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

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

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

Ubiquitin was extracted from testis of 4 mammals and purified to homogeneity by gel filtration chromatography. Amino acid compositions and NH2-terminal sequences were found to be identical in the 4 species and with calf thymus ubiquitin. Ubiquitin conformation was shown to be very sensitive to oxidation. Improved methods for radioimmunoassay of ubiquitin in tissue extracts are also discussed.
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PMID:Purification and characterization of ubiquitin from mammalian testis. 632 40

The ubiquitin-dependent pathway for protein degradation has been found to play a major role in controlling protein turnover in the cell. Ubiquitin is one of the most conserved proteins yet identified, and up until now it has been thought to be present only in eukaryotes and archaebacteria. This is the first report on the detection and purification of ubiquitin from a eubacterium, the cyanobacterium Anabaena variabilis. The purification procedure included a heat denaturing step, fractionated ammonium sulfate precipitation, two gel filtration runs (Sephadex G-50 and Superose 12), and a final hydroxylapatite chromatography. Comparisons with bovine ubiquitin showed a high similarity with respect to antigenicity to anti-ubiquitin (bovine), molecular mass (M(r) = 6,000), isoelectric point (pI 6.5), and NH2-terminal sequence. The existence of ubiquitin in A. variabilis was confirmed by Southern hybridization. In in vitro experiments both cyanobacterial and bovine ubiquitin were covalently attached to several target proteins from A. variabilis, respectively. Data are presented which suggest ubiquitination of dinitrogenase reductase, the Fe-protein subunit of nitrogenase. Our findings imply that ubiquitination equivalent to the eukaryotic system is instrumental in this organism.
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PMID:Ubiquitin in the prokaryote Anabaena variabilis. 787 11

We have successfully expressed the active tyrosinase of Streptomyces antibioticus in Escherichia coli under the control of the trp promoter by fusing the sequence to the ORF438 gene. Because our attempt to connect the polycistronic gene of ORF438 and tyrosinase directly to the trp promoter of E. coli resulted in the expression of functionally inactive tyrosinase, we decided to fuse the COOH-terminus of ubiquitin sequence to the NH2-terminus of ORF438. Ubiquitin fusion has been shown to augment the yield of cloned gene products in E. coli by increasing the stability or translational efficiency of the fusion proteins. As a result, E. coli transformants harboring a plasmid pTRUBF that contains the ubiquitin-fused ORF438 and the tyrosinase gene produced the strong black pigment of melanin. About 300 units of tyrosinase per liter of batch culture were detected when cultivated in M9 medium containing casamino acids, L-tyrosine, and copper supplements. The black pigment, however, was not seen when grown in LB medium, suggesting that the trp promoter is well regulated. When recombinant E. coli cells grown in LB medium were transferred to a tryptophan-deficient minimal medium with phenol, we observed that phenol was removed from the solution, and the color of the medium turned black. This is due to the fact that the tyrosinase has polyphenol oxidase properties. We expect to use this recombinant E. coli for the waste treatment of phenolic compounds.
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PMID:Tyrosinase production in recombinant E. coli containing trp promoter and ubiquitin sequence. 801 Jun 80

Ubiquitin-conjugating enzymes catalyze the covalent attachment of ubiquitin to cellular substrates. Here we describe the isolation of a novel ubiquitin-conjugating enzyme from human placenta and the cloning of the corresponding cDNA. DNA sequencing revealed that this gene, UbcH2, encodes a protein with significant sequence similarity to yeast UBC8. In contrast to a previous report (Qin, S., Nakajima, B., Nomura, M., and Arfin, S. M. (1991) J. Biol. Chem. 266, 15549-15554), we discovered that UBC8 is interrupted by a single intron bearing an unusual branch point sequence. The revised amino acid sequence of yeast UBC8 exhibits 54% amino acid sequence identity to human UbcH2. Moreover, full-length UbcH2 and UBC8 enzymes expressed from their cDNAs show similar enzymatic activities in vitro by catalyzing the ubiquitination of histones, suggesting that the two enzymes may fulfill similar functions in vivo. Interestingly, comparison of the enzymatic activities of a truncated UBC8 (Qin, S., Nakajima, B., Nomura, M. and Arfin, S. M. (1991) J. Biol. Chem. 266, 15549-15554) and of the full-length enzyme (this report) suggests, that the first 12 amino-terminal residues of UBC8 are required for ubiquitination of histones in vitro but not for thiolester formation with ubiquitin. This suggests that the NH2 terminus of UBC8 may be necessary either for substrate recognition or for the transfer of ubiquitin onto substrates. The UbcH2 gene is located on chromosome 7 and shows a complex expression pattern with at least five different mRNAs.
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PMID:A human ubiquitin-conjugating enzyme homologous to yeast UBC8. 813 13

Ubiquitin-carrier proteins (E2s, ubiquitin-conjugating enzymes, UBCs) participate in proteolysis by catalyzing transfer of activated ubiquitin to the protein substrates, which are bound to specific ubiquitin-protein ligases (E3s). Yeast UBC2 (RAD6) and the mammalian E2(14kDa) bind to the ligase that recognizes and is involved in the degradation of certain free amino-terminal substrates ("N-end rule" substrates). As such proteins are rather scarce, the role of these E2s in general proteolysis is probably limited. Here, we report the purification and characterization of a novel 18-kDa species of E2 from rabbit reticulocytes. Unlike most members of the E2 family, this enzyme does not adsorb to anion exchange resin in neutral pH, and it is purified from the unadsorbed material (Fraction 1). Thus, it is designated E2-F1. Like all members of the E2 family, it generates a thiol ester with ubiquitin that serves as an intermediate in the conjugation reaction. Sequence analysis revealed a significant homology to many known species of E2s. The enzyme generates multiply ubiquitinated proteins in the presence of an E3 that has not been characterized yet. Most importantly, the ubiquitination via this E2 leads to the degradation of certain non-"N-end rule" substrates such as glyceraldehyde-3-phosphate dehydrogenase (Val at the NH2 terminus) and to the ubiquitination and degradation of certain N-alpha-acetylated proteins such as histone H2A, actin, and alpha-crystallin. The enzyme is also involved in the conjugation and degradation of the tumor suppressor protein p53.
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PMID:Purification and characterization of a novel species of ubiquitin-carrier protein, E2, that is involved in degradation of non-"N-end rule" protein substrates. 814 44

We examined ubiquitination and ubiquitin-mediated degradation of glycated protein by rabbit reticulocyte lysate fraction II (ubiquitin-free preparation). Non-glycated lysozyme and three glycated lysozyme preparations with different glucose binding ratios were used as substrates. Glycation sites of the lysozyme were mostly the epsilon-NH2 group of lysine residues, since modification at the alpha-NH2 group of the amino terminal was not detectable. Ubiquitin was conjugated with three glycated lysozyme preparations, which contained 1.4, 2.8 and 4.5 mol glucose per mol, by fraction II supplemented with hemin. The extent of formed conjugates was reduced 81, 72 and 56% of those of ubiquitin-non-glycated lysozyme conjugates, respectively. Additionally, ubiquitin-mediated degradation of the resultant conjugates was reduced and their respective rates were 97, 56 and 19% of that of the non-glycated lysozyme. These results indicated that both ubiquitin conjugation and ubiquitin-mediated degradation of the lysozyme were inhibited by nonenzymatic glucose binding to the lysozyme.
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PMID:Inhibitory effect of nonenzymatic glycation on ubiquitination and ubiquitin-mediated degradation of lysozyme. 838 87

The cause of neurodegeneration in Huntington's disease (HD) is unknown. Patients with HD have an expanded NH2-terminal polyglutamine region in huntingtin. An NH2-terminal fragment of mutant huntingtin was localized to neuronal intranuclear inclusions (NIIs) and dystrophic neurites (DNs) in the HD cortex and striatum, which are affected in HD, and polyglutamine length influenced the extent of huntingtin accumulation in these structures. Ubiquitin was also found in NIIs and DNs, which suggests that abnormal huntingtin is targeted for proteolysis but is resistant to removal. The aggregation of mutant huntingtin may be part of the pathogenic mechanism in HD.
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PMID:Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. 930 93

The ubiquitin proteolytic pathway is a major system for selective protein degradation in eukaryotic cells. One of the first steps in the degradation of a protein via this pathway involves selective modification of epsilon-NH2 groups of internal lysine residues by ubiquitination. To date, this amino group has been the only known target for ubiquitination. Here we report that the N-terminal residue of MyoD is sufficient and necessary for promotion of conjugation and subsequent degradation of the protein. Substitution of all lysine residues in the protein did not affect significantly its conjugation and degradation either in vivo or in vitro. In cells, degradation of the lysine-less protein is inhibited by the proteasome inhibitors MG132 and lactacystin. Inhibition is accompanied by accumulation of high molecular mass ubiquitinated forms of the modified MyoD. In striking contrast, wild-type MyoD, in which all the internal Lys residues have been retained but the N-terminus has been extended by fusion of a short peptide, is stable both in vivo and in vitro. In a cell-free system, ATP and multiple ubiquitination are essential for degradation of the lysine-less protein. Specific chemical modifications have yielded similar results. Selective blocking of the alpha-NH2 group of wild-type protein renders it stable, while modification of the internal Lys residues with preservation of the free N-terminal group left the protein susceptible to degradation. Our data suggest that conjugation of MyoD occurs via a novel modification involving attachment of ubiquitin to the N-terminal residue. The polyubiquitin chain is then synthesized on an internal Lys residue of the linearly attached first ubiquitin moiety.
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PMID:A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein. 977 40


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