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)

A rubber particle protein with apparent molecular mass of 43 kD as determined by SDS-PAGE was purified. A degenerate oligonucleotide primer based on the N-terminal amino acid sequence of this purified protein was used to amplify a 1385 bp cDNA by 3' rapid amplification of cDNA ends (3'RACE). The cDNA contains five repeats in a head-to-tail arrangement without intervening sequences, each encoding a ubiquitin unit of 76 amino acids. The last ubiquitin unit is followed by an extraphenylanaline residue at the carboxyl-terminal end. The structure of the cDNA is consistent with the structure of other known polyubiquitin genes. Western blot demonstrated that 43 kD rubber particle protein might be a polyubiquitin. Southern blot analysis revealed that there were multiple copies of gene encoding 43 kD rubber particle protein in Hevea brasiliensis. The results of Northern blot analysis indicated that the gene was expressed in latex, young leaves and bark tissue.
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PMID:[Expression and cloning of cDNA encoding 43 kD rubber particle membrane protein of Hevea brasiliensis]. 1559 30

Concentrated conditioned medium (CM) fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni cells, eluting from a gel filtration column at around 10 kDa, were found to exhibit strong antibacterial activity against Bacillus megaterium and Escherichia coli. The B. megaterium cells incubated in the CM fraction from Sf9 cells rapidly lost viability: after 8 min the viability had decreased to 0.7%, as compared with the control. Addition of the CM fraction to E. coli cells resulted in a less drastic drop in viability: 65% viability was lost after 60 min of incubation. Further, exposure to the CM fraction caused a substantial leakage of intracellular proteins, as demonstrated by SDS-PAGE analysis. Cell lysis was confirmed by optical density measurements, microscopic investigations and flow cytometry. B. megaterium exposed to a CM fraction from T. ni cells lost 97% of their viability in about 40 min. Ubiquitin, thioredoxin and cyclophilin were identified in the antibacterial fraction from Sf9 cells by mass spectrometry and N-terminal amino acid sequencing. Other proteins in the fraction gave no matches in a database search. Since ubiquitin was shown not to cause the antimicrobial effect and thioredoxin and cyclophilin were likely not involved, the responsible agent may be an unknown protein, not yet registered in databases. The antimicrobial effect of the CM fraction from T. ni cells most probably comes from a lysozyme precursor protein.
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PMID:Antimicrobial activity of conditioned medium fractions from Spodoptera frugiperda Sf9 and Trichoplusia ni Hi5 insect cells. 1580 53

The organization of skeletal muscles in decapod crustaceans is significantly altered during molting and development. Prior to molting, the claw muscles atrophy dramatically, facilitating their removal from the base of the claw. During development, lobster claw muscles exhibit fiber switching over several molt cycles. Such processes may be influenced by the secretion of steroid molting hormones, known collectively as ecdysteroids. To assay the effects of these hormones, we used eyestalk ablation to trigger an elevation of circulating ecdysteroids and then quantified myofibrillar mRNA levels with real-time PCR and myofibrillar protein levels by SDS-PAGE. Levels of myosin heavy chain (MHC) and actin proteins and the mRNA encoding them were largely unaffected by eyestalk ablation, but in muscles from intact animals, myofibrillar gene expression was modestly elevated in premolt and postmolt animals. In contrast, polyubiquitin mRNA was significantly elevated (about 2-fold) in claw muscles from eyestalk-ablated animals with elevated circulating ecdysteroids. Moreover, patterns of MHC and actin gene expression are significantly different among slow and fast claw muscles. Consistent with these patterns, the three muscle types differed in the relative amounts of myosin heavy chain and actin proteins. All three muscles also co-expressed fast and slow myosin isoforms, even in fibers that are generally regarded as exclusively fast or slow. These results are consistent with other recent data demonstrating co-expression of myosin isoforms in lobster muscles.
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PMID:Eyestalk ablation has little effect on actin and myosin heavy chain gene expression in adult lobster skeletal muscles. 1583 62

A simple method is described to identify signature peptides derived from polyubiquitin (polyUb) chains. The method is based on MALDI-TOF MS/MS analysis after chemically assisted fragmentation, and works on peptides isolated from polyacrylamide gels. PolyUb chains branched at K48 and K63 were chosen as models for Ub-protein conjugates. They were resolved by SDS-PAGE, and their tryptic peptides (in-gel-trypsinolysis) derivatized with 3-sulfopropinic acid NHSester to obtain chemically assisted fragmentation during the MS/MS analysis. PolyUb-K63 produced a single peptide identified as (55)TLSDYNIQK(63) (GG)ESTLHLVLR(72). PolyUb-K48 produced two branched signature peptides identified as (43)LIFAGK(48)(GG)QLEDGR(54) and (43)LIFAGK(48)(LRGG)QLEDGR(54). The recovery of signature peptide with LRGG as branched chain underscores the need to take limited proteolysis into account in the search for detection of ubiquitinated peptides in proteomics studies. In conclusion, a simple method has been described allowing the identification of signature peptides, which are diagnostic markers of the majority of polyUb-conjugated proteins. In principle, the method should be applicable also for other more rare signature peptides.
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PMID:A method for simple identification of signature peptides derived from polyUb-K48 and K63 by MALDI-TOF MS and chemically assisted MS/MS fragmentation. 1604 92

The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) carries out N-terminal processing of the viral replicase polyprotein, and also exhibits Lys48-linked polyubiquitin chain debranching and ISG15 precursor processing activities in vitro. Here, we used SDS-PAGE and fluorescence-based assays to demonstrate that ISG15 derivatives are the preferred substrates for the deubiquitinating activity of the PLpro. With k(cat)/K(M) of 602,000 M(-1)s(-1), PLpro hydrolyzes ISG15-AMC 30- and 60-fold more efficiently than Ub-AMC and Nedd8-AMC, respectively. Data obtained with truncated ISG15 and hybrid Ub/ISG15 substrates indicate that both the N- and C-terminal Ub-like domains of ISG15 contribute to this preference. The enzyme also displays a preference for debranching Lys48- over Lys63-linked polyubiquitin chains. Our results demonstrate that SARS-CoV PLpro can differentiate between ubiquitin-like modifiers sharing a common C-terminal sequence, and that the debranching activity of the PLpro is linkage type selective. The potential structural basis for the demonstrated specificity of SARS-CoV PLpro is discussed.
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PMID:Selectivity in ISG15 and ubiquitin recognition by the SARS coronavirus papain-like protease. 1769 80

Post-translational modification of proteins via ubiquitination plays a crucial role in numerous vital functions of the cell. Polyubiquitination is one of the key regulatory processes involved in regulation of mitotic progression. Here we describe a differential proteomic screen dedicated to identification of novel proteins ubiquitinated upon mitotic exit in cell-free extract of Xenopus laevis embryo. Mutated recombinant His6-tagged ubiquitin (Ubi (K48R)) was added to mitotic extract from which we purified conjugated proteins, as well as associated proteins in nondenaturing conditions by cobalt affinity chromatography. Proteins eluted from Ubi (K48R) supplemented and control extracts were compared by LC-MS/MS analysis after monodimensional SDS-PAGE. A total of 144 proteins potentially ubiquitinated or associated with them were identified. Forty-one percent of these proteins were shown to be involved in ubiquitination and/or proteasomal degradation pathway confirming the specificity of the screen. Twelve proteins, among them ubiquitin itself, were shown to carry a "GG" or "LRGG" remnant tag indicating their direct ubiquitination. Interestingly, sequence analysis of ubiquitinated substrates carrying these tags indicated that in Xenopus cell-free embryo extract supplemented with Ubi (K48R) the majority of polyubiquitination occurred through lysine-11 specific ubiquitin chain polymerization. The potential interest in this atypical form of ubiquitination as well as usefulness of our method in analyzing atypical polyubiquitin species is discussed.
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PMID:Differential proteomic screen to evidence proteins ubiquitinated upon mitotic exit in cell-free extract of Xenopus laevis embryos. 1882 42

B cell activating factor (BAFF) belonging to the tumor necrosis factor (TNF) family is critical to B cell survival, proliferation, maturation, and immunoglobulin secretion and to T cell activation. In the present study, the full-length cDNA of BAFF (designated bBAFF) from the bat (Vespertilio superans Thomas) was cloned using RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of bBAFF consists of 986 bases including an 873 bp open reading frame encoding 290 amino acids. Sequence comparison indicated that the amino acid of bBAFF possessed the TNF signature, a transmembrane domain, a putative furin protease cleavage site and three cysteine residues. The predicted three-dimensional (3D) structure of the bsBAFF monomer, analyzed by comparative protein modeling, revealed that it was very similar to its counterparts. Real-time quantitative PCR (qPCR) analysis indicated that bBAFF mRNA was predominantly expressed in bat lymphoid tissue spleen. The SUMO (Small Ubiquitin-like Modifier)-bsBAFF was efficiently expressed in Escherichia coliBL21 (DE3) and confirmed by SDS-PAGE and Western blotting analysis. Laser scanning confocal microscopy analysis showed that bsBAFF could bind to its receptors on B cells. In vitro, the MTT assays indicated that SUMO-bsBAFF was not only able to promote survival/proliferation of bat lymphocytes but also able to stimulate survival/proliferation of mouse B cells. These findings indicate that bsBAFF plays an important role in the survival/proliferation of B cells and has functional cross-reactivity among mammalians. The present findings may provide valuable information for research into the immune system of the bat.
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PMID:Molecular cloning, expression, bioinformatics analysis and bioactivity characterization of TNF13B (BAFF) gene in bat (Vespertilio superans Thomas). 2223 85

Toxoplasmosis can cause serious disease in immunocompromised patients and to congenitally infected foetuses. Appropriate laboratory investigations in potential cases of acute Toxoplasma infection are important. Excretory secretory antigen (ESA) is immunogenic during both human and experimental infections, therefore is considered as a good candidate for investigation into new infection markers. In this study, ESA was prepared from in vitro cultures of Toxoplasma gondii to identify T. gondii ESA antigenic component(s) that is/are most reactive with serum samples from probable acute cases of toxoplasmosis. Serum samples were obtained from several categories of individuals with the following Toxoplasma serology: Group I: IgM+ IgG+ (low IgG avidity) or IgM+ IgG- from sera of patients who had clinical query of toxoplasmosis (n=35). Group II: IgM- IgG+ (high IgG avidity) from chronically infected individuals (n=30). Group III: normal/healthy individuals with anti-Toxoplasma IgMIgG- (n=20). Group IV: individuals with other infections who had anti-Toxoplasma IgM- IgG- (n=10). The ESA was subjected to SDS-PAGE, followed by Western blot analysis using the above sera and probed with peroxidase conjugated anti-human IgM and IgA antibodies. The blots were then developed using chemiluminescence substrate. The selected antigenic band was excised from the gel after two dimensional electrophoresis and sent for mass spectrometry analysis using MALDI TOF-TOF. The most promising antigenic band was a 10 kDa protein which showed sensitivity of 80% in both IgM and IgA blots, and specificity of 96.7% with sera from other infections and healthy controls. The two best identifications for the 10 kDa band were ubiquitin (ribosomal protein CEP52 fusion protein) and polyubiquitin.
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PMID:A Toxoplasma gondii 10 kDa in vitro excretory secretory antigen reactive with human IgM and IgA antibodies. 2243 90

Posttranslational modification (PTM) by the covalent conjugation of small ubiquitin-like modifier (SUMO) plays an important role in many biological processes, such as cell cycle progression, transcriptional regulation, subcellular transport, and other processes. An in-depth understanding of the function of SUMOylation requires the discovery of SUMO accepter sites. However, identification of endogenous SUMO-conjugated sites in higher eukaryotes by MS-based proteomic strategies is hampered by the low abundance of SUMO conjugates, the large tryptic fragments of SUMO1 or SUMO2/3 and the inability to match MS/MS spectra by protein database search engine. In this chapter, we describe a powerful method to overcome at least some of these challenges. To identify SUMO acceptor sites in endogenous SUMO1 conjugated protein, the SUMO1 conjugates are purified by immunoprecipitation with anti-SUMO1 antibodies followed by SDS-PAGE separation and in-gel tryptic digestion. The resulting peptides are either performed using standard data dependent acquisition (DDA) for protein identification or high mass DDA to enhance the sensitivity of detection on the LTQ-Orbitrap mass spectrometer. Finally, a Web-based database tool, ChopNSpice, coupled with a protein database search engine is introduced to ease the identification of SUMO1 attachment sites. Although this method was initially used to identify SUMO1 accepter sites, it can be readily adapted to study SUMO2/3 conjugates or even other Ubiquitin-like modifiers.
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PMID:Identification of endogenous SUMO1 accepter sites by mass spectrometry. 2266 16

Aspergillus fumigatus (Afu) causes allergic and invasive forms of diseases in humans. In order to identify genes relevant for pathogenesis, a total of 235 cDNA clones were randomly selected and sequenced from cDNA library of Afu. One of the partially sequenced cDNA clones was homologous to polyubiquitin. Sequencing of the complete cDNA clone showed an open reading frame of 912 bases. Comparison with genomic sequence of Afu using BlastN program, revealed that polyubiquitin gene comprises of 992 bases and contains one intron of 80 bases. The recombinant expression of fusion protein showed an approximately molecular weight of 43-kDa on SDS-PAGE. The translation product of the cDNA sequence showed four tandem repeats of 76 amino acid residues in a single polyubiquitin protein and showed 100% identity with polyubiquitin protein sequences of S. cerevisiae, N. crassa, C. albicans, S. pombe, and M. grisae. Polyubiquitin gene is known to play important role in a variety of cellular processes and recently have been implicated in fungal pathogenesis. Identification of polyubiquitin gene of Afu has opened up scope to study its role in understanding Aspergillus biology and pathogenesis.
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PMID:Identification and characterization of polyubiquitin gene from cDNA library of aspergillus fumigatus. 2310 24


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