring, into proteasome holoenzymes. Incorporation of immature CP into the proteasome depended on a proteasome-associated protein, Ecm29. Using ump1 mutants, we identified Ecm29 as a potent negative regulator of RP assembly and confirmed our previous findings that proper RP assembly requires the CP. Ecm29 was enriched on proteasomes of pocket lysine mutants, as well as those of rpt4-Δ1 and rpt6-Δ1 mutants, in which the C-terminal residue, thought to contact the pocket lysine, is deleted. In both rpt6-Δ1 and α6(K62A) proteasomes, Ecm29 suppressed opening of the CP substrate translocation channel, which is gated through interactions between Rpt C termini and the α pockets. The ubiquitin ligase Hul5 was recruited to these proteasomes together with Ecm29. Proteasome remodeling through the addition of Ecm29 and Hul5 suggests a new layer of the proteasome stress response and may be a common response to structurally aberrant proteasomes or deficient proteasome function. 相似文献
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Richard A. Moravec Martha A. O’Brien Michael A. Scurria Terry L. Riss 《Analytical biochemistry》2009,387(2):294-1657
A luminescent method to individually measure the chymotrypsin-like, trypsin-like, or caspase-like activities of the proteasome in cultured cells was developed. Each assay uses a specific luminogenic peptide substrate in a buffer optimized for cell permeabilization, proteasome activity, and luciferase activity. Luminescence is generated in a coupled-enzyme format in which proteasome cleavage of the peptide conjugated substrate generates aminoluciferin, which is a substrate for luciferase. The homogeneous method eliminates the need to prepare individual cell extracts as samples. Luminogenic proteasome substrates and buffer formulations enabled development of a single reagent addition method with adequate sensitivity for 96- and 384-well plate formats. Proteasome trypsin-like specificity was enhanced by incorporating a mixture of protease inhibitors that significantly reduce nonspecific serum and cellular backgrounds. The assays were used to determine EC50 values for the specific proteasome inhibitors epoxomicin and bortezomib for each of the catalytic sites using a variety of cancer lines. These cell-based proteasome assays are direct, simple, and sensitive, making them ideal for high-throughput screening. 相似文献
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Impaired proteasome function in Alzheimer's disease 总被引:9,自引:0,他引:9
Inhibition of proteasome activity is sufficient to induce neuron degeneration and death; however, altered proteasome activity in a neurodegenerative disorder has not been demonstrated. In the present study, we analyzed proteasome activity in short-postmortem-interval autopsied brains from 16 Alzheimer's disease (AD) and nine age- and sex-matched controls. A significant decrease in proteasome activity was observed in the hippocampus and parahippocampal gyrus (48%), superior and middle temporal gyri (38%), and inferior parietal lobule (28%) of AD patients compared with controls. In contrast, no significant decrease in proteasome activity was observed in either the occipital lobe or the cerebellum. The loss of proteasome activity was not associated with a decrease in proteasome expression, suggesting that the proteasome may become inhibited in AD by a posttranslational modification. Together, these data indicate a possible role for proteasome inhibition in the neurodegeneration associated with AD. 相似文献
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Favour A. Akinjiyan Seth Carbonneau Nathan T. Ross 《Bioorganic & medicinal chemistry letters》2017,27(20):4589-4596
Protein degradation is critical for proteostasis, and the addition of polyubiquitin chains to a substrate is necessary for its recognition by the 26S proteasome. Therapeutic intervention in the ubiquitin proteasome system has implications ranging from cancer to neurodegeneration. Novel screening methods and chemical biology tools for targeting E1-activating, E2-conjugating and deubiquitinating enzymes will be discussed in this review. Approaches for targeting E3 ligase-substrate interactions as well as the proteasome will also be covered, with a focus on recently described approaches. 相似文献
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Zhang NY Jacobson AD Macfadden A Liu CW 《The Journal of biological chemistry》2011,286(29):25540-25546
The 26 S proteasome possesses two distinct deubiquitinating activities. The ubiquitin (Ub) chain amputation activity removes the entire polyUb chain from the substrates. The Ub chain trimming activity progressively cleaves a polyUb chain from the distal end. The Ub chain amputation activity mediates degradation-coupled deubiquitination. The Ub chain trimming activity can play a supportive or an inhibitory role in degradation, likely depending on features of the substrates. How Ub chain trimming assists degradation is not clear. We find that inhibition of the chain trimming activity of the 26 S proteasome with Ub aldehyde significantly inhibits degradation of Ub4 (Lys-48)-UbcH10 and causes accumulation of free Ub4 (generated from chain amputation) that can be retained on the proteasome. Also, a non-trimmable Lys-48-mimic Ub4 efficiently targets UbcH10 to the 26 S proteasome, but it cannot support efficient degradation of UbcH10 compared with regular Lys-48 Ub4. These results indicate that polyUb chain trimming promotes proteasomal degradation of Lys-48-linked substrates. Mechanistically, we propose that Ub chain trimming cleaves the proteasome-bound Lys-48-linked polyUb chains, which vacates the Ub binding sites of the 26 S proteasome, thus allowing continuous substrate loading. 相似文献