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)

In budding yeast, cell division is initiated in late G1 phase once the Cdc28 cyclin-dependent kinase is activated by the G1 cyclins Cln1, Cln2, and Cln3. The extreme instability of the Cln proteins couples environmental signals, which regulate Cln synthesis, to cell division. We isolated Cdc53 as a Cln2-associated protein and show that Cdc53 is required for Cln2 instability and ubiquitination in vivo. The Cln2-Cdc53 interaction, Cln2 ubiquitination, and Cln2 instability all depend on phosphorylation of Cln2. Cdc53 also binds the E2 ubiquitin-conjugating enzyme, Cdc34. These findings suggest that Cdc53 is a component of a ubiquitin-protein ligase complex that targets phosphorylated G1 cyclins for degradation by the ubiquitin-proteasome pathway.
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PMID:Cdc53 targets phosphorylated G1 cyclins for degradation by the ubiquitin proteolytic pathway. 875 27

Ubiquitin is a covalent signal that targets cellular proteins to the 26 S proteasome. Multiple ubiquitins can be ligated together through the formation of isopeptide bonds between Lys48 and Gly76 of successive ubiquitins. Such a polyubiquitin chain constitutes a highly effective proteolytic targeting signal, but its mode of interaction with the proteasome is not well understood. Experiments to address this issue have been limited by difficulties in preparing useful quantities of polyubiquitin chains of uniform length. We report a simple method for large scale synthesis of Lys48-linked polyubiquitin chains of defined length. In the first round of synthesis, two ubiquitin derivatives (K48C-ubiquitin and Asp77-ubiquitin) were used as substrates for the well characterized ubiquitin-conjugating enzyme E2-25K. Diubiquitin blocked at the nascent proximal and distal chain termini was obtained in quantitative yield. Appropriately deblocked chains were then combined to synthesize higher order chains (tetramer and octamer in the present study). Deblocking was achieved either enzymatically (proximal terminus) or by chemical alkylation (distal terminus). Chains synthesized by this method were used to obtain the first quantitative information concerning the influence of polyubiquitin chain length on binding to the 26 S proteasome; this was done through comparison of different length (unanchored) polyubiquitin chains as inhibitors of ubiquitin-conjugate degradation. K0.5 was found to decrease approximately 90-fold, from 430 to 4.8 microM, as the chain was lengthened from two to eight ubiquitins. The implications of these results for the molecular basis of chain recognition are discussed.
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PMID:Inhibition of the 26 S proteasome by polyubiquitin chains synthesized to have defined lengths. 929 15

In chronic renal failure (CRF), the ATP-dependent, ubiquitin-proteasome proteolytic pathway is activated with concurrent increases in the transcription of genes encoding proteins of this pathway in muscle. We have shown that the stimuli for these responses include acidosis and glucocorticoids, but other endocrine abnormalities in CRF (e.g., insulin resistance) could contribute to these responses. In fact, a major effect of insulin in muscle is to suppress protein degradation. To examine whether insulin influences the ubiquitin-proteasome pathway, we measured protein degradation in incubated epitrochlearis muscles of diabetic and pair-fed control rats. Muscle proteolysis was increased in pathways that do not involve lysosomes or Ca(2+)-dependent proteases; but MG132, a protease inhibitor that blocks ATP synthesis, eliminated the accelerated rate of protein degradation in diabetic rat muscles. Diabetes mellitus also increased levels of mRNAs encoding ubiquitin (334%), E2 ubiquitin-conjugating enzyme (247%), and the C3 (320%), C5 (349%), and C9 (216%) proteasome subunits in muscle. Finally, transcription of the ubiquitin gene in diabetic rat muscles was increased. Diabetic rats were acidotic, but eliminating acidemia by giving NaHCO3 did not block the increase in muscle proteolysis. Giving diabetic rats insulin prevented the excessive muscle proteolysis, suggesting that insulin acts as a suppressor of the ubiquitin-proteasome pathway. Thus, the insulin resistance of uremia could contribute to muscle protein wasting in CRF.
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PMID:Signals regulating accelerated muscle protein catabolism in uremia. 938 16

Nuclear factor kappaB1 (NF-kappaB) is a heterodimeric complex that regulates transcription of many genes involved in immune and inflammatory responses. Its 50-kDa subunit (p50) is generated by the ubiquitin-proteasome pathway from a 105-kDa precursor (p105). We have reconstituted this proteolytic process in HeLa cell extracts and purified the responsible enzymes. Ubiquitination of p105 requires E1, and either of two types of E2s, E2-25K (for which p105 is the first proven substrate) or a member of the UBCH5 (UBC4) family. It also requires a new E3 of 50 kDa, which we call E3kappaB. This set of enzymes differs from the E2s and E3 reported by others to catalyze p105 ubiquitination in reticulocytes. The ubiquitinating enzymes purified here, together with 26S proteasomes, allowed formation of p50. Thus, the 26S proteasome provides all the proteolytic activities necessary for p105 processing. Interestingly, in the reconstituted system, as observed in cells, the C-terminally truncated form of p105, p97, was processed into p50 more efficiently than normal p105, even when both species were ubiquitinated to a similar extent. Therefore, some additional mechanism involving the C-terminal region of p105 influences the proteolytic processing of the ubiquitinated precursor.
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PMID:Enzymes catalyzing ubiquitination and proteolytic processing of the p105 precursor of nuclear factor kappaB1. 953 61

The NEDD8/Rub1 class of ubiquitin-like proteins has been implicated in progression of the cell cycle from G1 into S phase. These molecules undergo a metabolism that parallels that of ubiquitin and involves specific interactions with many different proteins. We report here the crystal structure of recombinant human NEDD8 refined at 1.6-A resolution to an R factor of 21.9%. As expected from the high sequence similarity (57% identical), the NEDD8 structure closely resembles that reported previously for ubiquitin. We also show that recombinant human NEDD8 protein is activated, albeit inefficiently, by the ubiquitin-activating (E1) enzyme and that NEDD8 can be transferred from E1 to the ubiquitin conjugating enzyme E2-25K. E2-25K adds NEDD8 to a polyubiquitin chain with an efficiency similar to that of ubiquitin. A chimeric tetramer composed of three ubiquitins and one histidine-tagged NEDD8 binds to the 26 S proteasome with an affinity similar to that of tetraubiquitin. Seven residues that differ from the corresponding residues in ubiquitin, but are conserved between NEDD8 orthologs, are candidates for mediating interactions with NEDD8-specific partners. One such residue, Ala-72 (Arg in ubiquitin), is shown to perform a key role in selecting against reaction with the ubiquitin E1 enzyme, thereby acting to prevent the inappropriate diversion of NEDD8 into ubiquitin-specific pathways.
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PMID:Crystal structure of the human ubiquitin-like protein NEDD8 and interactions with ubiquitin pathway enzymes. 985 30

Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes. As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis.
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PMID:SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis. 1058 39

For most substrates of ubiquitin (Ub)-dependent degradation, recognition by the proteasome is mediated by a covalently attached signal assembled from multiple ubiquitins linked to each other via the C terminus of one Ub and the epsilon-amine of Lys(48) of another Ub. Among Ub-conjugating enzymes, E2-25K is unique in its ability to synthesize in vitro unanchored Lys(48)-linked poly-Ub chains from mono- or poly-Ub, E1, and ATP; thus, E2-25K has distinct binding sites for donor and acceptor (poly)Ub. During studies of chain assembly by E2-25K, we observed that Lys(48)-linked tri-Ub was efficiently converted to a new species that upon SDS-polyacrylamide gel electrophoresis migrated between linear di-Ub and tri-Ub. Analysis of this product by mass spectrometry and tryptic digestion showed that it was a cyclic form of tri-Ub. Cyclization of tri-Ub requires E1, E2-25K, ATP, and that the linear substrate has a free Gly(76) C terminus on the proximal end Ub and a Lys(48) side chain available on the distal end Ub. E2-25K similarly can catalyze the cyclization of longer poly-Ub chains, including tetra- and penta-Ub. Although cyclic tri-Ub resists hydrolysis by the PA700 or isopeptidase T deubiquitinating enzymes, it can be disassembled to Ub monomers by isopeptidase(s) in a red blood cell extract. Thus, if cyclic poly-Ub forms in vivo, it will not accumulate as a dead-end product.
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PMID:Cyclization of polyubiquitin by the E2-25K ubiquitin conjugating enzyme. 1090 48

Mutations in the VHL tumor suppressor gene result in constitutive expression of many hypoxia-inducible genes, at least in part because of increases in the cellular level of hypoxia-inducible transcription factor HIF1alpha, which in normal cells is rapidly ubiquitinated and degraded by the proteasome under normoxic conditions. The recent observation that the VHL protein is a subunit of an Skp1-Cul1/Cdc53-F-box (SCF)-like E3 ubiquitin ligase raised the possibility that VHL may be directly responsible for regulating cellular levels of HIF1alpha by targeting it for ubiquitination and proteolysis. In this report, we test this hypothesis directly. We report development of methods for production of the purified recombinant VHL complex and present direct biochemical evidence that it can function with an E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzyme to activate HIF1alpha ubiquitination in vitro. Our findings provide new insight into the function of the VHL tumor suppressor protein, and they provide a foundation for future investigations of the mechanisms underlying VHL regulation of oxygen-dependent gene expression.
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PMID:Activation of HIF1alpha ubiquitination by a reconstituted von Hippel-Lindau (VHL) tumor suppressor complex. 1097 99

Many proteins are targeted to proteasome degradation by a family of E3 ubiquitin ligases, termed SCF complexes, that link substrate proteins to an E2 ubiquitin-conjugating enzyme. SCFs are composed of three core proteins-Skp1, Cdc53/Cull, Rbx1/Hrt1-and a substrate specific F-box protein. We have identified in Drosophila melanogaster the closest homologues to the human components of the SCF(betaTrCP) complex and the E2 ubiquitin-conjugating enzyme UbcH5. We show that putative Drosophila SCF core subunits dSkpA and dRbx1 both interact directly with dCu11 and the F-box protein Slmb. We also describe the direct interaction of the UbcH5 related protein UbcD1 with dCul1 and Slmb. In addition, a functional complementation test performed on a Saccharomyces cerevisiae Hrt1p-deficient mutant showed that Drosophila Rbx1 is able to restore the yeast cells viability. Our results suggest that dRbx1, dSkpA, dCullin1, and Slimb proteins are components of a Drosophila SCF complex that functions in combination with the ubiquitin conjugating enzyme UbcD1.
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PMID:Occurrence of a putative SCF ubiquitin ligase complex in Drosophila. 1150 45

Synaptophysin is an integral membrane protein of synaptic vesicles characterized by four transmembrane domains with both termini facing the cytoplasm. Although synaptophysin has been implicated in neurotransmitter release, and decreased synaptophysin levels have been associated with several neurodegenerative diseases, the molecular mechanism that regulates the degradation of synaptophysin remains unsolved. Using the cytoplasmic C terminus of synaptophysin as bait in a yeast two-hybrid screen, we identified two synaptophysin-binding proteins, Siah-1A and Siah-2, which are rat homologues of Drosophila Seven in Absentia. We demonstrated that Siah-1A and Siah-2 associate with synaptophysin both in vitro and in vivo and defined the binding domains of synaptophysin and Siah that mediate their association. Siah proteins exist in both cytosolic and membrane-associated pools and co-localize with synaptophysin on synaptic vesicles and early endosomes. In addition, Siah proteins interact specifically with the brain-enriched E2 ubiquitin-conjugating enzyme UbcH8 and facilitate the ubiquitination of synaptophysin. Furthermore, overexpression of Siah proteins promotes the degradation of synaptophysin via the ubiquitin-proteasome pathway. Our findings indicate that Siah proteins function as E3 ubiquitin-protein ligases to regulate the ubiquitination and degradation of synaptophysin.
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PMID:Regulation of synaptophysin degradation by mammalian homologues of seven in absentia. 1178 35


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