Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

An immunological hierarchy among three H-2Db-restricted cytotoxic T lymphocyte (CTL) determinants in simian virus 40 (SV40) large T antigen (Tag) was described previously: determinants I and II/III are immunodominant, whereas determinant V is immunorecessive. To assess the immunogenicity of each determinant individually and define mechanisms that contribute to the immunorecessive nature of determinant V, we constructed a panel of recombinant vaccinia viruses (rVVs) expressing minigenes encoding these determinants in various polypeptide contexts. We found the following. (i) Immunization of mice with an rVV encoding full-length SV40 Tag resulted in priming for CTL responses to determinants I and II/III but not determinant V. (ii) rVVs encoding peptide I or II/III in the cytosol or targeted to the endoplasmic reticulum (ER) were highly antigenic and immunogenic. (iii) rVVs encoding peptide V minigenes were antigenic and immunogenic if the peptide was targeted to the ER, expressed in the cytosol with short flanking sequences, or expressed from within a self-protein, murine dihydrofolate reductase. (iv) Presentation of the nonflanked peptide V (preceded by a Met codon only) could be enhanced by using a potent inhibitor of the proteasome. (v) H-2Db-epitope V peptide complexes decayed more rapidly than complexes containing epitope I or II/III peptides. In brefeldin A blocking experiments, functional epitope V complexes were detected longer on targets expressing ER-targeted epitope V than on targets expressing forms of epitope V dependent on the transporter associated with antigen processing. Therefore, limited formation of relatively unstable cell surface H-2Db complexes most likely contributes to the immunorecessive nature of epitope V within SV40 Tag. Increasing the delivery of epitope V peptide to the major histocompatibility complex class I presentation pathway by ER targeting dramatically enhanced the immunogenicity of epitope V.
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PMID:An endoplasmic reticulum-targeting signal sequence enhances the immunogenicity of an immunorecessive simian virus 40 large T antigen cytotoxic T-lymphocyte epitope. 944 50

We report here that amino acid analogs, which activate hsp70 promoter, are powerful transcriptional activators of human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR), an activation which was impaired when the two kappaB sites present in the LTR were mutated or deleted. Amino acid analogs also stimulated the transcription of a kappaB-controlled reporter gene. Upon treatment with amino acid analogs, the two NF-kappaB subunits (p65 and p50), which are characterized by a relatively long half-life, redistributed into the nucleus where they bound to kappaB elements. This phenomenon, which began to be detectable after 1 h of treatment, was concomitant with the degradation of the short lived inhibitory subunit IkappaB-alpha by the proteasome. However, contrasting with other NF-kappaB inducers that trigger IkappaB-alpha degradation through a phosphorylation step, amino acid analogs did not change IkappaB-alpha isoform composition. Antioxidant conditions inhibited amino acid analog stimulatory action toward NF-kappaB. This suggests that aberrant protein conformation probably generates a pro-oxidant state that is necessary for IkappaB-alpha proteolysis by the proteasome. Moreover, this activation of NF-kappaB appeared different from that mediated by endoplasmic reticulum overload as it was not inhibited by calcium chelation.
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PMID:Amino acid analogs activate NF-kappaB through redox-dependent IkappaB-alpha degradation by the proteasome without apparent IkappaB-alpha phosphorylation. Consequence on HIV-1 long terminal repeat activation. 945 29

The human immunodeficiency virus type 1 (HIV-1) vpu gene encodes a type I anchored integral membrane phosphoprotein with two independent functions. First, it regulates virus release from a post-endoplasmic reticulum (ER) compartment by an ion channel activity mediated by its transmembrane anchor. Second, it induces the selective down regulation of host cell receptor proteins (CD4 and major histocompatibility complex class I molecules) in a process involving its phosphorylated cytoplasmic tail. In the present work, we show that the Vpu-induced proteolysis of nascent CD4 can be completely blocked by peptide aldehydes that act as competitive inhibitors of proteasome function and also by lactacystin, which blocks proteasome activity by covalently binding to the catalytic beta subunits of proteasomes. The sensitivity of Vpu-induced CD4 degradation to proteasome inhibitors paralleled the inhibition of proteasome degradation of a model ubiquitinated substrate. Characterization of CD4-associated oligosaccharides indicated that CD4 rescued from Vpu-induced degradation by proteasome inhibitors is exported from the ER to the Golgi complex. This finding suggests that retranslocation of CD4 from the ER to the cytosol may be coupled to its proteasomal degradation. CD4 degradation mediated by Vpu does not require the ER chaperone calnexin and is dependent on an intact ubiquitin-conjugating system. This was demonstrated by inhibition of CD4 degradation (i) in cells expressing a thermally inactivated form of the ubiquitin-activating enzyme E1 or (ii) following expression of a mutant form of ubiquitin (Lys48 mutated to Arg48) known to compromise ubiquitin targeting by interfering with the formation of polyubiquitin complexes. CD4 degradation was also prevented by altering the four Lys residues in its cytosolic domain to Arg, suggesting a role for ubiquitination of one or more of these residues in the process of degradation. The results clearly demonstrate a role for the cytosolic ubiquitin-proteasome pathway in the process of Vpu-induced CD4 degradation. In contrast to other viral proteins (human cytomegalovirus US2 and US11), however, whose translocation of host ER molecules into the cytosol occurs in the presence of proteasome inhibitors, Vpu-targeted CD4 remains in the ER in a transport-competent form when proteasome activity is blocked.
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PMID:CD4 glycoprotein degradation induced by human immunodeficiency virus type 1 Vpu protein requires the function of proteasomes and the ubiquitin-conjugating pathway. 949 87

Expression of the T cell antigen receptor (TCR) on the surface of thymocytes and mature T cells is dependent on the assembly of receptor subunits into TCRs in the endoplasmic reticulum (ER) and their successful traversal of the secretory pathway to the plasma membrane. TCR subunits that fail to exit the ER for the Golgi complex are degraded by nonlysosomal processes that have been referred to as "ER degradation". The molecular basis for the loss of the TCR CD3-delta and TCR-alpha subunits from the ER was investigated in lymphocytes. For CD3-delta, we describe a process leading to its degradation that includes trimming of mannose residues from asparagine-linked (N-linked) oligosaccharides, generation of ubiquitinated membrane-bound intermediates, and proteasome-dependent removal from the ER membrane. When either mannosidase activity or the catalytic activity of proteasomes was inhibited, loss of CD3-delta was markedly curtailed and CD3-delta remained membrane bound in a complex with CD3-epsilon. TCR-alpha was also found to be degraded in a proteasome-dependent manner with ubiquitinated intermediates. However, no evidence of a role for mannosidases was found for TCR-alpha, and significant retrograde movement through the ER membrane took place even when proteasome function was inhibited. These findings provide new insights into mechanisms employed to regulate levels of TCRs, and underscore that cells use multiple mechanisms to target proteins from the ER to the cytosol for degradation.
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PMID:Novel aspects of degradation of T cell receptor subunits from the endoplasmic reticulum (ER) in T cells: importance of oligosaccharide processing, ubiquitination, and proteasome-dependent removal from ER membranes. 950 Jul 86

The identification and characterization of the CFTR gene and protein have provided not only a major impetus to the dissection of the molecular pathophysiology of cystic fibrosis (CF) but also a new perspective on the structure and function of the large superfamily of membrane transport proteins to which it belongs. While the mechanism of the active vectorial translocation of many hydrophobic substrates by several of these transporters remains nearly as perplexing as it has for several decades, considerable insight has been gained into the control of the bidirectional permeation of chloride ions through a single CFTR channel by the phosphorylation of the R-domain and ATP interactions at the two nucleotide binding domains. However, details of these catalytic and allosteric mechanisms remain to be elucidated and await the replacement of two-dimensional conceptualizations with three dimensional structure information. Secondary and tertiary structure determination is required both for the understanding of the mechanism of action of the molecule and to enable a more complete appreciation of the misfolding and misprocessing of mutant CFTR molecules. This is the primary cause of the disease in the majority of the patients and hence understanding the details of the cotranslational interactions with multiple molecular chaperones, the ubiquitin-proteasome pathway and other components of the quality control machinery at the endoplasmic reticulum could provide a basis for the development of new therapeutic interventions.
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PMID:Cystic fibrosis: channel, catalytic, and folding properties of the CFTR protein. 951 28

Proteasomes are large multicatalytic proteinase complexes which are responsible for the selective degradation of cellular proteins and the production of peptides for antigen presentation. Proteasomes are localized both in the nucleus and in the cytoplasm, where some are associated with the endoplasmic reticulum membrane. Recent studies have shown differences in the localization of proteasome subpopulations, demonstrated the functional importance of endoplasmic reticulum-associated proteasomes and investigated the role of putative nuclear localization signals and tyrosine phosphorylation on proteasome transport into the nucleus.
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PMID:Intracellular distribution of proteasomes. 952 20

Factor VIII (FVIII) and factor V (FV) are homologous coagulation cofactors sharing a similar domain organization (A1-A2-B-A3-C1-C2) and are both extensively glycosylated within their B-domains. In mammalian cell expression systems, compared with FV, the FVIII primary translation product is inefficiently transported out of the endoplasmic reticulum. Here we show that FVIII is degraded within the cell by a lactacystin-inhibitable pathway, implicating the cytosolic 20 S proteasome machinery. Protein chaperones calnexin (CNX) and calreticulin (CRT) preferentially interact with glycoproteins containing monoglucosylated N-linked oligosaccharides and are proposed to traffic proteins through degradative and/or secretory pathways. Utilizing co-immunoprecipitation assays, intracellular FVIII was detected in association with CNX maximally within 30 min to 1 h following synthesis, whereas FV could not be detected in association with CNX. In contrast, both FVIII and FV displayed interaction with CRT during transit through the secretory pathway. B-domain deleted FVIII significantly reduced the CNX and CRT interaction, indicating the B-domain may represent a primary CNX and CRT interaction site. In the presence of inhibitors of glucose trimming, the interactions of FVIII with CNX, and of FVIII and FV with CRT, were significantly reduced whereas the secretion of FVIII, and not FV, was inhibited. In addition, transfection in a glucosidase I-deficient Chinese hamster ovary cell line (Lec23) demonstrated that both degradation and secretion of FVIII were inhibited, with little effect on the secretion of FV. These results support that CNX and CRT binding, mediated at least in part by the B-domain of FVIII, is required for efficient FVIII degradation and secretion. In contrast, FV does not require CNX interaction for efficient secretion. The results suggest a unique requirement for carbohydrate processing and molecular chaperone interactions that may limit the productive secretion of FVIII.
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PMID:Differential interaction of coagulation factor VIII and factor V with protein chaperones calnexin and calreticulin. 952 69

The peptidase activities of eukaryotic proteasomes are markedly activated by the 11 S REG or PA28. The three identified REG subunits, designated alpha, beta, and gamma, differ significantly in sequence over a short span of 15-30 amino acids that we call homolog-specific inserts. These inserts were deleted from each REG to produce the mutant proteins REGalphaDeltai, REGbetaDeltai, and REGgammaDeltai. The purified recombinant proteins were then tested for their ability to oligomerize and activate the proteasome. Both REGalphaDeltai and REGgammaDeltai formed apparent heptamers and activated human red cell proteasomes to the same extent as their full-length counterparts. By contrast, REGbetaDeltai exhibited, at low protein concentrations, reduced proteasome activation when compared with the wild-type REGbeta protein. REGbetaDeltai was able to form hetero-oligomers with a single site, monomeric REGalpha mutant and with REGalphaDeltai. At low concentrations, the REGalphaDeltai/REGbetaDeltai hetero-oligomers stimulated the proteasome less than REGalpha/REGbeta oligomers formed from wild-type subunits, and the reduced activation by REGalphaDeltai/REGbetaDeltai was due to removal of the REGbeta insert, not the REGalpha insert. These studies demonstrate that the REGalpha and REGgamma inserts play virtually no role in oligomerization or in proteasome activation. By contrast, removal of REGbeta insert reduces binding of this subunit and REGalpha/REGbeta oligomers to proteasomes. On the whole, however, our findings show that REG inserts are not required for binding and activating the proteasome. We speculate that they serve to localize REG-proteasome complexes within cells, possibly by binding components in endoplasmic reticulum membranes.
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PMID:Proteasome activation by REG molecules lacking homolog-specific inserts. 954 78

In the endoplasmic reticulum (ER), an efficient "quality control system" operates to ensure that mutated and incorrectly folded proteins are selectively degraded. We are studying ER-associated degradation using a truncated variant of the rough ER-specific type I transmembrane glycoprotein, ribophorin I. The truncated polypeptide (RI332) consists of only the 332 amino-terminal amino acids of the protein corresponding to most of its luminal domain and, in contrast to the long-lived endogenous ribophorin I, is rapidly degraded. Here we show that the ubiquitin-proteasome pathway is involved in the destruction of the truncated ribophorin I. Thus, when RI332 that itself appears to be a substrate for ubiquitination was expressed in a mutant hamster cell line harboring a temperature-sensitive mutation in the ubiquitin-activating enzyme E1 affecting ubiquitin-dependent proteolysis, the protein is dramatically stabilized at the restrictive temperature. Moreover, inhibitors of proteasome function effectively block the degradation of RI332. Cell fractionation experiments indicate that RI332 accumulates in the cytosol when degradation is prevented by proteasome inhibitors but remains associated with the lumen of the ER under ubiquitination-deficient conditions, suggesting that the release of the protein into the cytosol is ubiquitination-dependent. Accordingly, when ubiquitination is impaired, a considerable amount of RI332 binds to the ER chaperone calnexin and to the Sec61 complex that could effect retro-translocation of the polypeptide to the cytosol. Before proteolysis of RI332, its N-linked oligosaccharide is cleaved in two distinct steps, the first of which might occur when the protein is still associated with the ER, as the trimmed glycoprotein intermediate efficiently interacts with calnexin and Sec61. From our data we conclude that the steps that lead a newly synthesized luminal ER glycoprotein to degradation by the proteasome are tightly coupled and that especially ubiquitination plays a crucial role in the retro-translocation of the substrate protein for proteolysis to the cytosol.
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PMID:Ubiquitination is required for the retro-translocation of a short-lived luminal endoplasmic reticulum glycoprotein to the cytosol for degradation by the proteasome. 954 9

For an effective CD8+ cytotoxic T cell response to occur during infection, MHC class I molecules must be loaded with antigenic peptides in the endoplasmic reticulum. The cytosolic factor responsible for peptide generation is believed to be the proteasome, with the TAP heterodimer mediating peptide transport into the endoplasmic reticulum. However, the rate-determining step(s) in this intracellular pathway of Ag presentation is currently unresolved. The availability of a specific and irreversible proteasome inhibitor called lactacystin has enabled us to determine the amount of proteasomes required for the peptide loading of MHC class I molecules in four cell types. In the absence of the IFN-gamma-inducible proteasome subunits LMP2 and LMP7, the trypsin-like (but not the chymotrypsin-like) activity of the proteasome is directly related to MHC class I peptide loading. However, IFN-gamma stimulation or assimilation of catalytic LMP2 and LMP7 subunits into proteasomes causes both chymotrypsin- and trypsin-like activities of the proteasome to become limiting for the loading of class I molecules. Our data suggest that upon full IFN-gamma stimulation, peptide supply by the proteasome is the limiting step in the assembly of MHC class I polypeptides. This mechanism may enable the cell to prevent competition between novel Ags and the pool of endogenous proteins for binding to MHC class I molecules.
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PMID:Proteasome activity limits the assembly of MHC class I molecules after IFN-gamma stimulation. 955 Mar 86


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