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)

Autophagy is a cellular process for bulk degradation of cytoplasmic components. The attachment of Apg12p, a modifier with no significant similarity to ubiquitin, to Apg5p is crucial for autophagy in yeast. This reaction proceeds in a ubiquitination-like manner, and requires Apg7p and Apg10p. Apg7p exhibits a considerable similarity to ubiquitin-activating enzyme (E1) and is found to activate Apg12p with ATP hydrolysis. Apg10p, on the other hand, shows no significant similarity to other proteins whose functions are known. Here, we show that after activation by Apg7p, Apg12p is transferred to the Cys-133 residue of Apg10p to form an Apg12p-Apg10p thioester. Cells expressing Apg10p(C133S) do not generate the Apg12p-Apg5p conjugate, which leads to defects in autophagy and cytoplasm-to-vacuole targeting of aminopeptidase I. These findings indicate that Apg10p is a new type of protein-conjugating enzyme that functions in the Apg12p-Apg5p conjugation pathway.
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PMID:Apg10p, a novel protein-conjugating enzyme essential for autophagy in yeast. 1050 57

Mutations in the PMA1 gene, encoding plasma membrane H+ -ATPase, were isolated that are able to suppress the temperature sensitivity (ts) phenotype of mdp1 mutations located in RSP5, the ubiquitin-protein ligase gene. The mdp1 mutants were previously found to change the mitochondrial/cytosolic distribution of Mod5p-I, the tRNA modifying enzyme, and to affect fluid phase endocytosis. The data presented reveal that mdp1 mutants are also pH sensitive, and hypersensitive to hygromycin B and paromomycin. The ts phenotype, hygromycin B and paromomycin sensitivity are suppressed by pmal-t, but the pH sensitivity, the effect of mdp1 on Mod5p-I cytoplasmic/mitochondrial localization and endocytosis are not. Characterization of pmal-t revealed the substitution of amino acid G(653)V in the ATP-binding domain of the H+ -ATPase. Our results indicate that Rsp5 ubiquitin-protein ligase may also influence, in addition to protein distribution, the functioning of plasma membrane H+ -ATPase and the response of cells to stress.
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PMID:The growth of mdp1/rsp5 mutants of Saccharomyces cerevisiae is affected by mutations in the ATP-binding domain of the plasma membrane H+ -ATPase. 1072 5

We report the synthesis of fluorescently labeled ubiquitin (Ub) and its use for following ubiquitin transfer to various proteins. Using Oregon green (Og) succinimidyl ester, we prepared a population of Ub mainly labeled by a single Og molecule; greater than 95% of the Og label is associated with Lys 6 of Ub. We demonstrate that Og-Ub is efficiently accepted by Ub-utilizing enzymes, such as the human ubiquitin-activating enzyme (E1). We used this fluorescent substrate to follow the steady-state kinetics of human E1-catalyzed Ub-transfer to the ubiquitin-carrier enzyme Ubc4. In this reaction, E1 uses three substrates: ATP, Ubc4, and Ub. The steady-state kinetics of Og-Ub utilization by E1 is presented. We have also used analytical ultracentrifugation methods to establish that E1 is monomeric under our assay condition (low salt) as well as under physiological condition (150 mM NaCl).
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PMID:Steady-state kinetic analysis of human ubiquitin-activating enzyme (E1) using a fluorescently labeled ubiquitin substrate. 1119 73

Oxidatively modified proteins are continuously produced in cells by reactive oxygen and nitrogen species generated as a consequence of aerobic metabolism. During periods of oxidative stress, protein oxidation is significantly increased and may become a threat to cell survival. In eucaryotic cells the proteasome has been shown (by purification of enzymatic activity, by immunoprecipitation, and by antisense oligonucleotide studies) to selectively recognize and degrade mildly oxidized proteins in the cytosol, nucleus, and endoplasmic reticulum, thus minimizing their cytotoxicity. From in vitro studies it is evident that the 20S proteasome complex actively recognizes and degrades oxidized proteins, but the 26S proteasome, even in the presence of ATP and a reconstituted functional ubiquitinylating system, is not very effective. Furthermore, relatively mild oxidative stress rapidly (but reversibly) inactivates both the ubiquitin activating/conjugating system and 26S proteasome activity in intact cells, but does not affect 20S proteasome activity. Since mild oxidative stress actually increases proteasome-dependent proteolysis (of oxidized protein substrates) the 20S 'core' proteasome complex would appear to be responsible. Finally, new experiments indicate that conditional mutational inactivation of the E1 ubiquitin-activating enzyme does not affect the degradation of oxidized proteins, further strengthening the hypothesis that oxidatively modified proteins are degraded in an ATP-independent, and ubiquitin-independent, manner by the 20S proteasome. More severe oxidative stress causes extensive protein oxidation, directly generating protein fragments, and cross-linked and aggregated proteins, that become progressively resistant to proteolytic digestion. In fact these aggregated, cross-linked, oxidized proteins actually bind to the 20S proteasome and act as irreversible inhibitors. It is proposed that aging, and various degenerative diseases, involve increased oxidative stress (largely from damaged and electron 'leaky' mitochondria), and elevated levels of protein oxidation, cross-linking, and aggregation. Since these products of severe oxidative stress inhibit the 20S proteasome, they cause a vicious cycle of progressively worsening accumulation of cytotoxic protein oxidation products.
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PMID:Degradation of oxidized proteins by the 20S proteasome. 1129 90

The involvement of red blood cell spectrin in the ubiquitination process was studied. Spectrin was found to form two ubiquitin-associated derivatives, a DTT-sensitive ubiquitin adduct and a DTT-insensitive conjugate, characteristic intermediate and final products of the ubiquitination reaction cascade. In addition to spectrin and ubiquitin, ubiquitin-activating enzyme (E1) and ATP were necessary and sufficient to form both the spectrin-ubiquitin adduct and conjugate. No exogenous ubiquitin-conjugating (E2) or ligase (E3) activities were required, suggesting that erythrocyte spectrin is an E2 ubiquitin-conjugating enzyme able to target itself. Both ubiquitin adduct and conjugate were linked to the alpha subunit of spectrin, suggesting that the ubiquitin-conjugating (UBC) domain and its target regions reside on the same subunit.
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PMID:Erythrocyte spectrin is an E2 ubiquitin conjugating enzyme. 1156 May 14

The tumor suppressor p53 is a labile protein whose level is known to be regulated by the Mdm-2-ubiquitin-proteasome degradation pathway. We have found another pathway for p53 proteasomal degradation regulated by NAD(P)H quinone oxidoreductase 1 (NQO1). Inhibition of NQO1 activity by dicoumarol induces p53 and p73 proteasomal degradation. A mutant p53 (p53([22,23])), which is resistant to Mdm-2-mediated degradation, was susceptible to dicoumarol-induced degradation. This finding indicates that the NQO1-regulated proteasomal p53 degradation is Mdm-2-independent. The tumor suppressor p14(ARF) and the viral oncogenes SV40 LT and adenovirus E1A that are known to stabilize p53 inhibited dicoumarol-induced p53 degradation. Unlike Mdm-2-mediated degradation, the NQO1-regulated p53 degradation pathway was not associated with accumulation of ubiquitin-conjugated p53. In vitro studies indicate that dicoumarol-induced p53 degradation was ubiquitin-independent and ATP-dependent. Inhibition of NQO1 activity in cells with a temperature-sensitive E1 ubiquitin-activating enzyme induced p53 degradation and inhibited apoptosis at the restrictive temperature without ubiquitination. Mdm-2 failed to induce p53 degradation under these conditions. Our results establish a Mdm-2- and ubiquitin-independent mechanism for proteasomal degradation of p53 that is regulated by NQO1. The lack of NQO1 activity that stabilizes a tumor suppressor such as p53 can explain why humans carrying a polymorphic inactive NQO1 are more susceptible to tumor development.
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PMID:Mdm-2 and ubiquitin-independent p53 proteasomal degradation regulated by NQO1. 1223 53

Human Nedd8-activating enzyme AppBp1-Uba3 was purified to apparent homogeneity from erythrocytes. In the presence of [2,8-3H]ATP and 125I-Nedd8, heterodimer rapidly forms a stable stoichiometric ternary complex composed of tightly bound Nedd8 [3H]adenylate and Uba3-125I-Nedd8 thiol ester. Isotope exchange kinetics show that the heterodimer follows a pseudo-ordered mechanism with ATP the leading and Nedd8 the trailing substrate. Human AppBp1-Uba3 follows hyperbolic kinetics for HsUbc12 transthiolation with 125I-Nedd8 (kcat = 3.5 +/- 0.2 s-1), yielding Km values for ATP (103 +/- 12 microm), 125I-Nedd8 (0.95 +/- 0.18 microm), and HsUbc12 (43 +/- 13 nm) similar to those for ubiquitin activation by Uba1. Wild type 125I-ubiquitin fails to support AppBp1-Uba3 catalyzed activation or HsUbc12 transthiolation. However, modest inhibition of 125I-Nedd8 ternary complex formation by unlabeled ubiquitin suggests a Kd > 300 microm for ubiquitin. Alanine 72 of Nedd8 is a critical specificity determinant for AppBp1-Uba3 binding because 125I-UbR72L undergoes heterodimer-catalyzed hyperbolic HsUbc12 transthiolation and yields Km = 20 +/- 9 microm and kcat = 0.9 +/- 0.3 s-1. These observations demonstrate remarkable conservation in the mechanism of AppBp1-Uba3 that mirrors its sequence conservation with the Uba1 ubiquitin-activating enzyme.
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PMID:Conservation in the mechanism of Nedd8 activation by the human AppBp1-Uba3 heterodimer. 1274 Mar 88

The endoplasmic reticulum (ER) enzyme, 3-hydroxy-3-methylglutaryl-CoA reductase, catalyzes the production of mevalonate, a rate-controlling step in cholesterol biosynthesis. Excess sterols promote ubiquitination and subsequent degradation of reductase as part of a negative feedback regulatory mechanism. To characterize the process in more detail, we here report the development of a permeabilized cell system that supports reductase ubiquitination stimulated by the addition of sterols in vitro. Sterol-dependent ubiquitination of reductase in permeabilized cells is dependent upon exogenous cytosol, ATP, and either Insig-1 or Insig-2, two membrane-bound ER proteins shown previously to mediate sterol regulation of reductase degradation in intact cells. Oxysterols, but not cholesterol, promote reductase ubiquitination under our conditions. Finally, we show that ubiquitin-activating enzyme (E1) can efficiently replace cytosol to ubiquitinate reductase in response to sterol treatment, suggesting that other molecules required for ubiquitination of reductase, such as the ubiquitin-conjugating and -ligating enzymes (E2 and E3), are localized to ER membranes.
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PMID:Ubiquitination of 3-hydroxy-3-methylglutaryl-CoA reductase in permeabilized cells mediated by cytosolic E1 and a putative membrane-bound ubiquitin ligase. 1509 May 40

It is established that neuronal nitric-oxide synthase (nNOS) is ubiquitylated and proteasomally degraded. The proteasomal degradation of nNOS is enhanced by suicide inactivation of nNOS or by the inhibition of hsp90, which is a chaperone found in a native complex with nNOS. In the current study, we have examined whether CHIP, a chaperone-dependent E3 ubiquitin-protein isopeptide ligase that is known to ubiquitylate other hsp90-chaperoned proteins, could act as an ubiquitin ligase for nNOS. We found with the use of HEK293T or COS-7 cells and transient transfection methods that CHIP overexpression causes a decrease in immunodetectable levels of nNOS. The extent of the loss of nNOS is dependent on the amount of CHIP cDNA used for transfection. Lactacystin (10 microM), a selective proteasome inhibitor, attenuates the loss of nNOS in part by causing the nNOS to be found in a detergent-insoluble form. Immunoprecipitation of the nNOS and subsequent Western blotting with an anti-ubiquitin IgG shows an increase in nNOS-ubiquitin conjugates because of CHIP. Moreover, incubation of nNOS with a purified system containing an E1 ubiquitin-activating enzyme, an E2 ubiquitin carrier protein conjugating enzyme (UbcH5a), CHIP, glutathione S-transferase-tagged ubiquitin, and an ATP-generating system leads to the ubiquitylation of nNOS. The addition of purified hsp70 and hsp40 to this in vitro system greatly enhances the amount of nNOS-ubiquitin conjugates, suggesting that CHIP is an E3 ligase for nNOS whose action is facilitated by (and possibly requires) its interaction with nNOS-bound hsp70.
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PMID:Ubiquitylation of neuronal nitric-oxide synthase by CHIP, a chaperone-dependent E3 ligase. 1546 72

Protein ubiquitinylation plays a key role in many important cellular processes. Ubiquitinylation requires the E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and, frequently, a substrate-specific E3 ubiquitin-protein ligase. In one class of E3 ubiquitin ligases, the catalytic domain contains a zinc-binding RING finger motif. ARD1 (ADP-ribosylation factor domain protein 1), with a RING finger domain in the N-terminal region, two predicted B-Boxes, and a coiled-coil protein interaction motif immediately preceding an ADP-ribosylation factor domain at the C terminus, belongs to the TRIM (Tripartite motif) or RBCC (RING, B-Box, coiled-coil) family. The region containing the B-Boxes and the coiled-coil motif acts as a GTPase-activating protein for the ADP-ribosylation factor domain of ARD1. We report here that full-length ARD1 or the RING finger domain (residues 1-110) produced polyubiquitinylated proteins in vitro in the presence of mammalian E1, an E2 enzyme (UbcH6 or UbcH5a, -5b, or -5c), ATP, and ubiquitin. Deletion of the RING region or point mutations within the RING sequence abolished ARD1 E3 ligase activity. All data are consistent with a potential function for ARD1 as an E3 ubiquitin ligase in cells.
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PMID:E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1). 1568 77


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