Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P62988 (Ubiquitin)
 4,326 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ubiquitin-protein conjugates in the hippocampus were analyzed by immunoblotting with a monoclonal anti-ubiquitin antibody. In the CA1 region, Triton X-100 insoluble ubiquitin-protein conjugates increased after 24 hr following 20 min of ischemia. When the total hippocampi were fractionated subcellularly, ubiquitin-protein conjugates increased in the particulate, especially in the mitochondrial fraction. The ubiquitin-protein conjugates were solubilized by SDS, or were partially solubilized by urea. The results indicate that insoluble ubiquitin-protein conjugates increase after ischemia.
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PMID:Subcellular distribution of ubiquitin-protein conjugates in the hippocampus following transient ischemia. 132 64

Ubiquitin has been purified to homogeneity, through a dialysis membrane having a NMW cutoff of 12 kDa, by taking advantage of its non-dialysable nature under these conditions. The dialysate was continuously recycled through a CM-52 cation exchange column at pH 4.5. The adsorbed fraction was eluted selectively at pH 7.2. Ubiquitin (25 mg) was obtained from 500 ml of packed RBCs. On SDS PAGE, ubiquitin showed varying mobility depending on the time of boiling in SDS. With 2 min of boiling, the molecular weight seemed to be 10.5 kDa, whereas 10 min of boiling resulted in a molecular weight of 8.5 kDa. Ubiquitin showed a slow intrinsic proteolytic activity against SDS-denatured beta-galactosidase in the absence of ATP. For the first 4 hr, there was no detectable degradation, but degradation was nearly complete after 8 hr. These data are not in agreement with those of Freid et al. [Proc. Natl Acad. Sci, USA, 84 (1987), 3685] who have reported a proteolytic activity comparable to that of other proteolytic enzymes.
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PMID:Ubiquitin with a non-ATP-dependent slow intrinsic proteolytic activity: a mild and rapid purification procedure. 132 85

Western blot analysis, using a polyclonal antibody to the 240-kDa endogenous inhibitor of the 20 S proteasome, revealed that the inhibitor is a component of the 26 S complex. Although isolated inhibitor displayed a single 40-kDa band on SDS-PAGE, the antibody detected a 55-kDa component in the 26 S proteasome complex. Ubiquitin polyclonal antibody recognized the same 55-kDa component but did not react with free 40-kDa inhibitor subunit. Addition of purified 40-kDa inhibitor to a ubiquitin ligating system also generated the 55-kDa species. In crude erythrocyte extracts, most of the inhibitor migrated at 55 kDa in the presence of ATP but shifted to 40 kDa in the absence of ATP, consistent with removal of ubiquitin. It is suggested that ubiquitination of the inhibitor may be involved in regulating assembly and/or activity of the 26 S proteasome complex.
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PMID:Ubiquitinated proteasome inhibitor is a component of the 26 S proteasome complex. 133 90

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

Ubiquitination of red blood cell (RBC) proteins was investigated by encapsulation of 125I-ubiquitin into human erythrocytes using a procedure of hypotonic dialysis, isotonic resealing, and reannealing. Incubation (37 degrees C, up to 2 h) of 125I-ubiquitin-loaded cells resulted in the recovery of 125I-ubiquitin with the cytosolic proteins (9.22 +/- 0.4 micrograms/ml RBC) and conjugated to membrane proteins (2.18 +/- 0.05 micrograms/ml RBC). This conjugation was time-dependent, and the predominant membrane protein band that became labeled showed an apparent molecular mass of 240 kDa on SDS-polyacrylamide gel electrophoresis (PAGE). Western blotting experiments with three different anti-ubiquitin antibodies revealed that this protein is also ubiquitinated in vivo. Cell-free experiments have shown that fraction II (a DEAE-bound protein fraction eluted by 0.5 M KCl) prepared from both mature erythrocytes and reticulocytes is able to conjugate ubiquitin to this protein. Ubiquitin conjugation was ATP-dependent (Km 0.09 mM), time-dependent, and fraction II-dependent (8 +/- 0.5 pmol of 125I-ubiquitin/h/mg of fraction II). Isolation of the major RBC membrane protein that is ubiquitinated was obtained by using biotinylated ubiquitin. Membrane proteins, once ubiquitinated with this derivative, were extracted and purified by affinity chromatography on immobilized avidin. The major components retained by the column were two peptides of molecular masses 220 and 240 kDa. Both proteins are recognized by a monoclonal anti-spectrin antibody, but only the 240-kDa component is detected by streptavidin peroxidase conjugate. That indeed the ubiquitinated membrane protein of 240-kDa is alpha-spectrin was confirmed by immunoaffinity chromatography using 125I-ubiquitin and a monoclonal anti-spectrin antibody. Antigen-antibody complexes were purified by protein A chromatography and analyzed by SDS-PAGE and autoradiography. Again two bands of 240 and 220 kDa were eluted (alpha- and beta-spectrin), but only one band corresponding to the electrophoretic mobility of alpha-spectrin was detected by autoradiography. Thus, alpha-spectrin is a substrate for the ATP-dependent ubiquitination system, suggesting that the cytoskeleton is covalently modified by ubiquitination both in reticulocytes and mature RBC.
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PMID:Ubiquitin is conjugated to the cytoskeletal protein alpha-spectrin in mature erythrocytes. 772 1

The ATP-ubiquitin-dependent proteolytic pathway (ubiquitin pathway) is believed to be involved in the formation of various neuronal inclusion bodies including Lewy bodies (LBs), a pathological hallmark of Parkinson disease and diffuse Lewy body disease (DLBD). Since multicatalytic proteinase (MCP) is involved in the ubiquitin pathway, an investigation of whether MCP is involved in neuronal inclusion bodies would provide a clue to the mechanism underlying the formation of neuronal inclusion bodies as well as to the pathogenesis of degenerative neurological disorders. In this study, we investigated detailed immunolocalization of MCP in LBs in DLBD brains using light and electron microscopy. We raised three different monoclonal antibodies against purified human MCP. Each of them recognized different sets of MCP subunits on Western blotting. Immunohistochemically, anti-MCP antibodies recognized all ubiquitin-positive cortical LBs in situ as well as those isolated from frozen DLBD cortices, suggesting that MCP is present in LBs as a whole molecule exhibiting protease activity. In electron microscopy, MCP immunoreactivity (MCP-IR) was exclusively localized on a characteristic oval structure with an approximate diameter of 100 nm. This structure was distributed throughout the LBs and was devoid of ubiquitin immunoreactivity. Treatment of isolated LBs with 2% SDS, but not with 0.5% Triton X-100, removed this structure from LBs in which fibrous materials predominated. Ubiquitin immunoreactivity was also decreased in isolated LBs treated with 2% SDS, suggesting that the fibrous structures in LBs were not ubiquitinated in situ. Thus, it is suggested that LBs are subjected to a proteolytic process in which MCP plays a role via processing of specific components of LBs.
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PMID:Multicatalytic proteinase is associated with characteristic oval structures in cortical Lewy bodies: an immunocytochemical study with light and electron microscopy. 802 94

Monoclonal nonspecific suppressor factor (MNSF), a lymphokine produced by a murine hybridoma, was originally found to inhibit the generation of LPS-induced immunoglobulin secreting cells. MNSF comprises of MNSF beta, an isoform of MNSF, and the other isoform, MNSF alpha. Ubiquitin-like segment (Ubi-L) of MNSF beta shows MNSF-like activity. Ubi-L (7.8 kDa) has 36% homology with 8.5 kDa ubiquitin. GST-Ubi-L was labeled with 125I by the chloramine T method and tested for its conjugation to acceptor in splenocyte lysates. 125I-GST-Ubi-L conjugation on SDS-PAGE showed heterogeneous bands including 95 kDa GST-Ubi-L conjugation in the splenocyte, but not reticulocyte lysates. The Ubi-L adduct appeared to be MNSF-related molecule because anti-MNSF monoclonal antibody (mAb) recognized the 95 kDa band. The pattern of the conjugations was different from that seen in ubiquitination. Unlabeled GST-Ubi-L inhibited the conjugations, while ubiquitin did not. alpha-Lactalbumin, one of the target proteins for ubiquitination, failed to conjugate to GST-Ubi-L. In addition, covalent conjugation of ubiquitin to reticulocyte lysates was also interfered by GST-Ubi-L. These results suggest that Ubi-L may conjugate to acceptor proteins in a similar, but not in the same way as ubiquitination, and might play an important role in lymphoid cells.
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PMID:Conjugation of ubiquitin-like polypeptide to intracellular acceptor proteins. 954 Aug 22

It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) with guanidine compounds, or inhibition of the hsp90-based chaperone system with geldanamycin, leads to the enhanced proteolytic degradation of nNOS. This regulated proteolysis is mediated, in part, by the proteasome. We show here with the use of human embryonic kidney 293 cells transfected with nNOS that inhibition of the proteasome with lactacystin leads to the accumulation of immunodetectable higher molecular mass forms of nNOS. Some of these higher molecular mass forms were immunoprecipitated by an anti-ubiquitin antibody, indicating that they are nNOS-polyubiquitin conjugates. Moreover, the predominant nNOS-ubiquitin conjugate detected in human embryonic kidney 293 cells, as well as in rat brain cytosol, migrates on SDS-polyacrylamide gels with a mobility near that for the native monomer of nNOS and likely represents a conjugate containing a few or perhaps one ubiquitin. Studies in vitro with the use of (125)I-ubiquitin and reticulocyte extracts could mimic this ubiquitination reaction, which was dependent on ATP. The heme-deficient monomeric form of nNOS is preferentially ubiquitinated over that of the heme-sufficient functionally active homodimer. Thus, we have shown for the first time that ubiquitination of nNOS occurs and is likely involved in the regulated proteolytic removal of non-functional enzyme.
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PMID:Ubiquitination of neuronal nitric-oxide synthase in vitro and in vivo. 1075 85

Ubiquitin-conjugated proteins in human colorectal cancer tissues were analyzed by the immunoprecipitation with the antibody FK2 against conjugated ubiquitin followed with SDS-PAGE. In these immunoprecipitable proteins, a 38-kDa protein was abundant in the tumor regions but almost absent in the adjacent normal regions in 17/26 patients, thus we attempted to purify it. Using immunoaffinity chromatography with the antibody FK2 followed by gel filtration and SDS-PAGE, approximately 10 pmol of this protein was separated from 34 g of the pooled cancerous tissue and transferred onto a PVDF membrane. The 38-kDa protein was further digested with Achromobacter protease I, resulting in several peptide fragments. Amino acid sequences of these peptides showed complete sequence identity to those derived from either ubiquitin or phosphoglycerate mutase-B, suggesting that the 38-kDa protein is monoubiquitinated phosphoglycerate mutase-B, whose calculated mass is 37,369 Da. Western blot using an antibody against phosphoglycerate mutase-B revealed the presence of the 38-kDa protein in the anti-ubiquitin immunoprecipitates derived from the tumor regions, but not from normal counterparts. In addition, part of non-ubiquitinated phosphoglycerate mutase-B (29 kDa) was also found in the anti-ubiquitin immunoprecipitates, whose levels were higher in the tumor regions than in the adjacent normal regions. These results suggest that monoubiquitination of phosphoglycerate mutase-B as well as formation of a noncovalent complex containing ubiquitin and phosphoglycerate mutase-B increases in colorectal cancer and novel modification of phosphoglycerate mutase-B might have a pathophysiological role.
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PMID:Purification and identification of monoubiquitin-phosphoglycerate mutase B complex from human colorectal cancer tissues. 1174 60

Ubiquitin and ubiquitin-like modifiers (UBLs) form covalent complexes with other proteins by isopeptide formation between their carboxyl (C)-termini and epsilon-amino groups of lysine residues of acceptor proteins. A hallmark of UBLs is a protruding C-terminal tail with a terminal glycine residue, which is required for ATP-dependent conjugation. Recently, the highly conserved protein HUB1 (homologous to ubiquitin 1) has been reported to function as a UBL following C-terminal processing. HUB1 exhibits sequence similarity with ubiquitin but lacks a C-terminal tail bearing a glycine residue. Here we show that HUB1 can form SDS-resistant complexes with cellular proteins, but provide evidence that these adducts are not formed through covalent C-terminal conjugation of HUB1 to substrates. The adducts are still formed when the C-terminus of HUB1 was altered by epitope tagging, amino-acid exchange or deletion, or when cells were depleted of ATP. We propose that HUB1 may act as a novel protein modulator through the formation of tight, possibly noncovalent interactions with target proteins.
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PMID:The ubiquitin-like protein HUB1 forms SDS-resistant complexes with cellular proteins in the absence of ATP. 1460 71


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