Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.25.1 (proteasome)
 28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

PA28, a protein activator of the 20 S proteasome, was previously identified in soluble extracts of bovine red blood cells (Ma, C.-P., Slaughter, C. A., and DeMartino, G. N. (1992) J. Biol. Chem. 267, 10515-10523). To determine whether this regulatory protein is as widely distributed as the proteasome, PA28 content and activity were examined in various eukaryotic tissues by immunoblot analysis and by functional assays of tissue extracts. PA28 protein was present in all sources examined. PA28 activity, however, was not detected in many of these sources, including those with the highest level of PA28 protein. To determine the biochemical basis of this result, PA28 was purified from extracts of rat liver, which had high levels of PA28 protein but no PA28 activity. The resulting purified PA28 had no detectable activity but had native and subunit molecular weights indistinguishable from the active PA28 of bovine red blood cells. Using the inactivation of purified PA28 as an assay, a protein that inactivated PA28 without altering its apparent molecular weight on SDS-polyacrylamide gel electrophoresis was identified, purified, and characterized from bovine liver. It had biochemical and catalytic characteristics similar to those of lysosomal carboxypeptidase B. When leupeptin, an inhibitor of lysosomal carboxypeptidase B, was included in the buffers used for the preparation of PA28, PA28 activity was detected in tissues which otherwise failed to demonstrate this activity. A similar result was obtained when extracts were prepared in a manner that minimized disruption of lysosomes. Other carboxypeptidases such as carboxypeptidase Y and pancreatic carboxypeptidase B also inactivated PA28 without altering its apparent molecular weight. Active PA28 binds to the proteasome to form a protease-activator complex that can be isolated after velocity sedimentation centrifugation through glycerol density gradients. Carboxypeptidase-inactivated PA28 failed to form such a complex, suggesting that the carboxyl terminus of PA28 is required for binding to the proteasome. These results indicate the importance of the carboxyl terminus of PA28 for proteasome activation.
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PMID:PA28, an activator of the 20 S proteasome, is inactivated by proteolytic modification at its carboxyl terminus. 822 60

PA28 is a protein activator of the 20S proteasome. It has a native molecular weight of approximately 200,000 and is composed of six 28,000-dalton subunits arranged in a ring-shaped complex. Purified preparations of PA28 contain two polypeptides, alpha and beta, which are about 50% identical in primary structure. It has been unclear whether native PA28 consists of two distinct homohexameric proteins or of a single protein containing both alpha and beta subunits. To distinguish between these possibilities, we prepared antibodies that reacted specifically with either the alpha or beta subunit and used these subunit-specific antibodies in two types of experiments designed to elucidate PA28 quaternary structure. In the first experiment, the alpha and beta subunits were completely co-immunoprecipitated by each subunit-specific antibody, indicating that both subunits were part of a single protein complex. In the second experiment, PA28 was chemically cross-linked using bis(sulfosuccinimidyl)suberate. When the cross-linked products were immunoblotted after SDS-polyacrylamide gel electrophoresis, indistinguishable patterns were obtained with each subunit-specific antibody. These results confirm that the alpha and beta subunits were part of the same protein complex. The pattern of cross-linked products also provided insight as to the relative abundance and arrangement of the subunits within the PA28 complex and indicated that the ring-shaped PA28 hexamer may be composed of alternating alpha and beta subunits with a stoichiometry of (alphabeta)3. PA28 was inactivated by treatment with carboxypeptidase Y, which cleaved Tyr and Ile residues from the carboxyl terminus of the alpha subunit but had very little effect on the beta subunit. This selective and limited proteolysis prevented binding of both alpha and beta subunits to the proteasome and therefore provides additional evidence of the heterodimeric nature of PA28. These results indicate that a short carboxyl-terminal sequence of the alpha subunit is critical for binding of native PA28 to the proteasome. To learn about the relative functions of the alpha and beta subunits, PA28alpha was expressed in Escherichia coli and purified to homogeneity. Purified PA28alpha stimulated proteasome activity but required 5-10-fold greater concentrations than the heterodimeric PA28 to achieve a given level of activity. These results suggest that the heterodimeric structure of PA28 is required for maximal proteasome activation.
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PMID:A model for the quaternary structure of the proteasome activator PA28. 882 98

Lactacystin, the most specific inhibitor of the proteasome, strongly inhibited at pH 5.5 the activity of human platelet lysosomal cathepsin A-like enzyme. At a concentration as low as 1-5 microM it almost completely decreased the hydrolysis rate of cathepsin A specific substrates: Cbz-Phe-Ala and FA-Phe-Phe. This inhibition was probably due to the lactacystin intermediate beta-lactone formed during 10 min hydrolysis at pH 8.0 since nonhydrolyzed inhibitor did not affect cathepsin A activity. Basing on similarities in the inhibitor sensitivity, pH optimum, and substrate preferences it is suggested that the cathepsin A-like activity may be involved in chymotrypsin-like activity of the proteasome.
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PMID:Lactacystin, a specific inhibitor of the proteasome, inhibits human platelet lysosomal cathepsin A-like enzyme. 917 83

MHC class I molecules display peptides selected from a poorly characterized pool of peptides available in the endoplasmic reticulum. We analyzed the diversity of peptides available to MHC class I molecules by monitoring the generation of an OVA-derived octapeptide, OVA257-264 (SL8), and its C-terminally extended analog, SL8-I. The poorly antigenic SL8-I could be detected in cell extracts only after its conversion to the readily detectable SL8 with carboxypeptidase Y. Analysis of extracts from cells expressing the minimal precursor Met-SL8-I by this method revealed the presence of SL8/Kb and the extended SL8-I/Kb complexes, indicating that the peptide pool contained both peptides. In contrast, cells expressing full length OVA generated only the SL8/Kb complex, demonstrating that the peptide pool generated from the full length precursor contained only a subset of potential MHC-binding peptides. Deletion analysis revealed that SL8-I was generated only from precursors lacking additional C-terminal flanking residues, suggesting that the generation of the C terminus of the SL8 peptide involves a specific endopeptidase cleavage. To investigate the protease responsible for this cleavage, we tested the effect of different protease inhibitors on the generation of the SL8 and SL8-I peptides. Only the proteasome inhibitors blocked generation of SL8, but not SL8-I. These findings demonstrate that the specificities of the proteases in the Ag-processing pathway, which include but are not limited to the proteasome, limit the diversity of peptides available for binding by MHC class I molecules in the endoplasmic reticulum.
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PMID:Specific proteolytic cleavages limit the diversity of the pool of peptides available to MHC class I molecules in living cells. 1020 12

Previous studies have described a human platelet cathepsin A-like enzyme with a number of similarities to the "acidic" and "neutral" chymotrypsin-like activities of the proteasome. This includes its strong inhibition by the highly specific proteasome inhibitor Lactacystin/beta-lactone, suggesting that either the Cbz-Phe-Ala-hydrolyzing activity attributed to cathepsin A was due to the chymotrypsin-like activity of the proteasome or that lactacystin was not a specific inhibitor of the proteasome. In the present study we discard the first possibility on the basis of the following findings: (a) human platelet cathepsin A, unlike proteasome, binds to concanavalin A, and does not bind to Heparin-Sepharose at pH 7.4; (b) neither the chymotrypsin-like activity of the proteasome, nor proteasome antigens are detected in the cathepsin A preparation; (c) purified proteasome does not exhibit Cbz-Phe-Ala-hydrolyzing activity; (d) Z-lle-Glu-(Ot-Bu)Ala-leucinal (PSI), a compound that selectively inhibits the chymotrypsin-like activity of the proteasome at a concentration of 10 microM has no inhibitory effect on the carboxypeptidase activity of cathepsin A; (e) cathepsin A, free of the proteasome, is completely inhibited by micromolar concentrations of lactacystin/beta-lactone. It is therefore concluded that lactacystin/beta-lactone is not a specific inhibitor of the proteasome.
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PMID:Separation of cathepsin A-like enzyme and the proteasome: evidence that lactacystin/beta-lactone is not a specific inhibitor of the proteasome. 1085 5

The endoplasmic reticulum (ER) has a mechanism to block the exit of misfolded or unassembled proteins from the ER for the downstream organelles in the secretory pathway. Misfolded proteins retained in the ER are subjected to proteasome-dependent degradation in the cytosol when they cannot achieve correct folding and/or assembly within an appropriate time window. Although specific mannose trimming of the protein-bound oligosaccharide is essential for the degradation of misfolded glycoproteins, the precise mechanism for this recognition remains obscure. Here we report a new alpha-mannosidase-like protein, Mnl1p (mannosidase-like protein), in the yeast ER. Mnl1p is unlikely to exhibit alpha1,2-mannosidase activity, because it lacks cysteine residues that are essential for alpha1,2-mannosidase. However deletion of the MNL1 gene causes retardation of the degradation of misfolded carboxypeptidase Y, but not of the unglycosylated mutant form of the yeast alpha-mating pheromone. Possible roles of Mnl1p in the degradation and in the ER-retention of misfolded glycoproteins are discussed.
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PMID:Mnl1p, an alpha -mannosidase-like protein in yeast Saccharomyces cerevisiae, is required for endoplasmic reticulum-associated degradation of glycoproteins. 1125 55

Misfolded proteins are recognized in the endoplasmic reticulum (ER), transported back to the cytoplasm and degraded by the proteasome. Processing intermediates of N-linked oligosaccharides on incompletely folded glycoproteins have an important role in their folding/refolding, and also in their targeting to proteolytic degradation. In Saccharomyces cerevisiae, we have identified a gene coding for a non-essential protein that is homologous to mannosidase I (HTM1) and that is required for degradation of glycoproteins. Deletion of the HTM1 gene does not affect oligosaccharide trimming. However, deletion of HTM1 does reduce the rate of degradation of the mutant glycoproteins such as carboxypeptidase Y, ABC-transporter Pdr5-26p and oligosaccharyltransferase subunit Stt3-7p, but not of mutant Sec61-2p, a non-glycoprotein. Our results indicate that although Htm1p is not involved in processing of N-linked oligosaccharides, it is required for their proteolytic degradation. We propose that this mannosidase homolog is a lectin that recognizes Man8GlcNAc2 oligosaccharides that serve as signals in the degradation pathway.
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PMID:Htm1p, a mannosidase-like protein, is involved in glycoprotein degradation in yeast. 1137 35

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro-alpha-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain-containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.
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PMID:Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation. 1138 Oct 90

We describe the inhibitory effect of the proteasome inhibitor, lactacystin, on cathepsin A activity in murine melanoma cell lines. In vitro lactacystin metabolite, beta-lactone, at a concentration of 1 microM, significantly suppressed cathepsin A activity in B78 melanoma cell lysates by about 50%. Exposure of three murine melanoma cell lines with different metastatic potential to lactacystin at a concentration of 5 microM for 6 h caused a significant reduction in the carboxypeptidase activity of this enzyme, while the inhibitory activity remained unchanged for at least 12 h. Other proteasome-specific inhibitors, e.g. epoxomicin and N-benzyloxycarbonyl-Ile-Glu(O-tert-Bu)-Ala-leucinal (PSI) at a concentration of 1 microM did not affect cathepsin A activity in melanoma cell line lysates. These data support our previous proposal that lactacystin is not a specific inhibitor of the proteasome. Since cathepsin A is also a tumor-associated enzyme, further research is needed to clarify its role and the significance of its inhibition by lactacystin in tumor biology.
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PMID:Lactacystin inhibits cathepsin A activity in melanoma cell lines. 1139 45

Cytochrome P450, CYP3A4, is the dominant human liver endoplasmic reticulum (ER) hemoprotein enzyme, responsible for the metabolism of over 60% of clinically relevant drugs. We have previously shown that mechanism-based suicide inactivation of CYP3A4 and its rat liver ER orthologs, CYPs 3A, via heme-modification of their protein moieties, results in their ubiquitin (Ub)-dependent 26S proteasomal degradation (Korsmeyer et al. (1999) Arch. Biochem. Biophys. 365, 31; Wang et al. (1999) Arch. Biochem. Biophys. 365, 45). This is not surprising given that the heme-modified CYP3A proteins are structurally damaged. To determine whether the turnover of the native enzyme similarly recruited this pathway, we heterologously expressed this protein in wild-type Saccharomyces cerevisiae and mutant strains (hrd1Delta, hrd2-1, and hrd3Delta) previously shown to be deficient in the Ub-dependent 26S proteasomal degradation of the polytopic ER protein 3-hydroxy-3-methylglutaryl-CoA reductase (isoform Hmg2p), the rate-limiting enzyme in sterol biosynthesis, as well as in strains deficient in ER-associated Ub-conjugating enzymes, Ubc6p and/or Ubc7p (Hampton et al. (1996) Mol. Biol. Cell 7, 2029; Hampton and Bhakta (1997) Proc. Natl. Acad. Sci. USA 94, 12,944). Our findings reveal that in common with the degradation of Hmg2p, that of native CYP3A4 also requires Hrd2p (a subunit of the 19S cap complex of the 26S proteasome) and Ubc7p, and to a much lesser extent Hrd3p, a component of the ER-associated Ub-ligase complex. In contrast to Hmg2p-degradation, that of native CYP3A4 does not appear to absolutely require Hrd1p, another component of the ER-associated Ub-ligase complex. Furthermore, studies in a S. cerevisiae pep4Delta strain proven to be deficient in the vacuolar degradation of carboxypeptidase Y indicated that CYP3A4 degradation is also largely independent of vacuolar (lysosomal) proteolytic function. The degradation of two other native ER proteins, Sec61p and Sec63p, normal components of the ER translocon, were also examined in parallel and found to be stabilized to some extent in HRD2- and UBC7-deficient strains. Together these findings attest to the remarkable mechanistic diversity in the normal degradation of ER proteins.
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PMID:Ubiquitin-dependent 26S proteasomal pathway: a role in the degradation of native human liver CYP3A4 expressed in Saccharomyces cerevisiae? 1151 67


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