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

---

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

The two isoforms of the 14-kDa ubiquitin carrier protein (E2(14k)) are unique among rabbit E2s in efficiently supporting ubiquitin-protein ligase (E3)-mediated ubiquitination of proteins destined for degradation. To begin determining the structural basis for this property, we have isolated a cDNA encoding the predominant reticulocyte isoform of the E2 from a rabbit skeletal muscle library. The sequence predicts a protein of 152 amino acids with a molecular weight of 17,293. Expression of the cDNA in Escherichia coli and purification of the recombinant protein revealed an E2 with high affinity for E3 and ubiquitin activating enzyme (E1). The latter high affinity interaction appears to be between the ubiquitin charged form of E1 and the uncharged form of E2 and does not result in a stable complex between these two enzymes. The predicted sequence shows regions of strong homology with other sequenced E2s, suggesting that these regions may be involved in binding to E1 and/or in ubiquitin transfer from E1, functions common to all E2s. Surprisingly, the E2(14k)) sequence is markedly more similar to Saccharomyces cerevisiae RAD6 (69% identity) than to its proposed homologs UBC4/UBC5 (38% identity). The sequence is identical to that recently reported for a human 17-kDa E2 which can complement rad6 mutants thereby identifying rabbit E2(14k) as a RAD6 homologue. The biochemical properties of this previously uncharacterized human 17-kDa E2 are now defined and its misassignment as a homologue of rabbit E2(17k) is corrected. Our findings resolve current confusion regarding relationships among E2s and define yeast RAD6, rabbit E2(14k), and the human 17-kDa E2 as a subclass of E2s which biochemically support E3-mediated conjugation and ubiquitin-dependent proteolysis and physiologically play a role in DNA repair.
...
PMID:A rabbit reticulocyte ubiquitin carrier protein that supports ubiquitin-dependent proteolysis (E214k) is homologous to the yeast DNA repair gene RAD6. 131 8

The c-mos proto-oncogene product, Mos, functions in both early (germinal vesicle breakdown) and late (metaphase II arrest) steps during meiotic maturation in Xenopus oocytes. In the early step, Mos is only partially phosphorylated and metabolically unstable, while in the late step it is fully phosphorylated and highly stable. Using a number of Mos mutants expressed in oocytes, we show here that the instability of Mos in the early step is determined primarily by its penultimate N-terminal residue, or by a rule referred to here as the 'second-codon rule'. We demonstrate that unstable Mos is degraded by the ubiquitin-dependent pathway. In the late step, on the other hand, Mos is stabilized by autophosphorylation at Ser3, which probably acts to prevent the N-terminus of Mos from being recognized by a ubiquitin-protein ligase. Moreover, we show that Ser3 phosphorylation is essential for Mos to exert its full cytostatic factor (CSF) activity in fully mature oocytes. Thus, a few N-terminal amino acids are primary determinants of both the metabolic stability and physiological activity of Mos during the meiotic cell cycle.
...
PMID:The 'second-codon rule' and autophosphorylation govern the stability and activity of Mos during the meiotic cell cycle in Xenopus oocytes. 132 Oct 32

Covalent ligation of multiubiquitin chains targets eukaryotic proteins for degradation. Ubiquitin-conjugating enzyme E2(25K) utilizes isolated ubiquitin as the substrate for synthesis of such chains, in which successive ubiquitin units are linked by isopeptide bonds involving the side chain of Lys-48 of one ubiquitin and the COOH group of Gly-76 of the next. During continuous synthesis of multiubiquitin chains in the presence of purified ubiquitin-activating enzyme and E2(25K), there was a slight discrimination against radioiodinated ubiquitin (2.3-fold reduction in specific radioactivity of diubiquitin relative to value expected for no discrimination). Single-turnover experiments employing stoichiometrically iodinated ubiquitin derivatives indicated that E2(25K) discriminates extremely strongly (greater than 20-fold reduction in kcat/Km for diubiquitin synthesis) against ubiquitin that is monoiodinated at Tyr-59. The modest overall selection effect observed in continuous reactions is in part due to the occurrence of discrimination only when iodotyrosylubiquitin is the acceptor (Lys-48 donor) in diubiquitin synthesis; iodotyrosylubiquitin is kinetically competent when it is the species being transferred to native ubiquitin. The competence as acceptor of a site-directed mutant form of ubiquitin bearing a Tyr to Phe substitution at position 59 indicated that discrimination against iodotyrosylubiquitin by E2(25K) is not due to loss of the hydrogen-bonding interactions of Tyr-59. Rather, iodotyrosylubiquitin may be unable to react with the ubiquitin adduct of E2(25K) for steric reasons. Discrimination against iodotyrosylubiquitin as acceptor is unique to E2(25K) among three enzymes surveyed: iodotyrosylubiquitin is a fully competent acceptor in diubiquitin synthesis catalyzed by E2(25K) and is also utilized for multiubiquitin chain synthesis by E2(14K) and ubiquitin-protein ligase. These findings should assist in the design of future studies concerning E2(25K) structure and function.
...
PMID:Iodination of tyrosine 59 of ubiquitin selectively blocks ubiquitin's acceptor activity in diubiquitin synthesis catalyzed by E2(25K). 132 Nov 47

The ubiquitin-activating enzyme, E1, is required for initiating a multi-step pathway for the covalent linkage of ubiquitin to target proteins. A CHO cell line containing a mutant thermolabile E1, ts20, has been shown to be defective in stress-induced degradation of proteins at restrictive temperature (Gropper et al., 1991. J. Biol. Chem. 266:3602-3610). Parental E36 cells responded to restrictive temperature by stimulating lysosome-mediated protein degradation twofold. Such a response was not observed in ts20 cells. The absence of accelerated degradation in these cells at 39.5 degrees C was accompanied by an accumulation of autolysosomes. The fractional volume of these degradative autophagic vacuoles was at least sixfold greater than that observed for either E36 cells at 30.5 degrees or 39.5 degrees C, or ts20 cells at 30.5 degrees C. These vacuoles were acidic and contained both acid phosphatase and cathepsin L, but, unlike the autolysosomes observed in E36 cells, ubiquitin-conjugated proteins were conspicuously absent. Combined, our results suggest that in ts20 cells, which are unable to generate ubiquitin-protein conjugates due to heat inactivation of E1, the formation and maturation of autophagosomes into autolysosomes is normal, but the conversion of autolysosomes into residual bodies is disrupted.
...
PMID:Ubiquitin-activating enzyme, E1, is associated with maturation of autophagic vacuoles. 132 Nov 57

Ubiquitin is involved in such fundamental cellular processes as cell cycle control, DNA repair, protein degradation and stress responses. We previously reported that cisplatin could inhibit the ubiquitin-ATP-dependent proteolysis and ubiquitination. We further investigated the effect of various antitumor agents on the ubiquitin system and found that aclarubicin (ACR) inhibits the ubiquitin-ATP-dependent proteolysis but not the ubiquitination process. We found that ACR as well as cisplatin inhibited the ubiquitin-ATP-dependent proteolytic activity of rabbit reticulocytes. The IC50 values of these agents were 52 and 90 microM, respectively. Although cisplatin inhibits the conjugation of ubiquitin to proteins through the inhibition of a ubiquitin-activating enzyme, ACR, at 120 microM, does not. Thus, the antitumor agents affecting the ubiquitin system could be classified into two groups; one is represented by cisplatin, which inhibits the ubiquitination of the proteins, and the other is ACR, which does not inhibit the ubiquitination but does inhibit the ubiquitin-ATP-dependent proteolysis. Mitomycin C belongs to the latter group.
...
PMID:Inhibition of different steps of the ubiquitin system by cisplatin and aclarubicin. 132 34

Aminoacyl-tRNA protein transferases post-translationally aminoacylate protein N-termini. At least in part, these enzymes function to allow a subset of cellular proteins to be targeted for protein degradation. A eukaryotic enzyme of this class, Arg aminoacyl-tRNA protein transferase, arginylates N-terminal Glu or Asp residues of proteins, allowing such proteins to be recognized by a specific ubiquitin-protein ligase. We showed previously that inorganic arsenite, a reagent expected to bind specifically to protein vicinal thiol groups, inhibited Arg aminoacyl-tRNA transferase activity in rabbit reticulocyte lysate (N. S. Klemperer and C. M. Pickart, 1989, J. Biol. Chem. 264, 19245-19252). We now report that a bifunctional arsenoxide reagent, p-[(bromoacetyl)-amino]phenylarsenoxide, is a potent and irreversible inactivator of the same enzyme (K0.5 = 11.5 microM). Bromoacetyl aniline, which lacks the arsenoxide moiety, has no effect. These results show that the transferase has a reactive nucleophile proximal to the site which binds arsenoxides. The related monofunctional arsenoxide reagent, p-aminophenylarsenoxide, is a reversible inhibitor whose potency (K0.5 = 7.7 microM) is 20-fold greater than that of inorganic arsenite. As expected for a mechanism in which p-aminophenylarsenoxide binds to vicinal thiol groups: (i) pretreatment of reticulocyte lysate with a thiol-blocking reagent prevents binding of the transferase to a phenylarsenoxide-Sepharose column; and (ii) inhibition by p-aminophenylarsenoxide is reversed by a competing chemical dithiol, but not by a monothiol reagent. Like the rabbit enzyme, Arg aminoacyl-tRNA protein transferase from the yeast Saccharomyces cerevisiae (expressed in Escherichia coli) is reversibly inhibited by the monofunctional phenylarsenoxide and irreversibly inactivated by the bifunctional phenylarsenoxide (but not by bromoacetylaniline). Thus, a reactive nucleophile proximal to vicinal thiol groups is a conserved feature of the activity of the transferase. We speculate that these groups are catalytic elements in the transferase active site.
...
PMID:A reactive nucleophile proximal to vicinal thiols is an evolutionarily conserved feature in the mechanism of Arg aminoacyl-tRNA protein transferase. 141 79

A cDNA encoding the ubiquitin-activating enzyme, E1, was isolated from the mouse mammary carcinoma cell line, FM3A, and shown to complement mutant mouse cells deficient in the enzyme. The 3495-bp cDNA encodes 1058 amino acids (aa), and shares extensive homology with the human E1 enzyme at both the nucleotide and aa sequence levels.
...
PMID:Cloning and sequence of a functionally active cDNA encoding the mouse ubiquitin-activating enzyme E1. 151 1

The induction of thermotolerance was studied in a temperature sensitive mouse cell line, ts85, and results were compared with those for the wild-type FM3A cells. At the nonpermissive temperature of 39 degrees C, ts85 cells are defective in the degradation of short-lived abnormal proteins, apparently because of loss of activity of a ubiquitin-activating enzyme. The failure of the ts85 cells to develop thermotolerance to 41-43 degrees C after incubation at the nonpermissive temperature of 39 degrees C correlated with the failure of the cells to degrade short-lived abnormal proteins at 39 degrees C. However, the failure of the ts85 cells to develop thermotolerance to 43 degrees C during incubation at 33 degrees C after either arsenite treatment or heating at 45.5 degrees C for 6 or 10 min did not correlate with protein degradation rates. Although the rate of degrading abnormal protein was reduced after heating at 45.5 degrees C for 10 min, the rates were normal after arsenite treatment or heating at 45.5 degrees C for 6 min. In addition, when protein synthesis was inhibited with cycloheximide both during incubation at 33 degrees C or 39 degrees C and during heating at 41-43 degrees C, resistance to heating was observed, but protein degradation rates at 39 degrees C or 43 degrees C were not altered by the cycloheximide treatment. Therefore, there is apparently no consistent relationship between rates of degrading abnormal proteins and the ability of cells to develop thermotolerance and resistance to heating in the presence of cycloheximide.
...
PMID:Relationship between thermal tolerance and protein degradation in temperature-sensitive mouse cells. 157 5

tsBN75 and tsBN423 are independently isolated temperature-sensitive (ts) mutants of the BHK21 cell line for cell growth. Both tsBN75 and tsBN423 belong to the same complementation group and show G2 block at the nonpermissive temperature. Both were efficiently transformed to ts+ cells with the mouse and human cDNA encoding the ubiquitin-activating enzyme, E1. While no transformants of tsBN423 cells had a DNA content greater than the parental 2C, several ts+ transformants of tsBN75 cells acquired a multiploid DNA content. These data thus demonstrate the function of the human and mouse E1 cDNAs and further suggest that E1 functions in more than one step in cell cycle progression.
...
PMID:tsBN75 and tsBN423, temperature-sensitive x-linked mutants of the BHK21 cell line, can be complemented by the ubiquitin-activating enzyme E1 cDNA. 157 20

Trivalent arsenoxides bind to vicinal thiol groups of proteins. We showed previously that the simplest trivalent arsenoxide, inorganic arsenite, inhibits ubiquitin-dependent protein degradation in rabbit reticulocyte lysate (Klemperer, N.S., and Pickart, C.M. (1989) J. Biol. Chem. 264, 19245-19242). We now show that, relative to arsenite, phenylarsenoxides are 10-165-fold more potent inhibitors of protein degradation in the same system (K0.5 for inhibition by p-aminophenylarsenoxide was 3.5-20 microM, depending on the substrate). In the ubiquitin-dependent proteolytic pathway, covalent ligation of ubiquitin to protein substrates targets the latter for degradation. In certain cases, specificity in ubiquitin-substrate conjugation depends critically upon the properties of ubiquitin-protein ligase or E3. Among other effects, p-aminophenylarsenoxide decreased the steady-state level of ubiquitinated human alpha-lactalbumin; this is a substrate which is acted upon directly by ubiquitin-protein ligase-alpha (E3-alpha). This finding suggests that phenylarsenoxides (unlike arsenite) inhibit E3. Several other lines of evidence confirm this conclusion. 1) A complex of E3-alpha and the 14-kDa ubiquitin-conjugating (E2) isozyme binds to phenylarsenoxide-Sepharose resin, with the E3 component of the complex mediating binding. 2) p-Aminophenylarsenoxide inhibited isolated E3 (K0.5 approximately 50 microM); inhibition was readily reversed by addition of dithiothreitol (which contains a competing vicinal thiol group), but not by beta-mercaptoethylamine (a monothiol). 3) A bifunctional phenylarsenoxide (bromoacetylaminophenylarsenoxide) rapidly and irreversibly inactivated E3; bromoacetyl aniline, which lacks an arsenoxide moiety, did not inhibit E3. These results suggest that E3 possesses essential vicinal thiol groups and that there is a reactive nucleophile proximal to the vicinal thiol site. The bifunctional phenylarsenoxide should be a useful tool for probing the relationship between structure and function in E3. As expected from prior results with arsenite, p-aminophenylarsenoxide was also a potent inhibitor of the turnover of ubiquitin-(human) alpha-lactalbumin conjugates.
...
PMID:Inhibition of ubiquitin-protein ligase (E3) by mono- and bifunctional phenylarsenoxides. Evidence for essential vicinal thiols and a proximal nucleophile. 164 25


1 2 3 4 5 6 7 8 9 10 Next >>

---