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Results for “posttranslational modification”

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PubMed2002

The cyclin-dependent kinase inhibitor roscovitine inhibits the transactivating activity and alters the posttranslational modification of herpes simplex virus type 1 ICP0.

The cyclin-dependent kinase (cdk) inhibitor Roscovitine (Rosco) reduces transcription of herpes simplex virus early genes significantly, even in the presence of wild-type levels of immediate-early (IE) viral proteins, suggesting that the transactivating functions of IE proteins may require the activities of one or more Rosco-sensitive cdk (L. M. Schang, A. Rosenberg, and P. A. Schaffer, J. Virol. 73:2161-2172, 1999). Based on this observation, we sought to determine whether Rosco alters the transactivating activity and posttranslational modification state of the IE protein, infected cell protein 0 (ICP0), in KOS6beta-infected Vero cells. KOS6beta is a KOS-derived recombinant virus containing an ICP0-inducible ICP6 promoter::lacZ cassette. To monitor ICP0's transactivating activity, KOS6beta-infected cells were released from a cycloheximide (CHX)-mediated protein synthesis block into medium with or without Rosco, and beta-galactosidase activity was measured. Rosco inhibited the ability of ICP0 to transactivate the ICP6 promoter by 50-fold. This inhibition was shown not to be a consequence of inhibition of ICP6 basal promoter activity or aberrant nuclear localization of ICP0. Rosco also altered the electrophoretic mobility of a portion of ICP0 molecules derived from KOS-infected cells following reversal of a CHX block. Notably, however, Rosco had only a minimal effect on the phosphorylation state of ICP0. We conclude that ICP0's transactivating activity requires Rosco-sensitive cdks and hypothesize that these cdks regulate the functions of cellular enzymes which modify ICP0, and are, consequently, required for its transactivating activity. Thus, we propose that Rosco regulates ICP0's posttranslational state by mechanisms other than, or in addition to, phosphorylation.

Animals↗

PubMed1995

Biochemical characterization and posttranslational modification of AlgU, a regulator of stress response in Pseudomonas aeruginosa.

AlgU is homologous to the extreme heat shock sigma factor sigma E from enteric bacteria. In this work, AlgU was overproduced and purified and its function investigated at the biochemical level. AlgU was shown to associate with RNA polymerase and direct transcription of a target promoter. AlgU also exhibited multiple isoforms detected by 2D gel analysis. Treatment with a Ser/Thr phosphatase shifted the distribution of isoforms towards the basic side on 2D gels, suggesting that posttranslational modifications of AlgU may involve phosphorylation. The underphosphorylated forms of AlgU copurified with RNA polymerase. It is possible that phosphorylation affects AlgU activity or its stability.

Amino Acid Sequence↗

PubMed2001

Posttranslational modifications to human bone sialoprotein determined by mass spectrometry.

Bone sialoprotein (BSP) is an acidic 301 amino acid protein expressed by osteoblasts and at a low level by hypertrophic chondrocytes. Its expression is highest during early stages of bone formation, and it is particularly abundant in the cells lining the surface of newly formed trabeculae. BSP contains numerous substituents which are anionic in nature and apparently essential for the function of the protein. Thus, the proposed role of BSP in hydroxyapatite nucleation and growth may depend on such modifying groups. The posttranslational modifications include several acidic oligosaccharides as well as phosphate and sulfate groups. This work combines matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry with selective enzyme treatment of BSP to provide new information on the precise distribution and structure of oligosaccharides, sulfate, and phosphate groups in BSP isolated from human bone. The results provide a high level of detail in the location of these modifying groups toward the end of providing a basis for further understanding the function of BSP in bone nucleation.

Adult↗

PubMed1984

Posttranslational modification of the carboxy-terminal region of the beta subunit of human chorionic gonadotropin.

The beta subunit of human chorionic gonadotropin (hCG) contains at its carboxy terminus an extension of 29 amino acids not found in the beta subunits of the other glycoprotein hormones. This region provides the sites of attachment of four serine-linked oligosaccharide chains. We have examined the synthesis of this subunit in a cell-free translation system derived from Krebs II ascites tumor cells. The primary translation product was found to undergo a temperature-dependent posttranslational modification which resulted in an increase in apparent molecular weight of 2000 on sodium dodecyl sulfate gel electrophoresis. This modification was specific for the beta subunit of hCG, since no changes were observed for the beta subunit of bovine luteinizing hormone or for the alpha subunits of either hormone. The increase in molecular weight occurred in the absence of microsomal membranes and was not due to the addition of N-linked carbohydrate. An identical shift was observed when pre-hCG beta was incubated with extracts of human placenta. The site of modification was localized by fingerprint analysis to a carboxy-terminal tryptic peptide which contains two of the four O-glycosylated serine residues in the mature form of the subunit. The modified protein was resistant to oligosaccharidase digestion and beta-elimination, indicating that it does not contain O-linked oligosaccharides of the type found on mature hCG beta. These results demonstrate that a specific modification of the carboxy-terminal segment of hCG beta synthesized in vitro occurs in the absence of O-linked glycosylation.

Amino Acid Sequence↗

PubMed1998

Posttranslational modifications of human inter-alpha-inhibitor: identification of glycans and disulfide bridges in heavy chains 1 and 2.

Inter-alpha-inhibitor (IalphaI) is a serine proteinase inhibitor found in high concentrations in human plasma. The protein is composed of a light inhibitory chain called bikunin and two heavy chains of unknown function. The three polypeptide chains are covalently assembled via a carbohydrate cross-link [Enghild, J. J., Salvesen, G., Hefta, S. A., Thogersen, I. B., Rutherfurd, S., & Pizzo, S. V. (1991) J. Biol. Chem. 266, 747-751]. The aim of this study was to complete the primary structure by characterizing additional covalent posttranslational modifications of the heavy chains. Analysis revealed three N-linked oligosaccharides located on Asn251 and Asn554 of heavy chain 1 and on Asn64 of heavy chain 2: all these were complex biantennary structures composed of (Asn)-GlcNAc2-Man-(Man-GlcNAc-Gal-SA)2. In addition, the IalphaI heavy chains carried several O-linked glycans located on Thr619 of heavy chain 1 and a cluster of four O-linked oligosaccharides on Thr612, Ser619, Thr621, and Thr637 of heavy chain 2. The oligosaccharides were short (Ser/Thr)-GalNAc-Gal-SA trisaccharides. The IalphaI heavy chains contain nine Cys residues, of which eight are involved in disulfide bridges. The unpaired Cys residue residing on heavy chain 1, Cys26, appears to be modified by dihexosylation. The other Cys residues exclusively form intrachain disulfide bridges. In heavy chain 1 the two disulfide bonds are formed between Cys210 and Cys213 and between Cys234 and Cys506, and in heavy chain 2, between Cys207 and Cys210 and between Cys596 and Cys597. Interestingly, three of these four disulfides are formed between Cys residues that are either adjacent or only two amino acid residues apart.

Alpha-Globulins↗

PubMed2006

Insulin-like peptide 6: characterization of secretory status and posttranslational modifications.

Insulin-like peptide 6 (Insl6) is a member of the insulin/relaxin superfamily with unknown biological function(s). In the current report, we establish that meiotic and postmeiotic germ cells of the testis are the principal sites of expression of Insl6. Analysis of stably or transiently transfected cells revealed that Insl6 is a secreted protein localized to the endoplasmic reticulum and Golgi. Secretion could be detected in both CHO and GC2 germ cells and was sensitive to brefeldin A treatment. In cell lysates, the predominant Insl6 band was approximately 28 kDa in size. In contrast, the predominant Insl6 species in the supernatant was 8 kDa in size, suggesting posttranslational processing of the precursor protein. Ectopically expressed Insl6 is processed and secreted in furin-deficient LoVo cells and in CHO cells treated with a furin inhibitor, although the size profile of the secreted protein is altered suggesting that Insl6 is a substrate for furin action. Furthermore, mutation of a putative furin cleavage site in the Insl6 peptide resulted in aberrant processing of the Insl6 peptide. Additional investigations of the structure of Insl6 protein provided evidence for posttranslational modifications of Insl6, including the presence of disulfide bonds, glycosylation, and ubiquitination. On the basis of the demonstrated secretory status of Insl6, we speculate that the physical proximity of the germ cell to the Sertoli cell renders the Sertoli cell a likely candidate for Insl6 action.

Amino Acid Sequence↗

PubMed1985

Small nuclear ribonucleoprotein particle assembly in vivo: demonstration of a 6S RNA-free core precursor and posttranslational modification.

The in vivo synthesis and assembly of human small nuclear ribonucleoproteins (snRNPs) have been studied using pulse/chase analysis. Antibodies derived from patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) recognize distinguishable subsets of pulse-labeled snRNP peptides. These antibodies were used to immunoprecipitate sucrose gradient fractionated pulse-labeled and pulse/chased snRNP proteins. The results indicate that assembly of the U RNA-containing snRNPs is a multistep process involving prior assembly of an RNA-free 6S core particle. This precursor contains snRNP peptides D, E, F, and G, which are common to all the different U RNA-containing particles. Furthermore, a posttranslational modification of one of the U1 snRNP-specific peptides has been observed, and the kinetics of this process indicates that the modification occurs after particle assembly. Functional and structural implications of a protein core for snRNP particles are discussed.

Antibodies↗

PubMed1982

Different forms of adrenal phenylethanolamine N-methyltransferase: species-specific posttranslational modification.

Phenylethanolamine N-methyltransferase was purified from rat and cow adrenal glands. The enzymes from the two species have the same molecular weight of 31,000, but differ in electrophoretic mobility. During polyacrylamide gel electrophoresis, the rat form migrates faster than the bovine form. Antibodies to bovine enzyme precipitated equally well the rat and cow form of the enzyme, but antibodies against rat enzyme precipitated poorly the bovine form. In contrast, both antibodies recognized a similar protein in the in vitro translation products of poly(A+)mRNA isolated from cow adrenal glands. The results suggest that the primary protein structure of rat and bovine enzyme is similar and that differences in electrophoretic mobility are due to posttranslational modification of the enzyme molecule.

Adrenal Glands↗

PubMed1994

Heparin decreases activator protein-1 binding to DNA in part by posttranslational modification of Jun B.

Heparin is a potent inhibitor of the proliferation and migration of vascular smooth muscle cells. This agent selectively inhibits the transcription of tissue-type plasminogen activator and interstitial collagenase, probably by decreasing the binding of activator protein-1 (AP-1) to phorbol ester-responsive elements in the promoters of these genes. Decreased AP-1 binding is not due to a direct inhibition by heparin, since heparinase digestion of nuclear extracts prepared from heparin-treated smooth muscle cells does not restore AP-1 binding activity. Treatment of cells with heparin suppresses the expression of Jun B, one of the components of AP-1. The major effect of heparin is at the level of posttranslational modification of Jun B. Results from pulse-chase labeling experiments show that the newly synthesized Jun B is rapidly converted to a higher-molecular-weight form and that conversion is suppressed by heparin. Evidence is presented suggesting that the heparin-inhibited event is phosphorylation of Jun B.

Animals↗

PubMed1986

Further characterization of the posttranslational modifications of core histones in response to heat and arsenite stress in Drosophila.

The effects of a heat shock or arsenite treatment on the methylation and acetylation of core histones have been investigated in Drosophila cultured cells. The decrease in H3 methylation, which is observed during a heat shock, is not a demethylation process, but results from methylation arrest. Two-dimensional gel electrophoresis leaves no ambiguity concerning the identity of H2B as a methylated protein, since H2B and D2, a nuclear nonhistone protein, which comigrate on one-dimensional gels, are well separated on these gels. Two-dimensional gel electrophoresis in the presence of Triton X-100 resolves each of the core histones into multiple forms resulting from posttranslational modifications. There are apparently, however, no histone variants in cultured Drosophila cells. At 23 degrees C, the various forms of the core histones resolved on two-dimensional gels are methylated. Under heat-shock or arsenite treatment, the methylation of all forms of H3 is decreased, while that of the various forms of H2B increase. These stress conditions also induce a generalized diminution in the acetylation of all forms of core histones. In the course of a heat shock, the synthesis of H2B is increased and this newly synthesized histone remains unacetylated during the shock. These changes in the patterns of core histone methylation and acetylation may be correlated with the reorganization of gene activity brought about by the heat shock.

Acetylation↗

PubMed2006

Altered mouse leukemia L1210 thymidylate synthase, associated with cell resistance to 5-fluoro-dUrd, is not mutated but rather reflects posttranslational modification.

Thymidylate synthase purified from 5-fluoro-dUrd-resistant mouse leukemia L1210 cells (TSr) was less sensitive to slow-binding inhibition by 5-fluoro-dUMP than the enzyme from the parental cells (TSp), both enzyme forms differing also in sensitivity to several other dump analogues, apparent molecular weights of monomer and dimer, and temperature dependence of the catalyzed reaction. Direct sequencing of products obtained from RT-PCR, performed on total RNA isolated from the parental and 5-fluoro-dUrd-resistant cells, proved both nucleotide sequences to be identical to the mouse thymidylate synthase coding sequence published earlier (NCBI protein database access no. NP_067263). This suggests that the altered properties of TSr are caused by a factor different than protein mutation, presumably posttranslational modification. As a possibility of rat thymidylate synthase phosphorylation has been recently demonstrated (Samsonoff et al. (1997) J Biol Chem 272: 13281), the mouse enzyme amino-acid sequence was analysed, revealing several potential phosphorylation sites. In order to test possible influence of the protein phosphorylation state on enzymatic properties, endogenous TSp and TSr were purified in the presence of inhibitors of phosphatases. Although both enzyme forms were phosphorylated, as shown by electrophoretical separation followed by phosphoprotein detection, the extent of phosphorylation was apparently similar. However, the same two purified enzyme preparations, compared to the corresponding preparations purified in the absence of phosphatase inhibitors, showed certain properties, including sensitivity to the slow-binding inhibition by FdUMP, altered. Thus properties dependence on phosphorylation was indicated.

Animals↗

PubMed1992

Posttranslational modification of Klebsiella pneumoniae flavodoxin by covalent attachment of coenzyme A, shown by 31P NMR and electrospray mass spectrometry, prevents electron transfer from the nifJ protein to nitrogenase. A possible new regulatory mechanism for biological nitrogen fixation.

A strain of Escherichia coli (71-18) that produces ca. 15% of its soluble cytoplasmic protein as a flavodoxin, the Klebsiella pneumoniae nifF gene product, has been constructed. The flavodoxin was purified using FPLC and resolved into two forms, designated KpFldI and KpFldII, which were shown to have identical N-terminal amino acid sequences (30 residues) in agreement with that predicted by the K. pneumoniae nifF DNA sequence. 31P NMR, electrospray mass spectrometry, UV-visible spectra, and thiol group estimations showed that the single cysteine residue (position 68) of KpFldI is posttranslationally modified in KpFldII by the covalent, mixed disulfide, attachment of coenzyme A. KpFldII was inactive as an electron carrier between the K. pneumoniae nifJ product (a pyruvate-flavodoxin oxidoreductase) and K. pneumoniae nifH product (the Fe-protein of nitrogenase). This novel posttranslational modification of a flavodoxin is discussed in terms of the regulation of nitrogenase activity in vivo in response to the level of dissolved O2 and the carbon status of diazotrophic cultures.

Amino Acid Sequence↗

PubMed2001

The US3 protein kinase of herpes simplex virus 1 mediates the posttranslational modification of BAD and prevents BAD-induced programmed cell death in the absence of other viral proteins.

Earlier studies have shown that the d120 mutant of herpes simplex virus 1, which lacks both copies of the alpha4 gene, induces apoptosis in all cell lines tested. In some cell lines d120-induced apoptosis, manifested by the release of cytochrome c, activation of caspase 3, and fragmentation of cellular DNA, is blocked by the overexpression of Bcl-2. In these cells viral protein kinase U(S)3 delivered in trans blocks apoptosis induced by the mutant virus at a premitochondrial stage. We report that the U(S)3 protein kinase targets the pro-apoptotic BAD member of the Bcl-2 family. Specifically, the U(S)3 protein kinase mediates a posttranslational modification of BAD and blocks its cleavage, which is reported to activate apoptosis. Thus, U(S)3 protein kinase is the sole viral protein required to block activation of caspase 3, prevent cleavage of poly(ADP-ribose) polymerase, and block fragmentation of cellular DNA induced by BAD.

Animals↗

PubMed1995

The posttranslational modification of phosphoglucomutase is regulated by galactose induction and glucose repression in Saccharomyces cerevisiae.

The enzyme phosphoglucomutase functions at a key point in carbohydrate metabolism. In this paper, we show that the synthesis of the major isoform of yeast phosphoglucomutase, encoded by the GAL5 (PGM2) gene, is regulated in a manner that is distinct from that previously described for other enzymes involved in galactose metabolism in the yeast Saccharomyces cerevisiae. Accumulation of this isoform increased four- to sixfold when the culture experienced either glucose depletion or heat shock. However, heat shock induction did not occur unless the cells were under glucose repression. This nonadditive increase in expression suggests that the regulatory mechanisms controlling the heat shock induction and glucose repression of the GAL5 gene are functionally related. We previously demonstrated that phosphoglucomutase is modified by a posttranslational Glc-phosphorylation reaction. We now show that this posttranslational modification, like phosphoglucomutase expression itself, is also regulated by galactose induction and glucose repression. Finally, no evidence was found to indicate that the Glc-phosphorylation of phosphoglucomutase alters its enzymatic activity under the conditions examined.

Base Sequence↗

PubMed1990

Probing the transglutaminase-mediated, posttranslational modification of proteins during development.

Sphaerechinus granularis eggs were fertilized in seawater in the presence of 0.2 mM dansylcadaverine, and development was allowed to take place with this compound in the medium. gamma-Glutamyldansylcadaverine, indicative of the utilization of the amine tracer by intrinsic transglutaminase, was isolated from the embryonic proteins, and identity of the product with the chemically synthesized gamma-glutamyl derivative of dansylcadaverine was confirmed. Covalent labeling of proteins occurring during development was examined by means of electrophoresis in NaDodSO4, followed by immunoblotting with an antibody that specifically recognized the dansyl hapten. There was an increase in the total uptake of the tracer at an essentially constant rate with each cell division, from 2- to 8- and 64-cell stages. Moreover, multiple protein labeling was evident in all specimens. The described concept of studying posttranslational modifications in vivo by transglutaminase through detection of the haptenic or specific ligand recognizable group of an incorporated small amine substrate will undoubtedly be of general utility for probing the functions of this family of enzymes in other cell types as well.

Animals↗

PubMedDate not supplied

Tubulins in Trichomonas vaginalis: molecular characterization of alpha-tubulin genes, posttranslational modifications, and homology modeling of the tubulin dimer.

We have isolated and analysed an alpha-tubulin-encoding gene (atub1) in an early-diverging eukaryote, Trichomonas vaginalis. The complete atub1 open reading frame included 1.356 bp encoding a polypeptide of 452 amino-acyl residues. A second alpha-tubulin gene (atub2) was amplified by PCR using primers derived from consensus alpha-tubulin amino acid sequences. Both T. vaginalis alpha-tubulin sequences showed high identity to those described in other parabasalids (94.4%-97.3%), and exhibited a high degree of similarity to sequences from Metazoa (such as pig brain) and diplomonads (such as Giardia). Despite large evolutionary distances previously observed between trichomonads and mammals, the three-dimensional model of the T. vaginalis tubulin dimer was very similar to that of pig brain. Possible correlations between alpha-tubulin sequences and posttranslational modifications (PTMs) were examined. Our observations corroborated previous data obtained in T. vaginalis using specific anti-PTMs antibodies. As described in the related species Tritrichomonas mobilensis, microtubules are likely acetylated, non-tyrosinated, glutamylated, and non-glycylated in T. vaginalis. Evolutionary considerations concerning the time of appearance of these tubulin PTMs are also discussed since trichomonads are potentially one of the earliest diverging eukaryotic lineages.

Amino Acid Sequence↗

PubMed2003

The H4b minor histocompatibility antigen is caused by a combination of genetically determined and posttranslational modifications.

Minor histocompatibility (H) Ag disparities result in graft-vs-host disease and chronic solid allograft rejection in MHC-identical donor-recipient combinations. Minor H Ags are self protein-derived peptides presented by MHC class I molecules. Most arise as a consequence of allelic variation in the bound peptide (p) that results in TCR recognizing the p/MHC as foreign. We used a combinational peptide screening approach to identify the immune dominant H2K(b)-restricted epitope defining the mouse H4(b) minor H Ag. H4(b) is a consequence of a P3 threonine to isoleucine change in the MHC-bound peptide derived from epithelial membrane protein-3. This allelic variation also leads to phosphorylation of the H4(b) but not the H4(a) epitope. Further, ex vivo CD8(+) T lymphocytes bind phosphorylated Ag tetramers with high efficiency. Although we document the above process in the minor H Ag system, posttranslational modifications made possible by subtle amino acid changes could also contribute to immunogenicity and immune dominance in tumor immunotherapeutic settings.

Amino Acid Substitution↗

PubMed1996

Expression and posttranslational modification of class III beta-tubulin during neuronal differentiation of P19 embryonal carcinoma cells.

We have used a combination of immunofluorescence microscopy, northern blotting, ELISA, and isoelectric focusing to characterize the expression of neuronal Class III beta-tubulin in P19 embryonal carcinoma cells induced to differentiate along a neuronal pathway by retinoic acid. Following 48 h differentiation, beta-III tubulin mRNA is evident and beta-III tubulin appears in the mitotic spindle of neuroblasts. Neurite outgrowth is obvious by day 3, and beta-III tubulin protein and mRNA levels increase concurrently until approximately day 7, when beta-III mRNA levels begin to decrease while protein levels remain high. In addition, increasingly acidic beta-III tubulin isoforms appear during neuronal differentiation. The expression of these isoelectric variants occurs concomitant with a temporal increase in the levels of beta-III tubulin present in the colchicine-stable microtubules. These results implicate posttranslational modifications of beta-III tubulin in the increased microtubule stability noted in differentiating P19 neurons.

Animals↗