共查询到20条相似文献,搜索用时 0 毫秒
1.
Mary J. Kunjappu Mark Hochstrasser 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》2014
The proteasome is a cellular protease responsible for the selective degradation of the majority of the intracellular proteome. It recognizes, unfolds, and cleaves proteins that are destined for removal, usually by prior attachment to polymers of ubiquitin. This macromolecular machine is composed of two subcomplexes, the 19S regulatory particle (RP) and the 20S core particle (CP), which together contain at least 33 different and precisely positioned subunits. How these subunits assemble into functional complexes is an area of active exploration. Here we describe the current status of studies on the assembly of the 20S proteasome (CP). The 28-subunit CP is found in all three domains of life and its cylindrical stack of four heptameric rings is well conserved. Though several CP subunits possess self-assembly properties, a consistent theme in recent years has been the need for dedicated assembly chaperones that promote on-pathway assembly. To date, a minimum of three accessory factors have been implicated in aiding the construction of the 20S proteasome. These chaperones interact with different assembling proteasomal precursors and usher subunits into specific slots in the growing structure. This review will focus largely on chaperone-dependent CP assembly and its regulation. 相似文献
2.
Ferrington DA Sun H Murray KK Costa J Williams TD Bigelow DJ Squier TC 《The Journal of biological chemistry》2001,276(2):937-943
We have investigated the mechanisms that target oxidized calmodulin for degradation by the proteasome. After methionine oxidation within calmodulin, rates of degradation by the 20 S proteasome are substantially enhanced. Mass spectrometry was used to identify the time course of the proteolytic fragments released from the proteasome. Oxidized calmodulin is initially degraded into large proteolytic fragments that are released from the proteasome and subsequently degraded into small peptides that vary in size from 6 to 12 amino acids. To investigate the molecular determinants that result in the selective degradation of oxidized calmodulin, we used circular dichroism and fluorescence spectroscopy to assess oxidant-induced structural changes. There is a linear correlation between decreases in secondary structure and the rate of degradation. Calcium binding or the repair of oxidized calmodulin by methionine sulfoxide reductase induces comparable changes in alpha-helical content and rates of degradation. In contrast, alterations in the surface hydrophobicity of oxidized calmodulin do not alter the rate of degradation by the proteasome, indicating that changes in surface hydrophobicity do not necessarily lead to enhanced proteolytic susceptibility. These results suggest that decreases in secondary structure expose proteolytically sensitive sites in oxidized calmodulin that are cleaved by the proteasome in a nonprocessive manner. 相似文献
3.
The proteasome maturation protein POMP facilitates major steps of 20S proteasome formation at the endoplasmic reticulum 总被引:2,自引:0,他引:2
The quality control of proteins mediated by the plasticity of the proteasome system is regulated by the timely and flexible formation of this multisubunit proteolytic enzyme complex. Adaptable biogenesis of the 20S proteasome core complex is therefore of vital importance for adjusting to changing proteolytic requirements. However, the molecular mechanism and the cellular sites of mammalian proteasome formation are still unresolved. By using precursor complex-specific antibodies, we now show that the main steps in 20S core complex formation take place at the endoplasmic reticulum (ER). Thereby, the proteasome maturation protein (POMP)--an essential factor of mammalian proteasome biogenesis--interacts with ER membranes, binds to alpha1-7 rings, recruits beta-subunits stepwise and mediates the association of mammalian precursor complexes with the ER. Thus, POMP facilitates the main steps in 20S core complex formation at the ER to coordinate the assembly process and to provide cells with freshly formed proteasomes at their site of function. 相似文献
4.
The proteasome is responsible for most intracellular protein degradation and is essential for cell survival. Previous research has shown that the proteasome can be inhibited by a number of oxidants, including 4-hydroxynonenal (HNE). The present study demonstrates that HNE rapidly inhibits the chymotrypsin-like activity of the 20S proteasome purified from liver. Subunits containing HNE-adducts were identified following 2D gel electrophoresis, Western immunoblotting, and analysis by MALDI-TOF MS. At a time when only the chymotrypsin-like activity was inhibited, the alpha 6/C2 subunit was uniquely modified. These results provide important molecular details regarding the catalytic site-specific inhibition of proteasome by HNE. 相似文献
5.
Polypyridyl pentadentate ligands N4Py (1) and Bn-TPEN (2), along with their respective iron complexes, have been investigated for their ability to inhibit the purified 20S proteasome. Results demonstrated that the iron complexes of both ligands are potent inhibitors of the 20S proteasome (IC(50) = 9.2 μM for [Fe(II)(OH(2))(N4Py)](2+) (3) and 4.0 μM for [Fe(II)(OH(2))(Bn-TPEN)](2+) (4)). Control experiments showed that ligand 1 or Fe(II) alone showed no inhibition, whereas 2 was moderately active (IC(50) = 96 μM), suggesting that iron, when bound to these ligands, plays a key role in proteasome inhibition. Results from time-dependent inactivation studies suggest different modes of action for the iron complexes. Time-dependent decay of proteasome activity was observed upon incubation in the presence of 4, which accelerated in the presence of DTT, suggesting reductive activation of O(2) and oxidation of the 20S proteasome as a mode of action. In contrast, loss of 20S proteasome activity was not observed with 3 over time, suggesting inhibition through direct binding of the iron complex to the enzyme. Inhibition of the 20S proteasome by 4 was not blocked by reactive oxygen species scavengers, consistent with a unique oxidant being responsible for the time-dependent inhibition observed. 相似文献
6.
Interactions with LC3 and polyubiquitin chains link nbr1 to autophagic protein turnover 总被引:1,自引:0,他引:1
Nbr1, a ubiquitous kinase scaffold protein, contains a PB1, and a ubiquitin-associated (UBA) domain. We show here that the nbr1 UBA domain binds to lysine-48 and -63 linked polyubiquitin-B chains. Nbr1 also binds to the autophagic effector protein LC3-A via a novel binding site. Ubiquitin-binding, but not PB1-mediated p62/SQSTM1 interaction, is required to target nbr1 to LC3 and polyubiquitin-positive bodies. Nbr1 binds additionally to proteins implicated in ubiquitin-mediated protein turnover and vesicle trafficking: ubiquitin-specific peptidases USP8, and the endosomal transport regulator p14/Robld3. Nbr1 thus contributes to specific steps in protein turnover regulation disrupted in several hereditary human diseases.
Structured summary
MINT-7034452: USP8 (uniprotkb:P40818) physically interacts (MI:0218) with NBR1 (uniprotkb:Q14596) by pull down (MI:0096)MINT-7034438: SQSTM1 (uniprotkb:Q13501) and LC3 (uniprotkb:Q9H492) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7034309: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with Ubiquitin (uniprotkb:P62988) by pull down (MI:0096)MINT-7034323: NBR1 (uniprotkb:P97432) physically interacts (MI:0218) with Ubiquitin (uniprotkb:P62988) by pull down (MI:0096)MINT-7034233: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with USP8 (uniprotkb:P40818) by two hybrid (MI:0018)MINT-7034207: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with Robld3 (uniprotkb:Q9JHS3) by two hybrid (MI:0018)MINT-7034400, MINT-7034418: NBR1 (uniprotkb:Q14596) and LC3 (uniprotkb:Q9H492) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7034167: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with Ubiquitin B (uniprotkb:Q78XY9) by two hybrid (MI:0018)MINT-7034470: NBR1 (uniprotkb:Q14596) and USP8 (uniprotkb:P40818) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7034194: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with LC3-A (uniprotkb:Q91VR7) by two hybrid (MI:0018)MINT-7034336: SQSTM1 (uniprotkb:Q13501) physically interacts (MI:0218) with Ubiquitin (uniprotkb:P62988) by pull down (MI:0096)MINT-7034375: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with LC3 (uniprotkb:Q9H492) by pull down (MI:0096)MINT-7034350: NBR1 (uniprotkb:Q14596) and Ubiquitin (uniprotkb:P62988) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7034181: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with Tmed10 (uniprotkb:Q9D1D4) by two hybrid (MI:0018)MINT-7034220: NBR1 (uniprotkb:Q14596) physically interacts (MI:0218) with ube2o (uniprotkb:Q6ZPJ3) by two hybrid (MI:0018) 相似文献7.
8.
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 the A chain passes 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. 相似文献
9.
10.
Intracellular proteolysis is an essential process. In eukaryotes, most proteins in the cytosol and nucleus are degraded by the ubiquitin (Ub)-proteasome pathway. A major component within this system is the 26S proteasome, a 2.5MDa molecular machine, built from more than 31 different subunits. This complex is formed by a cylinder-shaped multimeric complex referred to as the proteolytic 20S proteasome (core particle, CP) capped at each end by another multimeric component called the 19S complex (regulatory particle, RP) or PA700. Structure, assembly and enzymatic mechanism have been elucidated only for the CP, whereas the organization of the RP is less well understood. The CP is composed of 28 subunits, which are arranged as an alpha7beta7beta7alpha7-complex in four stacked rings. The interior of the free core particle, which harbors the active sites, is inaccessible for folded and unfolded substrates and represents a latent state. This inhibition is relieved upon binding of the RP to the CP by formation of the 26S proteasome holoenzyme. This review summarizes the current knowledge of the structural features of 20S proteasomes. 相似文献
11.
Whittier JE Xiong Y Rechsteiner MC Squier TC 《The Journal of biological chemistry》2004,279(44):46135-46142
The 20 S proteasome has been suggested to play a critical role in mediating the degradation of abnormal proteins under conditions of oxidative stress and has been found in tight association with the molecular chaperone Hsp90. To elucidate the role of Hsp90 in promoting the degradation of oxidized calmodulin (CaM(ox)), we have purified red blood cell 20 S proteasomes free of Hsp90 and assessed their ability to degrade CaM(ox) in the absence or presence of Hsp90. Purified 20 S proteasome does not degrade CaM(ox) unless Hsp90 is added. CaM(ox) degradation is sensitive to both proteasome and Hsp90-specific inhibitors and is further enhanced in the presence of 2 mm ATP. Irrespective of the presence of Hsp90, we find that unoxidized CaM is not significantly degraded. Direct binding measurements demonstrate that Hsp90 selectively associates with CaM(ox); essentially no binding is observed between Hsp90 and unoxidized CaM. These results indicate that Hsp90 in association with the 20 S proteasome can selectively associate with oxidized and partially unfolded CaM to promote degradation by the proteasome. 相似文献
12.
Tilman Grune Diana Botzen Peter Voss Tobias Jung Gennady Ermak 《Archives of biochemistry and biophysics》2010,500(2):181-188
Tau is the major protein exhibiting intracellular accumulation in Alzheimer disease. The mechanisms leading to its accumulation are not fully understood. It has been proposed that the proteasome is responsible for degrading tau but, since proteasomal inhibitors block both the ubiquitin-dependent 26S proteasome and the ubiqutin-independent 20S proteasome pathways, it is not clear which of these pathways is involved in tau degradation. Some involvement of the ubiquitin ligase, CHIP in tau degradation has also been postulated during stress. In the current studies, we utilized HT22 cells and tau-transfected E36 cells in order to test the relative importance or possible requirement of the ubiquitin-dependent 26S proteasomal system versus the ubiquitin-independent 20S proteasome, in tau degradation. By means of ATP-depletion, ubiquitinylation-deficient E36ts20 cells, a 19S proteasomal regulator subunit MSS1-siRNA approaches, and in vitro ubiquitinylation studies, we were able to demonstrate that ubiquitinylation is not required for normal tau degradation. 相似文献
13.
Eukaryotic 20S proteasomes are complex oligomeric proteins. The maturation process of the 14 different - and -subunits has to occur in a highly coordinate manner. In addition -subunits are synthesized as proproteins and correct processing has to be guaranteed during complex maturation. The structure formation can be subdivided in different phases. The knowledge of the individual phases is summarized in this publication. As a first step the newly synthesized monomers have to adopt the correct tertiary structure, a process that might be supported in the case of the -subunits by the intramolecular chaperone activity postulated for the prosequences. Subsequently the -subunits form ring-like structures thereby providing docking sites for the different -subunits. The result most likely is a double ring structure (13S precursor) representing half-proteasomes, which contain immature proproteins. Two 13S precursors associate to form the proteolytically inactive 16S assembly intermediate which still contains unprocessed -monomers. In addition the chaperone Hsc73 is present within these particles suggesting an essential role during the structure formation process. The processing of monomers with an N-terminal threonine occurs within the 16S particles and is achieved autocatalytically by two subsequent processing events finally leading to the mature, active 20S proteasome. 相似文献
14.
An inhibitory protein for the 20S proteasome (also known as macropain, the multicatalytic proteinase complex and 20S proteinase) has been purified from bovine red blood cells. The inhibitor has an apparent molecular weight of 31,000 on SDS-PAGE and appears to form multimers under nondenaturing conditions. This protein inhibited all three of the putatively distinct catalytic activities of proteasome A (the active form of the proteinase) characterized by the hydrolysis of synthetic peptides such as Z-VLR-MNA, Z-GGL-AMC or Suc-LLVY-AMC and Z-LLE-beta NA. The inhibitor also prevented the hydrolysis of large protein substrates such as casein, lysozyme and bovine serum albumin. Proteasome L (the latent form of the proteinase) does not degrade these large protein substrates, but does hydrolyze the three synthetic peptides at rates similar to those by proteasome A. The inhibitor inhibited only two of these peptidase activities of proteasome L (hydrolysis of Z-GGL-AMC and of Z-LLE-beta NA or Suc-LLVY-AMC); it had no effect on the hydrolysis of Z-VLR-MNA. The inhibitor was specific for inhibition of the proteasome and had no effect on the activity of any other proteinase tested including trypsin, chymotrypsin, papain, subtilisin and both isoforms of calpain. Kinetic analysis indicates that the inhibitor interacted with the proteasome by a mechanism involving tight-binding. Because the proteasome appears to be a key component of the ATP/ubiquitin-dependent pathway of intracellular protein degradation, the inhibitor may represent an important regulatory protein of this pathway. 相似文献
15.
Growth-associated protein of 43 kDa (GAP-43) is cleaved nonprocessively by the 20S proteasome. 总被引:2,自引:0,他引:2
John B Denny 《European journal of biochemistry》2004,271(12):2480-2493
Purified, nonubiquitinated growth-associated protein of 43 kDa (GAP-43) was attacked by purified reticulocyte 20S proteasome but not by the 26S proteasome. Cleavage yielded 12 N-terminally labelled GAP-43 fragments that could be resolved by SDS/PAGE. Inhibitor experiments suggested that proteasome beta1 activity yielded the resolved bands and that proteasomebeta5 activity generated nonresolvable fragments. Processive degradation, yielding only nonresolvable fragments, therefore did not occur. Most of the resolved fragments co-migrated with fragments formed in the reticulocyte lysate translation mixture used for GAP-43 synthesis, which suggested that the fragments were also produced in the translation mixture by the endogenous reticulocyte lysate proteasome. Consistent with this idea, the addition of proteasome inhibitors to translation mixtures blocked fragment production. Ubiquitinated GAP-43 appeared to be the source of the fragments in the presence of ATP, and nonubiquitinated GAP-43 the source in the absence of ATP. The results therefore suggest that the lack of processing seen with the 20S proteasome is not an artefact arising from the way in which the 20S proteasome was purified. In one purification protocol, the GAP-43 fragments formed in translation mixtures co-purified with full-length GAP-43. These fragments were digested to nonresolvable products upon addition of purified 20S proteasome. Addition of calmodulin or G-actin blocked the consumption of both full-length GAP-43 and the co-purified GAP-43 fragments. This showed that the resolved fragments can re-enter the proteasome and be cleaved to nonresolvable products, indicating that the lack of processivity is not a result of their resistance to further proteasome attack. The difficult step therefore appears to be the transfer of the large fragments within the proteasome from the beta1 to the beta5 activity for further attack. 相似文献
16.
M Orlowski 《Biochemistry》2001,40(50):15318-15326
Two distinct activities cleaving bonds after hydrophobic amino acids have been identified in the bovine pituitary 20 S proteasome. One, expressed by the X subunit, that cleaves bonds after aromatic and branched chain amino acids was designated as chymotrypsin-like (ChT-L).(1) The second, expressed by the Y subunit, that cleaves bonds after acidic amino acids was designated as peptidylglutamyl-peptide hydrolyzing (PGPH) but also cleaves bonds after branched chain amino acids. Low micromolar concentrations of the arginine-rich histone H3 (H3) are shown to induce changes in the specificity of the proteasome by selectively activating cleavages after branched chain and acidic amino acids while inhibiting cleavage of peptidyl-arylamide bonds in synthetic substrates. H3 activates 15-fold cleavage after leucine but not phenylalanine residues in model synthetic substrates. The activation is associated with a decrease in K(m) and an increase in V(max), suggesting positive allosteric activation. H3 activates more than 60-fold degradation of the oxidized B-chain of insulin, by cleaving mainly bonds after acidic and branched chain amino acids, and accelerates the degradation of casein and lysozyme, the latter in the presence of dithiothreitol. The degradation of lysozyme in the presence of H3 generates fragments that differ from those in its absence, indicating H3-induced specificity changes. H3 inhibits cleavage of the Trp3-Ser4 and Tyr5-Gly6 bonds in gonadotropin releasing hormone, bonds cleaved by the ChT-L activity in the absence of H3. The results suggest H3-selective activation of the Y subunit and specificity changes that could potentially affect proteasomal function in the nuclear compartment. 相似文献
17.
Frankia is an actinomycete that fixes atmospheric nitrogen in symbiotic association with the root systems of a variety of non-leguminous plants, denominated actinorhizal plants. Information on the biology of proteolysis in Frankia is almost non-existent as it is extremely difficult to grow this organism. We have purified 20S proteasomes from Frankia strain ACN14a/ts-r. It is composed of one alpha-subunit and one beta-subunit, which assemble into the canonical structure of four rings of seven subunits each. The enzyme displayed a chymotrypsin-like activity against synthetic substrates and was sensitive to lactacystin, a specific proteasome inhibitor. Analysis of the structural genes and the flanking regions revealed a similar organization to Rhodococcus erythropolis, Mycobacterium tuberculosis and Streptomyces coelicolor and showed that the beta-subunit is encoded with a 52-amino-acid propeptide that is cleaved off in the course of the assembly. We report also for the first time the in vitro assembly of chimeric proteasomes composed of Frankia and Rhodococcus erythropolis subunits, which are correctly assembled and proteolytically active. 相似文献
18.
Glucose-6-phosphate dehydrogenase (G6PD) was treated with various concentrations of hypochlorite, which is produced by myeloperoxidase and is one of the most important oxidants during inflammatory processes. Inhibition of enzymatic activity, protein fragmentation, and proteolytic susceptibility toward the isolated 20S proteasome of G6PD were investigated. With rising hypochlorite concentrations, an increased proteasomal degradation of G6PD was measured. This occurred at higher hypochlorite concentrations than G6PD inactivation and at lower levels than G6PD fragmentation. The proteolytic activities of the 20S proteasome itself was determined by degradation of oxidized model proteins and cleavage of the synthetic proteasome substrate suc-LLVY-MCA. Proteasome activities remained intact at hypochlorite concentrations in which G6PD is maximally susceptible to proteasomal degradation. Only higher hypochlorite concentrations could decrease the proteolytic activities of the proteasome, which was accompanied by disintegration and fragmentation of the proteasome and proteasome subunits. Therefore, we conclude that the 20S proteasome can degrade proteins moderately damaged by hypochlorite and could contribute to an increased protein turnover in cells exposed to inflammatory stress. 相似文献
19.
Wang CC Bozdech Z Liu CL Shipway A Backes BJ Harris JL Bogyo M 《The Journal of biological chemistry》2003,278(18):15800-15808
We describe here biochemical characterization of the 20 S proteasome from the parasitic protozoan Trypanosoma brucei. Similar to the mammalian proteasome, the T. brucei proteasome is made up of seven alpha- and seven beta-subunits. Of the seven beta-type subunits, five contain pro-sequences that are proteolytically removed during assembly, and three of them are predicted to be catalytic based on primary sequence. Affinity labeling studies revealed that, unlike the mammalian proteasome where three beta-subunits were labeled by the affinity reagents, only two beta-subunits of the T. brucei proteasome were labeled in the complex. These two subunits corresponded to beta2 and beta5 subunits responsible for the trypsin-like and chymotrypsin-like proteolytic activities, respectively. Screening of a library of 137,180 tetrapeptide fluorogenic substrates against the T. brucei 20 S proteasome confirmed the nominal beta1-subunit (caspase-like or PGPH) activity and identified an overall substrate preference for hydrophobic residues at the P1 to P4 positions in a substrate. This overall stringency is relaxed in the 11 S regulator (PA26)-20 S proteasome complex, which shows both appreciable activities for cleavage after acidic amino acids and a broadened activity for cleavage after basic amino acids. The 20 S proteasome from T. brucei also shows appreciable activity for cleavage after P1-Gln that is minimally observed in the human counterpart. These results demonstrate the importance of substrate sequence specificity of the T. brucei proteasome and highlight its biochemical divergence from the human enzyme. 相似文献
20.
Kun-Peng Liu Dan Zhou Dong-Yun Ouyang Li-Hui Xu Yao Wang Li-Xian Wang Hao Pan Xian-Hui He 《Biochemical and biophysical research communications》2013
Autophagy is a conserved mechanism for controlling the degradation of misfolded proteins and damaged organelles in eukaryotes and can be induced by nutrient withdrawal, including serum starvation. Although differential acetylation of autophagy-related proteins has been reported to be involved in autophagic flux, the regulation of acetylated microtubule-associated protein 1 light chain 3 (LC3) is incompletely understood. In this study, we found that the acetylation levels of phosphotidylethanolamine (PE)-conjugated LC3B (LC3B-II), which is a critical component of double-membrane autophagosome, were profoundly decreased in HeLa cells upon autophagy induction by serum starvation. Pretreatment with lysosomal inhibitor chloroquine did not attenuate such deacetylation. Under normal culture medium, we observed increased levels of acetylated LC3B-II in cells treated with tubacin, a specific inhibitor of histone deacetylase 6 (HDAC6). However, tubacin only partially suppressed serum-starvation-induced LC3B-II deacetylation, suggesting that HDAC6 is not the only deacetylase acting on LC3B-II during serum-starvation-induced autophagy. Interestingly, tubacin-induced increase in LC3B-II acetylation was associated with p62/SQSTM1 accumulation upon serum starvation. HDAC6 knockdown did not influence autophagosome formation but resulted in impaired degradation of p62/SQSTM1 during serum starvation. Collectively, our data indicated that LC3B-II deacetylation, which was partly mediated by HDAC6, is involved in autophagic degradation during serum starvation. 相似文献