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:6.3.2.19 (ubiquitin-protein ligase)
799 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mechanisms that regulate ubiquitin-mediated degradation of proteins such as the mitotic cyclins at defined stages of the cell cycle are poorly understood. The initial step in the conjugation of ubiquitin to substrate proteins involves the activation of ubiquitin by the ubiquitin-activating enzyme, E1. Previously we have described the subcellular localization of this enzyme to both nuclear and cytoplasmic compartments. In the present study, we have used the 1C5 anti-E1 monoclonal antibody in immunofluorescent-microscopy and subcellular-fractionation techniques to examine the distribution of E1 during the HeLa cell cycle. E1 is both cytoskeletal and nuclear during the G1-phase. As the cells progress into S-phase, E1 is exclusively cytoskeletal and has a perinuclear distribution. During G2-phase, E1 reappears in the nucleus before breakdown of the nuclear envelope. In mitotic cells, E1 localizes to both the mitotic spindle and the cytosol, but is absent from the chromosomes. Immunoblot analysis reveals multiple forms of E1 in HeLa whole cell extract. This heterogeneity is not a result of polyubiquitination and may represent inactive pools of E1. Only the characteristic E1 doublet is able to activate ubiquitin. Cell-fractionation studies reveal a differential distribution of specific E1 isoforms throughout the cell cycle. Therefore we propose that the subcellular localization of E1 may play a role in regulating cell-cycle-dependent conjugation of ubiquitin to target proteins.
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PMID:Nuclear localization of the ubiquitin-activating enzyme, E1, is cell-cycle-dependent. 801 Sep 51

The wild-type tumor suppressor protein p53 is a short-lived protein that plays important roles in regulation of cell cycle, differentiation, and survival. Mutations that inactivate or alter the tumor suppressor activity of the protein seem to be the most common genetic change in human cancer and are frequently associated with changes in its stability. The ubiquitin system has been implicated in the degradation of p53 both in vivo and in vitro. A mutant cell line that harbors a thermolabile ubiquitin-activating enzyme, E1, fails to degrade p53 at the nonpermissive temperature. Studies in cell-free extracts have shown that covalent attachment of ubiquitin to the protein requires the three conjugating enzymes: E1, a novel species of ubiquitin-carrier protein (ubiquitin-conjugating enzyme; UBC),E2-F1, and an ubiquitin-protein ligase, E3. Recognition of p53 by the ligase is facilitated by formation of a complex between the protein and the human papillomavirus (HPV) oncoprotein E6. Therefore, the ligase has been designated E6-associated protein (E6-AP). However, these in vitro studies have not demonstrated that the conjugates serve as essential intermediates in the proteolytic process. In fact, in many cases, conjugation of ubiquitin to the target protein does not signal its degradation. Thus, it is essential to demonstrate that p53-ubiquitin adducts serve as essential proteolytic intermediates and are recognized and degraded by the 26S protease complex, the proteolytic arm of the ubiquitin pathway. In this study, we demonstrate that conjugates of p53 generated in the presence of purified, E1, E2, E6-AP, E6, ubiquitin and ATP, are specifically recognized by the 26S protease complex and degraded. In contrast, unconjugated p53 remains stable. The ability to reconstitute the system from purified components will enable detailed analysis of the recognition process and the structural motifs involved in targeting the protein for degradation.
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PMID:Complete reconstitution of conjugation and subsequent degradation of the tumor suppressor protein p53 by purified components of the ubiquitin proteolytic system. 803 27

Targeting of different cellular proteins for conjugation and subsequent degradation via the ubiquitin pathway involves diverse recognition signals and distinct enzymatic factors. A few proteins are recognized via their N-terminal amino acid residue and conjugated by a ubiquitin-protein ligase that recognizes this residue. Most substrates, including the N alpha-acetylated proteins that constitute the vast majority of cellular proteins, are targeted by different signals and are recognized by yet unknown ligases. We have previously shown that degradation of N-terminally blocked proteins requires a specific factor, designated FH, and that the factor acts along with the 26S protease complex to degrade ubiquitin-conjugated proteins. Here, we demonstrate that FH is the protein synthesis elongation factor EF-1 alpha. (a) Partial sequence analysis reveals 100% identity to EF-1 alpha. (b) Like EF-1 alpha, FH binds to immobilized GTP (or GDP) and can be purified in one step using the corresponding nucleotide for elution. (c) Guanine nucleotides that bind to EF-1 alpha protect the ubiquitin system-related activity of FH from heat inactivation, and nucleotides that do not bind do not exert this effect. (d) EF-Tu, the homologous bacterial elongation factor, can substitute for FH/EF-1 alpha in the proteolytic system. This last finding is of particular interest since the ubiquitin system has not been identified in prokaryotes. The activities of both EF-1 alpha and EF-Tu are strongly and specifically inhibited by ubiquitin-aldehyde, a specific inhibitor of ubiquitin isopeptidases. It appears, therefore, that EF-1 alpha may be involved in releasing ubiquitin from multiubiquitin chains, thus rendering the conjugates susceptible to the action of the 26S protease complex.
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PMID:Protein synthesis elongation factor EF-1 alpha is essential for ubiquitin-dependent degradation of certain N alpha-acetylated proteins and may be substituted for by the bacterial elongation factor EF-Tu. 805 36

The E6 protein of the oncogenic human papillomavirus types 16 and 18 facilitates the rapid degradation of the tumor-suppressor protein p53 via the ubiquitin-dependent proteolytic pathway. The E6 protein binds to a cellular protein of 100 kDa termed E6-AP. The complex of E6 and E6-AP specifically interacts with p53 and induces the ubiquitination of p53 in a reaction which requires the ubiquitin-activating enzyme (E1) and a cellular fraction thought to contain a mammalian ubiquitin-conjugating enzyme (E2). This mammalian E2 activity could be replaced with bacterially expressed UBC8 from Arabidopsis thaliana, which belongs to a subfamily of E2s including yeast UBC4 and UBC5 which are highly conserved at the amino acid level. In this paper we describe the cloning of a human cDNA encoding a human E2 that we have designated UbcH5 and that is related to Arabidopsis UBC8 and the other members of this subfamily. We demonstrate that UbcH5 can function in the E6/E6-AP-induced ubiquitination of p53.
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PMID:Identification of a human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53. 809 Jul 26

Cyclin B, a positive regulatory subunit of the cdc2 protein kinase complex, is synthesized across the cell cycle and then rapidly degraded at the end of mitosis. Degradation of cyclin B is triggered by increased levels of active cdc2 and is required for exit from mitosis. It was shown previously that cyclin degradation is carried out by the ubiquitin system, but the components responsible for the specificity and regulation of cyclin-ubiquitin ligation have not been identified. The formation of ubiquitin-protein conjugates usually requires the sequential action of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-carrier protein (E2), and a ubiquitin-protein ligase (E3). In this work we employed a fractionation approach to identify the components of a clam oocyte system responsible for specific ubiquitination of cyclin and to determine which components are regulated by cdc2. Experimental conditions were established under which a fusion protein containing an amino-terminal fragment of cyclin B is ligated to ubiquitin only in extracts from M-phase but not from interphase cells. Fractionation of M-phase extracts by DEAE-cellulose and high speed centrifugation yielded three fractions that were all required for cell cycle stage-specific cyclin-ubiquitin ligation. Only one of these fractions could be replaced by a previously known enzyme of the ubiquitin system, E1. A second fraction contained a novel species of E2, termed E2-C, which acts in the ligation of ubiquitin to cyclin but not to other endogenous proteins. A third component is associated with particulate material. Whereas E2-C from either M-phase or interphase extracts is active, the particulate component is active only in M-phase. Incubation of the particulate fraction from interphase cells with the protein kinase cdc2 activates it for cyclin-ubiquitin ligation, after a lag of about 30 min. These findings suggest that the particulate fraction may contain an E3 enzyme that acts on cyclin, as well as additional factors activated by cdc2.
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PMID:Components of a system that ligates cyclin to ubiquitin and their regulation by the protein kinase cdc2. 810 68

The tumor suppressor protein p53 is extremely unstable in most cell lines. In contrast, many mutant and oncogenic species of the protein are stable. The degradation of p53 in vivo requires metabolic energy; however, the proteolytic system(s) involved have not been identified. The ubiquitin system has been implicated in the degradation of p53 in vitro. The degradation is stimulated significantly by the human papillomavirus (HPV) oncoprotein E6 that associates with p53 and facilitates conjugate formation and subsequent degradation. Complex formation between E6 and p53 is promoted by a cellular protein designated E6-associated protein (E6-AP). Initial dissection of the conjugation process have demonstrated a role for the ubiquitin-activating enzyme, E1, but the ubiquitin-carrier protein (E2, UBC) and the ubiquitin protein ligase, E3, have not been identified. In this study, we report that a novel species of ubiquitin-carrier protein designated E2-F1 (Blumenfeld, N., Gonen, H., Mayer, A., Smith, C., Siegel, N.R., Schwartz, A.L., and Ciechanover, A. (1994) J. Biol. Chem. 269, 9574-9581) is involved in the conjugation and degradation of p53. This E2 enzyme recognizes non-"N-end rule" protein substrates and appears to mediate their conjugation via a novel species of E3. The process of recognition appears to be selective; E2-F1 is not required for the conjugation and degradation of human N-myc. The involvement of E2-F1 in the in vitro process appears to be physiologically meaningful and to reproduce the in vivo process; mutant species of p53 that do not interact with E6 and are stable in vivo are not recognized by the cell free system.
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PMID:Degradation of the tumor suppressor protein p53 by the ubiquitin-mediated proteolytic system requires a novel species of ubiquitin-carrier protein, E2. 814 45

The ubiquitin-dependent proteolytic pathway plays a major role in selective protein degradation. Ubiquitination of proteins requires the sequential action of the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (E2), and in some cases ubiquitin-protein ligases (E3s). The oncogenic human papillomavirus (HPV) types 16 and 18 utilize this cellular proteolytic system to target the tumor suppressor protein p53. The HPV E6 oncoprotein binds to a cellular protein of 100 kd, termed E6-associated protein (E6-AP). The E6-E6-AP complex specifically interacts with p53, resulting in the rapid ubiquitin-dependent degradation of p53. Here we report the purification and identification of the factors necessary for the E6-E6-AP-mediated ubiquitination of p53. The ubiquitination of p53 requires the E1 enzyme and a novel E2 in mammalian cells, while E3 activity is conferred by the E6-E6-AP complex. Furthermore, E6-AP appears to have ubiquitin-protein ligase activity in the absence of E6.
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PMID:The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. 822 89

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

A temperature-sensitive mutant defective in DNA replication, tsFT5, has been isolated from the mouse mammary carcinoma cell line FM3A. DNA synthesis in tsFT5 cells at a restrictive temperature (39 degrees C) has been characterized in detail. Incorporation of [3H]thymidine decreased rapidly after an increase in temperature to 39 degrees C and the incorporation was less than 20% and 10% of the initial level after 4 and 8 h, respectively. Analysis by DNA fiber autoradiography revealed that the initiation of DNA replication at the origin of the replicons was impaired in tsFT5 cells but that the DNA chain elongation rate of the mutant cells did not decrease at the nonpermissive temperature. tsFT5 cells were confirmed to belong to the complementation group which includes ts85 cells arrested mainly in the G2 phase at the nonpermissive temperature. It has been observed that the amount of ubiquintin-conjugated histone H2A (uH2A) in ts85 cells decreases at the nonpermissive temperature (Marunouchi, T., Yasuda, H., Matsumoto, Y., and Yamada, M. (1980) Biochem. Biophys. Res. Commun. 95, 126-131). The amount of uH2A in tsFT5 cells also decreased rapidly at 39 degrees C. This decrease occurred at the same time as or slightly preceding to reduction in DNA synthesis, and the reappearance of uH2A was followed by the restoration of DNA synthesis after the temperature was reduced. A similar temporal relationship between decrease in the amount of uH2A and reduction in DNA synthesis was observed in ts85 cells cultured at 39 degrees C. However, the rates of the decrease of uH2A and of the reduction in DNA synthesis in ts85 cells were slower than those observed in tsFT5 cells. A comparison of the thermolability of purified ubiquitin-activating enzyme E1s revealed that the E1 from ts85 cells had a thermolability intermediate between those of the E1 from tsFT5 cells and of the wild-type cells. A reduction in the phosphorylation of histone H1 was observed in tsFT5 cells cultured at 39 degrees C, but the reduction occurred several hours after the decrease in uH2A and the reduction in DNA synthesis.
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PMID:Characterization of DNA synthesis at a restrictive temperature in the temperature-sensitive mutants, tsFT5 cells, that belong to the complementation group of ts85 cells containing a thermolabile ubiquitin-activating enzyme E1. Involvement of the ubiquitin-conjugating system in DNA replication. 834 58

Covalent attachment of ubiquitin marks substrates for proteolysis, but features that identify ubiquitination targets such as chicken egg white lysozyme are poorly understood. Recognition of lysozyme first requires reduction of Cys-6 Cys-127, one of its four native disulfide bonds, and Cys-6,Cys-127-carboxymethylated (6,127-rcm) lysozyme can mimic this three-disulfide intermediate. The 6,127-rcm form of lysozyme is known to retain a substantially native-like conformation in solution, and we demonstrate that it is this folded structure that is recognized for ubiquitination. Because native lysozyme is not a substrate, differences between the native and three-disulfide structures must include features responsible for selective ubiquitination. The 1.9-A resolution crystal structure of 6,127-rcm-lysozyme, reported here, affords a view of this ubiquitin-dependent degradation substrate. Two conformers of 6,127-rcm-lysozyme were obtained in the crystal. These differ uniquely from crystal forms of native lysozyme by displacement of the C-terminal residues. The structures suggest that localized unfolding at the C terminus of three-disulfide lysozyme allows the complex of E3 alpha (ubiquitin-protein ligase) and E2 (ubiquitin-carrier protein) to bind to a surface that includes Lys-1 and the putative ubiquitination site Lys-13. From this we infer that the N-terminal and internal substrate recognition sites on the E3 alpha.E2 complex are separated by approximately 20 A.
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PMID:Crystal structure of a ubiquitin-dependent degradation substrate: a three-disulfide form of lysozyme. 838 11


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