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Query: UNIPROT:P62988 (Ubiquitin)
 4,326 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

The picornavirus 3C proteases are required for the processing of viral polyproteins during infections of host cells. Here we report that the 3C protease of the hepatitis A virus, like that of the encephalomyocarditis virus, is a substrate for rapid, ubiquitin-mediated degradation in vitro. Ubiquitin was shown to stimulate the turnover of the hepatitis virus 3C protease, and labeled protease was found to become incorporated into a mixture of high molecular weight species, which is characteristic of conjugation with polyubiquitin chains. In the presence of methylated ubiquitin, a new 33 kDa species formed, consistent with the generation of a monoubiquitin-3C protease conjugate. The rate of degradation of the 3C protease was reduced by inhibitors of the 26S proteasome. A similar evaluation of the 3C protease of poliovirus revealed that it is stable protein and is not conjugated with ubiquitin. It was also determined that the hepatitis A and encephalomyocarditis virus 3C proteases compete with each other for conjugation with ubiquitin and for degradation. This suggests that the two 3C proteases are both recognized by the same ubiquitin system enzyme, or enzymes, responsible for selecting them as targets for destruction.
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PMID:Evaluation of the susceptibility of the 3C proteases of hepatitis A virus and poliovirus to degradation by the ubiquitin-mediated proteolytic system. 929 63

Degradation of many eukaryotic proteins requires their prior ligation to polyubiquitin chains, which target substrates to the 26S proteasome, an abundant cellular protease. We describe a yeast deubiquitinating enzyme, Ubp14, that specifically disassembles unanchored ('free') ubiquitin chains in vitro, a specificity shared by mammalian isopeptidase T. Correspondingly, deletion of the UBP14 gene from yeast cells results in a striking accumulation of free ubiquitin chains, which correlates with defects in ubiquitin-dependent proteolysis. Increasing the steady-state levels of ubiquitin chains in wild-type cells (by expressing a derivative of ubiquitin with an altered C-terminus) inhibits protein degradation to a degree comparable with that observed in ubp14delta cells. Inhibition of degradation is also seen when an active site mutant of Ubp14 is overproduced in vivo. Surprisingly, overproduction of wild-type Ubp14 can inhibit degradation of some proteins as well. Finally, Ubp14 and human isopeptidase T are shown to be functional homologs by complementation analysis. We propose that Ubp14 and isopeptidase T facilitate proteolysis in vivo by preventing unanchored ubiquitin chains from competitively inhibiting polyubiquitin-substrate binding to the 26S proteasome.
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PMID:In vivo disassembly of free polyubiquitin chains by yeast Ubp14 modulates rates of protein degradation by the proteasome. 930 25

We have recently reported that the yeast plasma membrane uracil permease undergoes cell-surface ubiquitination, which is dependent on the Npi1/Rsp5 ubiquitin-protein ligase. Ubiquitination of this permease, like that of some other transporters and receptors, signals endocytosis of the protein, leading to its subsequent vacuolar degradation. This process does not involve the proteasome, which binds and degrades ubiquitin-protein conjugates carrying Lys48-linked ubiquitin chains. The data presented here show that ubiquitination and endocytosis of uracil permease are impaired in yeast cells lacking the Doa4p ubiquitin-isopeptidase. Both processes were rescued by overexpression of wild-type ubiquitin. Mutant ubiquitins carrying Lys-->Arg mutations at Lys29 and Lys48 restored normal permease ubiquitination. In contrast, a ubiquitin mutated at Lys63 did not restore permease polyubiquitination. Ubiquitin-permease conjugates are therefore extended through the Lys63 of ubiquitin. When polyubiquitination through Lys63 is blocked, the permease still undergoes endocytosis, but at a reduced rate. We have thus identified a natural target of Lys63-linked ubiquitin chains. We have also shown that monoubiquitination is sufficient to induce permease endocytosis, but that Lys63-linked ubiquitin chains appear to stimulate this process.
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PMID:Ubiquitin lys63 is involved in ubiquitination of a yeast plasma membrane protein. 931 43

The Drosophila fat facets gene encodes a deubiquitinating enzyme that regulates a cell communication pathway essential early during eye development to inhibit the determination of excess photoreceptors. Ubiquitin is a small polypeptide that tags proteins for degradation by a multisubunit proteolytic complex called the proteasome. The FAT FACETS protein is thought to be required to remove ubiquitin from a particular protein, thereby rescuing if from proteolysis. In order to identify the genes encoding the substrate of FAT FACETS and other components of the neural inhibition pathway, a mutagenesis screen for dominant enhancers of the fat facets mutant eye phenotype was performed. Several genes were identified, one of which is an excellent candidate for encoding a component of the pathway regulated by FAT FACETS. Three different eye phenotypes were observed when the fat facets mutants were dominantly enhanced by different mutations, suggesting that fat facets has other functions in addition to its critical role early in eye development.
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PMID:Mutagenesis screens for interacting genes reveal three roles for fat facets during Drosophila eye development. 933 74

The ubiquitin-proteasome pathway is the principal mechanism for the turnover of short-lived proteins in eukaryotic cells. In this pathway, the covalent ligation of ubiquitin to the substrate is a signal for recognition by the 26S proteasome. Recent studies indicate that targeting of substrates of the ubiquitin pathway to the proteasome is usually accomplished by the ligation of a polyubiquitin chain assembled through K48-G76 isopeptide bonds, rather than by ligation of monoubiquitin. In addition to providing benefits in signal generation, recognition, and persistence, assigning the proteolytic targeting function to a specific specific type of polyubiquitin chain may allow monoubiquitin or polyubiquitin chains of novel structures to serve distinct targeting functions. Besides polyubiquitinated substrates, the proteasome also degrades an unknown number of proteins that are recognized without undergoing ubiquitination. Ornithine decarboxylase is the prototype ubiquitin-independent substrate; it is targeted to the proteasome through noncovalent interaction with a specific protein factor known as antizyme. The existence of ubiquitin-independent substrates of the proteasome raises important questions about the nature of the substrate- and proteasome-based elements that cooperate to bring about the targeting of substrates to this novel proteolytic complex.
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PMID:Targeting of substrates to the 26S proteasome. 936 41

The modification of cytosolic proteins with polyubiquitin chains targets them for recognition and degradation by the multisubunit proteolytic particle, the 26S proteasome. Membrane proteins are also substrates for ubiquitination. Integral membrane proteins of the endoplasmic reticulum are ubiquitinated and destroyed by the proteasome. However, it has been shown recently that the ubiquitination of Saccharomyces cerevisiae plasma membrane proteins signals their degradation by the proteolytic system in the lysosome-like vacuole. Ubiquitination of several different classes of cell surface proteins serves as a signal for their entry into the endocytic pathway; this leads to their transport to the vacuole, where they are permanently inactivated by degradation. In yeast, ubiquitin has been implicated as an internalization signal for most, if not all, endogenous plasma membrane proteins that are known to be endocytosed. Ubiquitin-dependent internalization has been best characterized for two proteins: the mating pheromone alpha-factor receptor and the uracil permease. Some mammalian cell surface receptors are also ubiquitinated at the plasma membrane. Ubiquitination machinery is required for ligand-induced endocytosis of the growth hormone receptor, suggesting that ubiquitin-dependent endocytosis and sorting is also an important regulatory process in mammalian cells. Mammalian receptors may also be down-regulated through the degradation of their cytosolic domains by a proteasome-dependent pathway.
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PMID:Ubiquitin-dependent internalization and down-regulation of plasma membrane proteins. 940 40

The principal targeting signal used in the ubiquitin-proteasome degradation pathway is a homopolymeric, K48-linked polyubiquitin chain: the chain is recognized by a specific factor(s) in the 19S regulatory complex of the 26S proteasome, while the substrate is degraded by the 20S catalytic complex. We have previously presented evidence implicating the side chains of L8, I44, and V70 in the recognition of K48-linked chains. In the crystal structure of tetraubiquitin, these side chains form a repeating, surface-exposed hydrophobic patch. To test the hypothesis that a close-packing interaction involving this patch is important for the chain recognition, residue 8 was mutated to a series of smaller aliphatic amino acids (G, A, V). The effects of these mutations were first investigated in rabbit reticulocyte fraction II; even the severest truncating mutation (L8G) had only a modest inhibitory effect on the degradation of a model substrate (125I-lactalbumin). We show that these steady-state degradation data substantially underestimate the deleterious effects of these mutations on chain recognition by the proteasome, because the recognition step does not contribute to rate limitation in the fraction II system. Much stronger inhibition was observed when chain binding was measured in a competition assay using purified 26S proteasomes, and the change in binding free energy depended linearly on the surface area of the side chain. This behavior is consistent with a mode of binding in which the hydrophobic effect makes a favorable contribution; i.e., one or more L8 side chains is shielded from solvent when the chain binds to the 19S complex. A similar linear dependence of binding energy on side chain area was observed for chain binding to the 19S subunit known as S5a (as assayed using recombinant S5a bound to nickel beads). Octa-ubiquitin (K0.5 = 1.6 microM) bound to S5a 4.2-fold more tightly than tetra-ubiquitin; this is similar to the factor of 5. 8-fold relating the affinities of the same two chains for the 26S proteasome. Altogether, these findings indicate that the interaction of K48-linked chains with the 19S complex is substantially similar to the interaction of chains with isolated S5a. The results further suggest that the hydrophobic patch is part of a minimum element which allows for specific recognition of the polyubiquitin degradation signal by the 26S proteasome.
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PMID:The hydrophobic effect contributes to polyubiquitin chain recognition. 948 44

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

Ubiquitin conjugation is known to target protein substrates primarily to degradation by the proteasome or via the endocytic route. Here we describe a novel protein modification pathway in yeast which mediates the conjugation of RUB1, a ubiquitin-like protein displaying 53% amino acid identity to ubiquitin. We show that RUB1 conjugation requires at least three proteins in vivo. ULA1 and UBA3 are related to the N- and C-terminal domains of the E1 ubiquitin-activating enzyme, respectively, and together fulfil E1-like functions for RUB1 activation. RUB1 conjugation also requires UBC12, a protein related to E2 ubiquitin-conjugating enzymes, which functions analogously to E2 enzymes in RUB1-protein conjugate formation. Conjugation of RUB1 is not essential for normal cell growth and appears to be selective for a small set of substrates. Remarkably, CDC53/cullin, a common subunit of the multifunctional SCF ubiquitin ligase, was found to be a major substrate for RUB1 conjugation. This suggests that the RUB1 conjugation pathway is functionally affiliated to the ubiquitin-proteasome system and may play a regulatory role.
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PMID:A novel protein modification pathway related to the ubiquitin system. 954 34


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