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Query: UNIPROT:P62988 (Ubiquitin)
 4,326 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The low molecular weight polypeptide required for energy-dependent proteolysis, ubiquitin, is rapidly inactivated by 100,000 X g supernatants of rabbit liver extracts. Ubiquitin inactivation results from limited proteolysis by an endogenous contaminating lysosomal thiol protease having trypsin-like specificity. Evidence for this includes a pH optimum of 5.0 for the first order constant of ubiquitin inactivation and observation that inactivation is inhibited by EDTA, o-phenanthroline, iodoacetamide, p-chloromercuribenzoic acid, phenylmethylsulfonyl fluoride, N alpha-p-tosyl-L-lysine chloromethyl ketone, leupeptin, soybean trypsin inhibitor, and aprotinin. Metals stimulate but are not required for ubiquitin inactivation with the effect apparently mediated by a low molecular weight heat-labile component of crude extracts. When this heat-labile component is removed by gel exclusion chromatography a number of metals inhibit ubiquitin inactivation. In the presence of excess dithiothreitol, inhibition is relatively specific for Zn(II). Inhibition by Zn(II) is specifically overcome competitively by Cd(II) or by a concentration of ubiquitin in excess of Zn(II). The responsible cathepsin possesses a molecular mass of 35 kDa by gel exclusion chromatography and shows marked thermal lability at neutral pH but stability at acid pH. Proteolytic inactivation of ubiquitin results from limited cleavage of the carboxyl-terminal glycine dipeptide required for isopeptide bond formation and is supported by data on isoelectric point changes on subsequent digestion with carboxypeptidase B and by direct amino acid analysis. When the responsible cathepsin is inactivated, liver extracts display ATP,ubiquitin-dependent proteolysis that cannot be ascribed to contaminating erythrocytes. Thus the previous inability to demonstrate energy-dependent proteolysis in liver extracts is accounted for by the artifactual inactivation of ubiquitin.
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PMID:The inactivation of ubiquitin accounts for the inability to demonstrate ATP, ubiquitin-dependent proteolysis in liver extracts. 298 63

Ubiquitin, a 76 residue protein, occurs in eukaryotic cells either free or covalently joined via its carboxyl terminus to epsilon-amino groups of lysine residues in a wide variety of protein species. Previous work has shown that ubiquitin-protein conjugates are preferred substrates in vitro for a non-lysosomal ATP-dependent proteolytic pathway, suggesting that ubiquitin may function as a signal for attack by proteinases specific for ubiquitin-protein conjugates. One strategy to define the potential significance of the ubiquitin-dependent proteolytic pathway is to identify conditional mutants in the pathway. ts85 is a mouse derived cell-cycle mutant which has been shown to lose uH2A, a specific ubiquitin-histone H2A conjugate, at the nonpermissive temperature. We show that the loss of uH2A from ts85 cells is due to reduced ubiquitin-protein conjugation. We further show that the reduced conjugation is due to the specific thermolability of ubiquitin activating enzyme, E1, one of the three enzymic components of the ubiquitin-protein ligase system. We therefore proceeded to test whether the degradation of short-lived proteins is also temperature-sensitive in ts85 cells. Indeed, while more than 70% of the prelabeled abnormal (amino acid analog-containing) proteins or puromycyl peptides are degraded within 4 hours at the permissive temperature in the mutant (ts85), wild type (FM3A), and revertant (ts85R-MN3) cells, less than 15% of these proteins are degraded in ts85 cells at the nonpermissive temperature. In contrast, the rate of degradation of these proteins does not change significantly in either wild-type or revertant cells between permissive and nonpermissive temperatures. Degradation of normal short-lived proteins is also specifically temperature-sensitive in ts85 cells. Immunochemical analysis shows a strong and specific reduction in ubiquitin-protein conjugate levels in vivo at the nonpermissive temperature in ts85 cells. Taken together, our in vitro and in vivo findings with ts85 cells demonstrate that the degradation of the bulk of short-lived proteins in this higher eukaryotic cell is accomplished through a ubiquitin-mediated pathway.
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PMID:Mammalian cell cycle mutant defective in intracellular protein degradation and ubiquitin-protein conjugation. 299 83

Ubiquitin is a highly conserved, 76-amino acid polypeptide recently demonstrated to be involved in ATP-dependent protein degradation in mammalian cells. From immunoblot analyses with anti-human-ubiquitin antibodies we have detected the presence of free ubiquitin in green leaves, etiolated shoots, and dry seeds of the higher plant, oats (Avena sativa L.). We also find that crude oat extracts contain protease(s) that rapidly degrade both oat and human ubiquitin (t1/2 approximately 10 min at 27 degrees C). This proteolysis apparently cleaves ubiquitin at the carboxyl-terminal glycine dipeptide and results in inactivation of the molecule with respect to ligation but does not affect its mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using homogenization conditions that preclude this proteolysis (low pH and the addition of the protease inhibitor p-chloromercuribenzoate) and immunoblotting as an assay for the protein, a procedure for the purification of ubiquitin from etiolated oat shoots was developed. Characterization of purified oat ubiquitin by absorption spectra, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, radioimmunoassay with anti-human-ubiquitin antibodies, and kinetic analyses using the ubiquitin activating enzyme isolated from rabbit liver indicates that this protein is remarkably similar to the mammalian form. Small differences between the oat and human proteins have been observed by amino acid compositional analyses indicating that the two forms are not totally homologous. Immunoblotting of crude oat extracts has revealed the presence of high molecular weight proteins recognized by anti-ubiquitin antibodies that represent ubiquitin-protein conjugates formed in vivo. Taken together, these data provide evidence that higher plants contain a ubiquitin-dependent proteolytic pathway that is mechanistically identical to that present in animals.
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PMID:Purification and initial characterization of ubiquitin from the higher plant, Avena sativa. 299 56

Ubiquitin-lysozyme conjugates have been used as substrates to identify an ATP-dependent protease from rabbit reticulocyte lysates. The enzyme, which has been partially purified by DEAE chromatography and glycerol gradient centrifugation, has an apparent molecular weight greater than 600,000 based on sedimentation and gel filtration. Whereas it degrades conjugated lysozyme molecules in the presence of ATP, the protease does not degrade free lysozyme molecules even upon addition of ubiquitin, lysozyme-ubiquitin conjugates, and ATP. Degradation of lysozyme conjugates is independent of added ubiquitin and occurs in fractions incapable of ubiquitin conjugation. Proteolysis is maximal at pH 7.8, inhibited by hemin, N-ethylmaleimide, or aurintricarboxylic acid, and proceeds with an apparent Arrhenius activation energy in the range of 27 +/- 5 kcal/mol. These properties are similar to those observed for the degradation of lysozyme conjugates in lysates indicating that the partially purified protease catalyzes the "second" ATP-utilizing reaction identified previously (Hough, R., and Rechsteiner, M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 90-94; Hershko, A., Leshinsky, E., Ganoth, D., and Heller, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1619-1623; Tanaka, K., Waxman, L., and Goldberg, A. L. (1983) J. Cell Biol. 96, 1580-1585).
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PMID:Ubiquitin-lysozyme conjugates. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte lysates. 300 14

Degradation of intracellular proteins via the ubiquitin- and ATP-dependent proteolytic pathway involves several steps. In the initial event, ubiquitin, an abundant 76-residue polypeptide is covalently linked to the protein substrate in an ATP-requiring reaction. Proteins marked by ubiquitin are selectively proteolyzed in a reaction that also requires ATP. Ubiquitin conjugation to proteins appears also to be involved in regulation of cell cycle and cell division, and probably in the regulation of gene expression at the level of chromatin structure. We have previously shown (Ciechanover, A., Wolin, S. L., Steitz, J. A., and Lodish, H. F. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1341-1345) that transfer RNA is an essential component of the ubiquitin pathway. Ribonucleases strongly and specifically inhibited the degradation of 125I-labeled bovine serum albumin, while tRNA purified from reticulocyte extract could restore the proteolytic activity. Specifically, pure tRNAHis isolated by immunoprecipitation with human autoimmune serum could restore the proteolytic activity. Here we demonstrate that tRNA is required for conjugation of ubiquitin to some but not all proteolytic substrates of the ubiquitin mediated pathway. Conjugation of 125I-labeled ubiquitin to reduced carboxymethylated bovine serum albumin, alpha-lactalbumin, and soybean trypsin inhibitor was strongly and specifically inhibited by ribonucleases. Consequently, the ATP-dependent degradation of these substrates in the cell-free ubiquitin-dependent reticulocyte system was inhibited as well. Addition of tRNA to the ribonuclease inhibited system (following inhibition of the ribonuclease) restored both the conjugation activity and the ubiquitin- and ATP-dependent degradation of these substrates. Conjugation of ubiquitin to some endogenous reticulocyte proteins was also inhibited by ribonucleases and could be restored by the addition of tRNA. In striking contrast, the conjugation of radiolabeled ubiquitin to lysozyme, oxidized RNase A, alpha-casein, and beta-lactoglobulin was not affected by the ribonuclease treatment, and the degradation of these substrates was significantly accelerated by the ribonucleases. These findings indicate that there are at least two distinct ubiquitin conjugation systems. One requires tRNA, and the other is tRNA independent. These pathways, however, must share some common component(s) of the system, since the inhibition of one system accelerates the other. The possible function of tRNA in the selective conjugation reaction and the possible role of the two distinct ubiquitin marking mechanisms are discussed.
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PMID:Transfer RNA is required for conjugation of ubiquitin to selective substrates of the ubiquitin- and ATP-dependent proteolytic system. 300 81

[125I]Ubiquitin introduced into permeabilized hepatoma tissue culture (HTC) cells rapidly forms conjugates with endogenous proteins. A characteristic pattern of low mol. wt conjugates is obtained which includes the ubiquitinated histone, uH2A, and unknown molecular species with MrS of 14, 23, 26 (two bands) and 29 kd. A broad spectrum of higher mol. wt conjugates is also produced. The formation of all conjugates is absolutely dependent on ATP, and upon depletion of ATP they are rapidly broken down. The 14, 23 and 29 kd species are found in all subcellular fractions examined. uH2A is located exclusively in the nuclear fraction. The pair of 26 kd bands is specifically associated with the ribosome fraction. A considerable percentage of the higher mol. wt conjugates sediments with the small particle (100,000 g) fraction in the ultracentrifuge but is solubilized with deoxycholate, indicating that there are many membrane-associated conjugates. The pattern of ubiquitin conjugation in interphase and metaphase cells was compared. The incorporation of ubiquitin into uH2A was markedly reduced in metaphase cells whereas its incorporation into other low mol. wt conjugates and into high mol. wt conjugates was affected slightly, if at all. This shows that the known decrease of uH2A levels in metaphase is due to a specific effect on histone ubiquitination and not to a general decrease in ubiquitination activity or increase of isopeptidase activity. Changes in the levels of uH2A during mitosis measured by immunoblotting were similar to those estimated in permeabilized cells. These experiments indicate that permeabilized cells provide a useful approach to the study of rapidly turning over ubiquitin conjugates in mammalian cells.
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PMID:Conjugation of [125I]ubiquitin to cellular proteins in permeabilized mammalian cells: comparison of mitotic and interphase cells. 301 20

Ubiquitin is an extremely conserved protein, with an identical sequence throughout the animal kingdom. However, the gene sequence of the yeast protein [Ozkaynak, E., Finley, D., & Varshavsky, A. (1984) Nature (London) 312, 663-666] predicts three amino acid differences. This implies that some functions or binding interactions of ubiquitin are different in yeast and animal cells. In an effort to define these differences, ubiquitin has been purified to homogeneity from bakers' yeast and characterized. Amino acid analysis of the protein and the isolated tryptic peptides confirms the primary structure of this protein as predicted from the gene sequence. This result indicates that the gene sequenced is the transcriptionally active gene from yeast. The conformation of yeast ubiquitin is similar to human ubiquitin as judged by circular dichroism, sensitivity to trypsin, and Stokes radius. Yeast and animal ubiquitins show identical activities in supporting ubiquitin-dependent protein degradation and in the ATP-pyrophosphate exchange reaction catalyzed by the purified ubiquitin-adenylating enzyme. Thus, the three conservative amino acid differences between yeast and animal ubiquitins have very little effect on the structure of ubiquitin or its activity in the ubiquitin-dependent proteolytic system. These results suggest that at least some of the evolutionary pressure preventing sequence variation among animal ubiquitins stems from one or more of its nonproteolytic functions.
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PMID:Structure and activities of a variant ubiquitin sequence from bakers' yeast. 302 Dec 9

A human erythroleukemia cell line, K-562 T1, was adapted to a protein-free chemically defined medium; that is, the medium does not contain any proteins such as exogenous hormones, growth factors, serum and serum albumin. The K-562 T1 cells which can proliferate in a protein-free medium are one of the model systems suitably supporting the autocrine hypothesis, which claims that cancer cells produce and respond to their own growth factors. The K-562 T1 cells were cultured in a protein-free medium at large scale and the growth factors were purified from the conditioned medium. It was found that K-562 T1 cells produce at least two growth factors; one is LGF-I (leukemia-derived growth factor-I) which can stimulate the proliferation of a wide range of human leukemia cell lines and the other is LGF-II (leukemia-derived growth factor-II), which can contribute to the growth of fibroblasts. LGF-I was purified using QAE-Sephadex, Bio Gel P-60 and Mono S FPLC. The purified protein was found to be homogenous by SDS-polyacrylamide gel electrophoresis and NH2-terminal sequence analysis. The molecular weight of LGF-I was 20,000 by SDS-polyacrylamide gel electrophoresis. The 30 NH2-terminal residues of LGF-I are the same as that of ubiquitin. Ubiquitin is a protein found in eukaryotic cells with molecular weight of 8,600. In the nucleus ubiquitin is conjugated to histone 2A to form the nuclear protein A24 which may play a role in regulation of chromatin structure, and in the cytoplasm is part of an ATP-dependent non-lysosomal proteolytic pathway. However, its physiological significance has not yet been fully resolved. Ubiquitin purified from bovine thymus did not show cell proliferating activity for any cells tested. The results suggest that LGF-I is a new autocrine growth factor with a molecular weight of 20,000 daltons, containing ubiquitin at the NH2-terminal end.
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PMID:N-terminal amino acid sequence of leukemia derived growth factor (LGF) from human erythroleukemia cell culture. 303 91

Ubiquitin is a protein of 76 amino acids found in every eukaryotic cell. Although ubiquitin is implicated in ATP-dependent nonlysosomal protein degradation and is also conjugated to specific cellular proteins, the role played by ubiquitin in cellular events has not been defined. We report that purified ubiquitin has intrinsic proteolytic activity and demonstrate that this activity is comparable to that of other well-characterized proteases. Monoclonal antibodies specific to ubiquitin inhibit proteolysis. Ubiquitin has protease activity over a broad pH range with an optimum at pH 8.0. It is stimulated by Ca2+ and is inhibited by high concentrations of phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate. Ubiquitin will cleave proteins at a limited number of sites. We propose that the ubiquitination of a protein can convert that protein into an ad hoc specific protease and models are presented as to how this can play a role in regulating a variety of cellular events.
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PMID:Ubiquitin has intrinsic proteolytic activity: implications for cellular regulation. 303 47

Degradation of intracellular proteins via the ubiquitin pathway involves several steps. In the initial event, ubiquitin becomes covalently linked to the protein substrate in an ATP-requiring reaction. Following ubiquitin conjugation, the protein moiety of the adduct is selectively degraded with the release of free and reusable ubiquitin. Ubiquitin modification of a variety of protein targets in the cell plays a role in basic cellular functions. Modification of core nucleosomal histones is probably involved in regulation of gene expression at the level of chromatin structure. Ubiquitin attachment to cell surface proteins may play roles in processes of cell-cell interaction and adhesion, and conjugation of ubiquitin to other yet to be identified protein(s) could be involved in the progression of cells through the cell cycle. Despite the considerable progress that has been made in the elucidation of the mode of action and cellular roles of the ubiquitin pathway, many major problems remain unsolved. A problem of central importance is the specificity in the ubiquitin ligation system. Why are certain proteins conjugated and committed for degradation, whereas other proteins are not? A free alpha-NH2 group is an important feature of the protein structure recognized by the ubiquitin conjugation system, and tRNA is required for the conjugation of ubiquitin to selective proteolytic substrates and for their subsequent degradation. These findings can shed light on some of the features of a substrate that render it susceptible to ubiquitin-mediated degradation.
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PMID:Regulation of the ubiquitin-mediated proteolytic pathway: role of the substrate alpha-NH2 group and of transfer RNA. 303 96


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