Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.3.2.19 (ubiquitin-protein ligase)
 799 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A prerequisite for the selective degradation of intracellular proteins by the ubiquitin-dependent proteolytic pathway is the attachment of a chain of ubiquitin monomers to the targeted protein. In these multiubiquitin chains, the carboxyl-terminal glycine 76 of ubiquitin is linked via an isopeptide bond to the epsilon-amino group of lysine 48 in the adjacent ubiquitin. It remains to be determined whether these chains are preassembled and attached en masse to the target, are made by sequential conjugation of ubiquitin monomers to ubiquitins already linked to the protein, or both. Using the 20-kDa ubiquitin-conjugating enzyme TaUBC7 from wheat, we have generated free, glycine 76-->lysine 48-linked multiubiquitin chains (Ubqn), and have individually purified Ubqn species (n < or = 6) by anion-exchange, high pressure liquid chromatography. The migration of these chains during SDS-polyacrylamide gel electrophoresis was indistinguishable from those of major ubiquitin immunoreactive proteins in cell lysates from a variety of eukaryotes suggesting that free, multiubiquitin chains are abundant in vivo. One of these chain members (Ubq2) was purified from wheat and was demonstrated via amino acid sequence analysis of tryptic fragments to consist of two ubiquitin monomers joined via a glycine 76-->lysine 48 linkage. We also show in vitro that purified Ubq2 and Ubq4 are competent in activation by ubiquitin-activating enzyme (E1), in transfer to E2s, and in ubiquitin-protein ligase (E3)-independent conjugation to other Ubqn species and to histones H2A/B. These data demonstrate that multiubiquitin chains exist as free, functional structures in vivo and support the possibility that at least a portion of free ubiquitin is preassembled into multiubiquitin chains prior to its attachment to proteolytic substrates.
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PMID:Multiubiquitin chains linked through lysine 48 are abundant in vivo and are competent intermediates in the ubiquitin proteolytic pathway. 822 36

In corroboration of the hypothesized regulation of phototransduction proteins by the ubiquitin-dependent pathway, we identified free ubiquitin (8 kDa) and ubiquitin-protein conjugates (50 to >200 kDa; pI 5.3-6.8 by two-dimensional electrophoresis) in bovine rod outer segments (ROS). A 38-kDa ubiquitinylated protein and transducin (Gt) were eluted together from light-adapted ROS membranes with GTP. When ROS were dark-adapted, this 38-kDa ubiquitinylated species and Gt were readily solubilized in buffer lacking GTP. These data are consistent with ubiquitinylation of Gt and corroborate previous cell-free experiments identifying Gt as a substrate for ubiquitin-dependent proteolysis (Obin, M. S., Nowell, T., and Taylor, A. (1994) Biochem. Biophys. Res. Commun. 200, 1169-1176). Evidence for ubiquitinylation of rhodopsin (36 kDa), the (photo)receptor coupled to Gt, included (i) the presence in ROS membranes "stripped" of peripheral membrane proteins of numerous ubiquitin-protein conjugates, including two whose masses (44 and 50 kDa) are consistent with mono- and diubiquitinylated rhodopsin; (ii) catalysis by permeabilized ROS of 125I-labeled ubiquitin-protein conjugates whose masses (42, 50, and 58 kDa) suggest a "ladder" of mono-, di-, and triubiquitinylated rhodopsin; (iii) parallel mobility shifts on SDS-polyacrylamide gels of rhodopsin and these 125I-labeled ubiquitin-protein conjugates; and (iv) generation of enhanced levels of 125I-labeled ubiquitin-protein conjugates when stripped, detergent-solubilized ROS membranes (95% rhodopsin) were incubated with reticulocyte lysate. A functional ubiquitin-dependent pathway in ROS is demonstrated by the presence of (i) the ubiquitin-activating enzyme (E1); (ii) four ubiquitin carrier proteins (E214K, E220K, E225K, and E235K) and pronounced activity of E214K, an enzyme required for "N-end rule" proteolysis; (iii) ATP-dependent 26 S proteasome activity that rapidly degrades high mass 125I-labeled ubiquitin-ROS protein conjugates; and (iv) distinct ubiquitin C-terminal isopeptidase/hydrolase activities, including potent ubiquitin-aldehyde-insensitive activity directed at high mass ubiquitinylated moieties. Considered together, the data support a novel role for the ubiquitin-dependent pathway in the regulation of mammalian phototransduction protein levels and/or activities and provide the first identification of a non-calpain proteolytic system in photoreceptors.
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PMID:Ubiquitinylation and ubiquitin-dependent proteolysis in vertebrate photoreceptors (rod outer segments). Evidence for ubiquitinylation of Gt and rhodopsin. 866 97

The ubiquitin-activating enzyme exists as two isoforms: E1a, localized predominantly in the nucleus, and E1b, localized in the cytoplasm. Previously we generated hemagglutinin (HA) epitope-tagged cDNA constructs, HA1-E1 (epitope tag placed after the first methionine) and HA2-E1 (epitope tag placed after the second methionine) (Handley-Gearhart, P. M., Stephen, A. G., Trausch-Azar, J. S., Ciechanover, A., and Schwartz, A. L. (1994) J. Biol. Chem. 269, 33171-33178), which represent the native isoforms. HA1-E1 is exclusively nuclear, whereas HA2-E1 is found predominantly in the cytoplasm. Using high resolution isoelectric focusing and SDS-polyacrylamide gel electrophoresis, we confirm that these epitope-tagged constructs HA1-E1 and HA2-E1 represent the two isoforms E1a and E1b. HA1-E1/E1a exists as one non-phosphorylated and four phosphorylated forms, and HA2-E1/E1b exists as one predominant non-phosphorylated form and two minor phosphorylated forms. We demonstrate that the first 11 amino acids are essential for phosphorylation and exclusive nuclear localization of HA1-E1. Within this region are four serine residues and a putative nuclear localization sequence (NLS; 5PLSKKRR). Removal of these four serine residues reduced phosphorylation levels by 60% but had no effect on nuclear localization of HA1-E1. Each serine residue was independently mutated to an alanine and analyzed by two-dimensional electrophoresis; only serine 4 was phosphorylated. Disruption of the basic amino acids within the NLS resulted in loss of exclusive nuclear localization and a 90-95% decrease in the phosphorylation of HA1-E1. This putative NLS was able to confer nuclear import on a non-nuclear protein in digitonin-permeabilized cells in a temperature- and ATP-dependent manner. Thus the predominant requirement for efficient phosphorylation of HA1-E1/E1a is a functional NLS, suggesting that E1a may be phosphorylated within the nucleus.
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PMID:Identification of a region within the ubiquitin-activating enzyme required for nuclear targeting and phosphorylation. 909 46

Topoisomerase I (TOP1) relaxes superhelical DNA through a breakage/rejoining reaction in which the active site tyrosine links covalently to a 3' phosphate at the break site as a transient intermediate. The antitumor drug camptothecin (CPT) and its analogs inhibit the rejoining step of the breakage/rejoining reaction, which traps the enzyme in covalent linkage with DNA (the cleavable complex). Little is known about the fate of cellular TOP1 trapped in the cleavable complex. We have analyzed TOP1 in mammalian cell lines treated with CPT. When CPT-treated cells were lysed with either SDS or alkali and analyzed by Western blotting, greater than 90% of the TOP1 was linked to DNA. Nuclease treatment of the cell lysate to remove the covalently linked DNA from TOP1 revealed a distinct ladder of higher molecular weight bands having properties indicative of multi-ubiquitin (Ub) conjugates of TOP1. Approximately 5-10% of TOP1 was present as these conjugates within minutes of CPT treatment. Consistent with ubiquitination, TOP1 was not modified in ts85 cells at the restrictive temperature for its thermolabile ubiquitin-activating enzyme (E1). Because conjugation with ubiquitin can mark proteins for destruction by the 26S proteasome, we analyzed TOP1 protein levels during prolonged CPT treatment. TOP1 protein levels were reduced to about 25% during CPT treatments of 2-4 h resulting from increased destruction, with the half-life dropping from 10-16 h down to 1-2 h. The destruction of TOP1, like the formation of Ub-TOP1 conjugates, was not observed in ts85 cells at the restrictive temperature. The destruction of TOP1 was also prevented in cells treated with MG-132 and lactacystin, specific inhibitors of the 26S proteasome. Finally, the multi-Ub conjugates of TOP1 were observed whether or not aphidicolin was included in cotreatment with CPT, indicating that replication fork activity was not involved in making TOP1 a substrate for ubiquitination. These results demonstrate that independent of DNA replication, the TOP1 cleavable complex is ubiquitinated and destroyed in cells treated with antitumor drugs that block the religation step of the TOP1 reaction.
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PMID:Ubiquitin-dependent destruction of topoisomerase I is stimulated by the antitumor drug camptothecin. 930 65