Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Our previous work has shown that the amino-terminal residue of a short-lived protein is a distinct component of the protein's degradation signal. To define the complete signal, otherwise identical dihydrofolate reductase test proteins bearing different extensions and either a "stabilizing" or a "destabilizing" amino-terminal residue were expressed in the yeast S. cerevisiae and their in vivo half-lives compared. The amino-terminal degradation signal is shown to comprise two distinct determinants. One, discovered previously, is the protein's amino-terminal residue. The second determinant, identified in the present work, is a specific lysine residue whose function in the degradation signal is not dependent on the unique amino acid sequences in the vicinity of the residue. The mechanistic significance of the second determinant is illuminated by the finding that in a targeted, short-lived protein, a chain of branched ubiquitin-ubiquitin conjugates is confined to a lysine residue that has been identified in the present work as the second determinant of the degradation signal.
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PMID:The degradation signal in a short-lived protein. 253 46

Previous work has shown that a fusion protein bearing a "nonremovable" N-terminal ubiquitin (Ub) moiety is short-lived in vivo, the fusion's Ub functioning as a degradation signal. The proteolytic system involved, termed the UFD pathway (Ub fusion degradation), was dissected in the yeast Saccharomyces cerevisiae by analyzing mutations that perturb the pathway. Two of the five genes thus identified, UFD1 and UFD5, function at post-ubiquitination steps in the UFD pathway. UFD3 plays a role in controlling the concentration of Ub in a cell: ufd3 mutants have greatly reduced levels of free Ub, and the degradation of Ub fusions in these mutants can be restored by overexpressing Ub. UFD2 and UFD4 appear to influence the formation and topology of a multi-Ub chain linked to the fusion's Ub moiety. UFD1, UFD2, and UFD4 encode previously undescribed proteins of 40, 110, and 170 kDa, respectively. The sequence of the last approximately 280 residues of Ufd4p is similar to that of E6AP, a human protein that binds to both the E6 protein of oncogenic papilloma viruses and the tumor suppressor protein p53, whose Ub-dependent degradation involves E6AP. UFD5 is identical to the previously identified SON1, isolated as an extragenic suppressor of sec63 alleles that impair the transport of proteins into the nucleus. UFD5 is essential for activity of both the UFD and N-end rule pathways (the latter system degrades proteins that bear certain N-terminal residues). We also show that a Lys --> Arg conversion at either position 29 or position 48 in the fusion's Ub moiety greatly reduces ubiquitination and degradation of Ub fusions to beta-galactosidase. By contrast, the ubiquitination and degradation of Ub fusions to dihydrofolate reductase are inhibited by the UbR29 but not by the UbR48 moiety. ufd4 mutants are unable to ubiquitinate the fusion's Ub moiety at Lys29, whereas ufd2 mutants are impaired in the ubiquitination at Lys48. These and related findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve not only the previously identified Lys48 but also Lys29 of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation.
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PMID:A proteolytic pathway that recognizes ubiquitin as a degradation signal. 761 50

A new method for assaying ubiquitin C-terminal hydrolases was developed using a 125I-labeled ubiquitin-alpha NH-MHISPPEPESEEEEEHYC was substrate. Since the peptide portion was almost exclusively radiolabeled, the enzymes could be assayed directly by simple measurement of the radioactivity released into acid-soluble products. Using this assay protocol, we identified at least 10 ubiquitin C-terminal hydrolase activities from the extract of chick skeletal muscle, which were tentatively named UCHs 1 through 10. Of these, UCH-6 was purified to apparent homogeneity. Purified UCH-6 behaved as a dimer of 27-kDa subunits. The apparent molecular masses of the other partially purified UCHs ranged from 35 to 810 kDa as determined under a non-denaturing condition. Muscle UCHs, except UCH-1, were activated dramatically by poly-L-Lys but with an unknown mechanism. All of the UCHs were sensitive to inhibition by sulfhydryl-blocking agents such as iodoacetamide. In addition, all of the UCHs were capable of releasing free ubiquitin from a ubiquitin-alpha NH-carboxyl extension protein of 80 amino acids and from ubiquitin-alpha NH-dihydrofolate reductase. Five of the enzymes, UCHs 1 through 5, were also capable of generating free ubiquitin from poly-His-tagged diubiquitin. In addition, UCH-1 and UCH-7 could remove ubiquitin that had been ligated covalently by an isopeptide linkage to a ubiquitin (RGA)-alpha NH-peptide, the peptide portion of which consists of the 20 amino acids of the calmodulin binding domain of myosin light chain kinase. These results suggest that the 10 UCH activities isolated from chick skeletal muscle appear to be distinct from each other at least in their chromatographic behavior, size, and substrate specificity.
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PMID:Multiple ubiquitin C-terminal hydrolases from chick skeletal muscle. 764 26

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. In eukaryotes, the N-end rule pathway is a ubiquitin-dependent, proteasome-based system that targets and processively degrades proteins bearing certain N-terminal residues. Arg-DHFR, a modified dihydrofolate reductase bearing an N-terminal arginine (destabilizing residue in the N-end rule), is short lived in ATP-supplemented reticulocyte extract. It is shown here that methotrexate, which is a folic acid analog and high affinity ligand of DHFR, inhibits the degradation but not ubiquitination of Arg-DHFR by the N-end rule pathway. The degradation of other N-end rule substrates is not affected by methotrexate. We discuss implications of these results for the mechanism of proteasome-mediated protein degradation.
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PMID:Methotrexate inhibits proteolysis of dihydrofolate reductase by the N-end rule pathway. 771 22

We have previously shown that chick muscle extracts contain at least 10 different ubiquitin C-terminal hydrolases (UCHs). In the present studies, one of the enzymes, called UCH-1 was partially purified by conventional chromatographic procedures using (125)I-labeled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a substrate. The purified enzyme behaved as a 35-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consisted of a single polypeptide chain. It was maximally active at pHs between 8 and 9, but showed little or no activity at pH below 6 and above 11. Like other UCHs, its activity was strongly inhibited by sulfhydryl blocking reagents, such as iodoacetamide, and by ubiquitin-aldehyde. In addition to Ub-PESTc, UCH-1 hydrolyzed ubiquitin-alphaNH-protein extensions, including ubiquitin-alphaNH-carboxyl extension protein of 80 amino acids, ubiquitin-alphaNH-dihydrofolate reductase, and poly-His-tagged di-ubiquitin. This enzyme was also capable of generating free ubiquitin from mono-ubiquitin-epsilonNH-protein conjugates and from branched poly-ubiquitin chains that are ligated to proteins through epsilon NH-isopeptide bonds. These results suggest that UCH-1 may play an important role in the generation of free ubiquitin from ubiquitin-ribosomal protein fusions and linear poly-ubiquitin, as well as in recycling of Ub molecules after degradation of poly-ubiquitinated protein conjugates by the 26S proteasome.
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PMID:Purification and characterization of a new ubiquitin C-terminal hydrolase (UCH-1) with isopeptidase activity from chick skeletal muscle. 916 18

We have previously shown that chick muscle extracts contained at least 10 different ubiquitin C-terminal hydrolases (UCHs). Here we report the purification and characterization of one of the UCHs, called UCH-8, with 125I-labelled ubiquitin-alpha-NH-MHISPPEPESEEEEEHYC as a substrate. The purified UCH-8 behaved as a 240 kDa protein on a Superdex-200 column under non-denaturing conditions but as a 130 kDa polypeptide on analysis by PAGE under denaturing conditions, suggesting that the enzyme consists of two identical subunits. Thus this enzyme seems to be distinct in its dimeric nature from other purified UCHs that consist of a single polypeptide, except that UCH-6 is also a homodimer of 27 kDa subunits. UCH-8 was maximally active between pH 7.5 and 8, but showed little or no activity below pH 7 and above pH 9. Like other UCHs it was sensitive to inhibition by thiol-blocking agents such as N-ethylmaleimide, and by ubiquitin aldehyde. The purified UCH-8 hydrolysed not only ubiquitin-alpha-NH-protein extensions, including ubiquitin-alpha-NH-carboxy extension protein of 80 amino acid residues and ubiquitin-alpha-NH-dihydrofolate reductase, but also branched poly-ubiquitin that are ligated to proteins through epsilon-NH-isopeptide bonds. However, it showed little or no activity against poly-His-tagged di-ubiquitin, suggesting that UCH-8 is not involved in the generation of free ubiquitin from the linear poly-ubiquitin precursors. These results suggest that UCH-8 might have an important role in the production of free ubiquitin and ribosomal proteins from their conjugates as well as in the recycling of ubiquitin molecules after the degradation of poly-ubiquitinated protein conjugates by the 26 S proteasome.
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PMID:New de-ubiquitinating enzyme, ubiquitin C-terminal hydrolase 8, in chick skeletal muscle. 923 Jan 10

Ubiquitin-specific protease-6 (UBP6) in Saccharomyces cerevisiae was expressed in Escherichia coli and purified from the cells using 125I-labeled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a model substrate. The purified UBP6 behaved as a 58-kDa under both nondenaturing and denaturing conditions, indicating that the enzyme comprises a single polypeptide. It was maximally active at pH levels between 8.5 and 9, but showed little or no activity at pH below 7 and above 9.5. As with other UBPs, its activity was strongly inhibited by sulfhydryl-blocking reagents, such as N-ethylmaleimide, and by ubiquitin-aldehyde. In addition to the model substrate, UBP6 hydrolyzed ubiquitin-alphaNH-protein extensions, such as the ubiquitin-alphaNH-carboxyl extension protein of 80 amino acids and ubiquitin-alphaNH-dihydrofolate reductase, but not poly-His-tagged diubiquitin. It was also capable of releasing free ubiquitin from branched polyubiquitin chains that are ligated to proteins through epsilonNH-isopeptide bonds, although to a limited extent. These results suggest that UBP6 may play an important role in the generation of free ubiquitins and certain ribosomal proteins from ubiquitin-ribosomal fusion proteins as well as in deubiquitination of certain polyubiquitinated proteins targeted for degradation by the 26S proteasomes.
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PMID:Purification and characterization of UBP6, a new ubiquitin-specific protease in Saccharomyces cerevisiae. 934 67

The N-end rule pathway is a ubiquitin-dependent proteolytic system, the targets of which include proteins that bear destabilizing N-terminal residues. The latter are a part of the degradation signal called the N-degron. Arg-DHFRts, an engineered N-end rule substrate, bears N-terminal arginine (a destabilizing residue) and DHFRts [a temperature-sensitive mouse dihydrofolate reductase (DHFR) moiety]. Previous work has shown that Arg-DHFRts is long-lived at 23 degreesC but short-lived at 37 degreesC in the yeast Saccharomyces cerevisiae. In the present work, we extended this analysis, and found that the degradation of Arg-DHFRts can be nearly completely inhibited in vivo by methotrexate (MTX), a low-Mr ligand of DHFR. In S. cerevisiae, Arg-DHFRts is degraded at 37 degreesC exclusively by the N-end rule pathway, whereas in mouse cells the same protein at the same temperature is also targeted by another proteolytic system, through a degron in the conformationally perturbed DHFRts moiety. In mouse cells, MTX completely inhibits the degradation of Arg-DHFRts through its degron within the DHFRts moiety, but only partially inhibits degradation through the N-degron. When the N-terminus of Arg-DHFRts was extended with a 42-residue lysine-lacking extension, termed eDeltaK, the resulting Arg-eDeltaK-DHFRts was rapidly degraded at both 23 degreesC and 37 degreesC. Moreover, the degradation of Arg-eDeltaK-DHFRts, in contrast with that of Arg-DHFRts, could not be inhibited by MTX, suggesting that the metabolic stability of Arg-DHFRts at 23 degreesC results, at least in part, from steric inaccessibility of its N-terminal arginine. The N-degron of Arg-DHFRts is the first example of a portable degradation signal the activity of which can be modulated in vivo by a cell-penetrating compound. We discuss implications of this advance and the mechanics of targeting by the ubiquitin system.
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PMID:Analysis of a conditional degradation signal in yeast and mammalian cells. 991 99

Ubiquitin-dependent proteolysis is a major proteolytic pathway in the cytoplasm and nucleus of eukaryotic cells. We introduced a gene encoding a substrate for this pathway into the genome of Arabidopsis thaliana. The transgene codes for a hybrid protein consisting of dihydrofolate reductase (DHFR, EC 1.5.1.3) fused to a degradation signal that is specifically recognized by components of the ubiquitin-dependent proteolysis pathway. Elevated concentrations of the DHFR protein confer resistance to the drug methotrexate, but rapid degradation prevents accumulation of the protein in the plant. Therefore, transgenic A. thaliana lines expressing the DHFR fusion protein are methotrexate-sensitive. Selection for mutants resistant to methotrexate resulted in plants impaired in degradation of the DHFR model substrate, as shown by an increase in protein level in the mutants.
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PMID:Use of a reporter transgene to generate arabidopsis mutants in ubiquitin-dependent protein degradation. 1160 48

Yersinia enterocolitica organisms secrete Yop proteins via the type III pathway. Translational fusion of yop genes to ubiquitin or dihydrofolate reductase results in hybrid proteins that cannot be secreted. The folding of hybrids prevents their own transport, but it does not hinder the type III secretion of other Yops.
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PMID:Yop fusions to tightly folded protein domains and their effects on Yersinia enterocolitica type III secretion. 1205 71


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