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Results for “posttranslational modification”

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PubMed2006

Methylation of Smad6 by protein arginine N-methyltransferase 1.

Signal transduction pathways utilize posttranslational modifications to regulate the activity of their components in a temporal-spatial and efficient fashion. Arginine methylation is one of the posttranslational modifications that can result in monomethylated-, asymmetric dimethylated- and/or symmetric dimethylated-arginine residues in proteins. Here we demonstrate that inhibitory-Smads (Smad6 and Smad7), but not receptor-regulated- (R-)Smads and the common-partner Smad4, can be methylated by protein arginine N-methyltransferase (PRMT)1. Using mass-spectrometric analysis, we found that PRMT1 dimethylates arginine(74) (Arg(74)) in mouse Smad6. PRMT1 interacts with the N-terminal domain of Smad6 in which Arg(74) residue is located. Assays examined so far have shown no significant differences between the functions of Smad6 and those of methylation-defective Smad6 (Smad6R74A). Both wild-type and Smad6R74A were equally efficient in blocking BMP-induced growth arrest upon their ectopic expression in HS-72 mouse B-cell hybridoma cells.

Alanine↗

PubMed2007

High-mobility group box-1 isoforms as potential therapeutic targets in sepsis.

High-mobility group box-1 (HMGB1) protein was originally described as a nuclear DNA-binding protein that functions as a structural cofactor critical for proper transcriptional regulation and gene expression. Recent studies indicate that damaged, necrotic cells liberate HMGB1 into the extracellular milieu where it functions as a proinflammatory cytokine. Indeed, HMGB1 represents a novel family of inflammatory cytokines composed of intracellular proteins that can be recognized by the innate immune system as a signal of tissue damage. Posttranslational modifications of HMGB 1 determine its interactions with other proteins and modulate its biological activity. However, very little is known about how these posttranslational modifications of HMGB1 affect its extracellular inflammatory activity and pathological potential. These studies can provide more efficient therapeutic strategies directed against specific HMGB1 isoforms. Therapeutic strategies against these specific HMGB1 isoforms can serve as models for more efficient therapeutic strategies against rheumatoid arthritis or sepsis. This article reviews the recent studies on HMGB1 regulation and their impact on the inflammatory activity and pathological contribution of HMGB 1 to infectious and inflammatory disorders.

Animals↗

PubMed1999

Molecular definition and characterization of recombinant bovine CB8 and CB10: immunogenicity and arthritogenicity.

Theoretically, the ability to produce recombinant type II collagen (CII) peptide fragments in a prokaryotic expression system would be extremely useful for preparing adequate amounts of CII peptides suitable for therapeutic uses. Bacteria do not contain the enzymes involved in the extensive posttranslational modifications that occur during the biosynthesis of CII, such as the hydroxylation of prolyl and lysyl residues and glycosylation of hydroxylysyl residues. As these posttranslational modifications may play a role in the immune and arthritogenic response to CII, it was unclear whether collagen expressed in Escherichia coli would be immunologically comparable to tissue-derived CII. Therefore, we prepared recombinant proteins for CB8 and CB10 by cloning CB8 (CII 403-551) and CB10 (CII 552-897) genes from bovine chondrocytes by RT-PCR technique and expressing them in an E. coli expression system. Characterization of these recombinant proteins revealed that both rCB8 and rCB10 stimulated T cell proliferation in a T cell determinant-specific manner. The T cells from mice immunized with rCB8 respond specifically to a synthetic peptide, CII 445-453, the CB8 T cell determinant. Conversely, rCB10-primed T cells respond strongly to CII 610-618, the CB10 T cell determinant. Recombinant CB8-induced autoantibodies that bound to mouse CB8 as effectively and in the same topographic distribution as tissue-derived CB8. Finally, when rCB8 and rCB10 proteins were used to immunize B10.RIII (H-2(r)) mice, rCB8 induced arthritis in 33% of the mice, very similar to the incidence induced by tissue-derived CB8 peptide. As was found to be the case with tissue-derived CB10, rCB10 was completely ineffective in inducing arthritis. Pathological changes of arthritic joints in the mice immunized with rCB8 were similar to those observed in mice immunized with tissue-derived CB8. Thus, these recombinant CII peptides expressed in E. coli can induce an effective immunologic response and suggest that functionally useful CII peptides can be generated by the prokaryotic expression system.

Amino Acid Sequence↗

PubMed2004

Strategies for drug discovery by targeting sulfation pathways.

Posttranslational modifications of proteins such as phosphorylation have been recognized as pivotal modulators of biological activity in healthy and diseased tissues. Sulfation is a key posttranslational modification the role of which in physiology and pathology is only now becoming appreciated. Whereas phosphorylation is central to intracellular signal transduction, sulfation modulates cell-cell and cell-matrix communication. Sulfation involves a class of enzymes known as sulfotransferases, which transfer sulfate from the ATP-like sulfate donor 3'phosphoadenosine-5'phosphosulate to glycoproteins, glycolipids or metabolites. This review focuses on Golgi-localized sulfotransferases, their molecular biology and biochemistry, and strategies towards discovery of sulfotransferase inhibitors that could have potential as therapeutics in inflammation, cancer and infectious diseases.

Animals↗

PubMed1980

Genetics of the apolipoprotein E system in man.

The polymorphism of apolipoprotein E (Apo E) in man is controlled by two codominant alleles, Apo E(n) and Apo E(d), at the Apo E-N/D locus and by two alleles, the dominant, Apo E4(+), and the recessive, Apo E4(o), at the Apo E4 locus. Frequency distribution analysis of Apo E phenotypes demonstrated a highly significant association between both systems (P approximately 1%). The Apo E4-(+) variant was about twice as frequent in phenotype Apo E-N (30.1%) than in phenotype Apo E-ND (16.4%). The phenotypic combination Apo E-D/-E4(+) was not observed. The segregation of Apo E phenotypes in informative matings is consistent with a close linkage of both loci. The results may be explained by different models. On the basis of the present data, these models cannot be distinguished by formal genetic criteria. (1) Haplotypes Apo E(n)/E4(+), Apo E(n)/E4(o), and Apo E(d)/E4(o) determine the different phenotypes, and a linkage disequilibrium exists of Delta = .0147 between the E-N/D and E4 loci. (2) The fourth haplotype, Apo E(d)/E4(+), exists, but the gene E4(+) is not expressed in coupling with Apo E(d). The four-haplotype model seems more attractive in view of Apo E-N/D polymorphism's quantitative character and of biochemical results, which show that phenotypes Apo E-N and Apo E-D differ in the apparent molecular weight (M(r)) of the respective major Apo E polymorphic form. Hence, the Apo E-N/D locus may control structural genes involved in the posttranslational modification of Apo E. (3) Finally, there may exist only one Apo E structural gene locus but with mutations at two sites susceptible to posttranslational modification.

Adult↗

PubMed2001

Bioinformatics and mass spectrometry for microorganism identification: proteome-wide post-translational modifications and database search algorithms for characterization of intact H. pylori.

MALDI-TOF mass spectrometry has been coupled with Internet-based proteome database search algorithms in an approach for direct microorganism identification. This approach is applied here to characterize intact H. pylori (strain 26695) Gram-negative bacteria, the most ubiquitous human pathogen. A procedure for including a specific and common posttranslational modification, N-terminal Met cleavage, in the search algorithm is described. Accounting for posttranslational modifications in putative protein biomarkers improves the identification reliability by at least an order of magnitude. The influence of other factors, such as number of detected biomarker peaks, proteome size, spectral calibration, and mass accuracy, on the microorganism identification success rate is illustrated as well.

Algorithms↗

PubMed2004

Recombinant expression systems in the pharmaceutical industry.

In terms of downstream processing efficiency, secretory expression systems offer potential advantages for the production of recombinant proteins, compared with inclusion body forming cytosolic systems. However, for high-volume therapeutics like insulin, the product yields of the majority of the potentially available secretory systems is not yet fully competitive. Current strategies to improve productivity and secretion efficiency comprise: (1) enhancement of gene expression rates, (2) optimization of secretion signal sequences, (3) coexpression of chaperones and foldases, (4) creation of protease deficient mutants to avoid premature product degradation and (5) subsequent breeding and mutagenesis. For the production of non-glycosylated proteins and proteins, which are natively glycosylated but are also pharmacologically active without glycosylation, prokaryotes, which usually lack metabolic pathways for glycosylation, are theoretically the most suitable organisms and offer two alternatives: either Escherichia coli strains are conditioned to be efficient secreters or efficient native secreters like Bacillus species are accordingly developed. To fully exploit the secretory capacity of fungal species, a deeper understanding of their posttranslational modification physiology will be necessary to steer the degree and pattern of glycosylation, which influences both folding and secretion efficiency. Insect and mammalian cells display posttranslational modification patterns very similar or identical to humans, but in view of the entailed expenditures, their employment can only be justified if their modification machinery is required to ensure a desired pharmacological activity.

Animals↗

PubMedDate not supplied

The possible role of alpha-crystallins in human senile cataractogenesis.

alpha-Crystallins possess molecular chaperone properties and are one of the most abundant of the lenticular proteins. Posttranslational modifications of these proteins have been implicated as a possible etiology of human cataracts. This article will review current knowledge concerning the effects of known posttranslational modifications upon the molecular chaperone properties and aggregation behavior of alpha-A and alpha-B crystallin. Based upon these effects, experimental approaches will be discussed that may be useful in the development of reagents that may selectively inhibit the cataractogenic process in the aging human lens.

Aged↗

PubMed2001

Phosphorylation of xanthine dehydrogenase/oxidase in hypoxia.

The enzyme xanthine oxidase (XO) has been implicated in the pathogenesis of several disease processes, such as ischemia-reperfusion injury, because of its ability to generate reactive oxygen species. The expression of XO and its precursor xanthine dehydrogenase (XDH) is regulated at pre- and posttranslational levels by agents such as lipopolysaccharide and hypoxia. Posttranslational modification of the protein, for example through thiol oxidation or proteolysis, has been shown to be important in converting XDH to XO. The possibility of posttranslational modification of XDH/XO through phosphorylation has not been adequately investigated in mammalian cells, and studies have reported conflicting results. The present report demonstrates that XDH/XO is phosphorylated in rat pulmonary microvascular endothelial cells (RPMEC) and that phosphorylation is greatly increased ( approximately 50-fold) in response to acute hypoxia (4 h). XDH/XO phosphorylation appears to be mediated, at least in part, by casein kinase II and p38 kinase as inhibitors of these kinases partially prevent XDH/XO phosphorylation. In addition, the results indicate that p38 kinase, a stress-activated kinase, becomes activated in response to hypoxia (an approximately 4-fold increase after 1 h of exposure of RPMEC to hypoxia) further supporting a role for this kinase in hypoxia-stimulated XDH/XO phosphorylation. Finally, hypoxia-induced XDH/XO phosphorylation is accompanied by a 2-fold increase in XDH/XO activity, which is prevented by inhibitors of phosphorylation. In summary, this study shows that XDH/XO is phosphorylated in hypoxic RPMEC through a mechanism involving p38 kinase and casein kinase II and that phosphorylation is necessary for hypoxia-induced enzymatic activation.

Animals↗

PubMed2005

[Analysis of gene expression profile of G1/S transition in gastric cancer cell cycle].

OBJECTIVE: To detect the variation of gene expression profile of G1/S transition and elucidate the role of related genes regulating cell cycle from G1 to S phase in gastric cancer. METHODS: Nocodazole-thymidine and double thymidine methods were used to synchronize gastric cells at G2/M and G1/S point, cDNA microarray chips was applied to examine the gene expression profile at G1 early and late phase, S early and late phase during the cell cycle, hierarchy analysis was conducted by a professional software package. RESULTS: A total of 2001 genes were detected available, 959 genes appeared to be upregulated or downregulated, including 147 genes upregulated at G1 late phase. These 147 genes are involved in DNA metabolism, transcription and translation,posttranslational modification, ubiquitination, signal transduction etc, which all affected cell cycle from different aspects. CONCLUSION: Complex multiple gene processes, such as DNA metabolism, transcription and translation, posttranslational modification, ubiquitination, signal transduction etc,are implicated in and also essential for G1/S transition during gastric cancer cell cycle, part of these genes are significantly associate with overproliferation in gastric cancer.

Cell Division↗

PubMed1997

Tubulin synthesis and assembly in differentiating neurons.

Neurons are highly polarized cells that extend long processes, the axons and dendrites, to form contacts with target cells. The formation and maintenance of this specialized morphology relies on the assembly of an organized microtubule array that is the predominant component of the neuronal cytoskeleton. During this process there is an evolution in the composition and dynamics of microtubules, resulting in stable microtubule bundles that provide structural support and function in intracellular transport along the axon. In this essay we provide an overview of the mechanisms regulating the synthesis and assembly of tubulin in differentiating neurons with particular attention to the roles of multiple tubulin isotypes, posttranslational modifications of tubulin, and microtubule-associated proteins. We conclude that, ultimately, the developmental regulation of microtubules in neurons may require the coordinated expression and posttranslational modifications of tubulin and microtubule-associated proteins to provide biochemical forms that favour specific interactions, each combination conferring distinctive dynamic and functional properties.

Animals↗

PubMed2002

Mycobacterial heparin-binding hemagglutinin and laminin-binding protein share antigenic methyllysines that confer resistance to proteolysis.

Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin produce a heparin-binding hemagglutinin adhesin (HBHA) required for extrapulmonary dissemination and a laminin-binding protein (LBP) involved in cytoadherence through laminin recognition. These adhesins bear posttranslational modifications that are not present when the proteins are produced in a recombinant (r) form in Escherichia coli. Mass spectrometry analysis of HBHA revealed that the posttranslational modifications are borne by the C-terminal moiety, which comprises the heparin-binding domain made of repeated lysine-rich motifs. Amino acid sequencing showed that these modifications consist of mono- and dimethyllysines within these motifs. The methyllysine-containing repeats were recognized by mAb 4057D2 and were also detected in LBP, which is equally recognized by mAb 4057D2. This Ab does not recognize the recombinant forms of these proteins. However, when rHBHA and rLBP were subjected to NaBH(4) and formalin treatment to induce lysine methylation, reactivity with mAb 4057D2 was recovered. Methylated rHBHA displayed enhanced resistance to proteolysis compared with rHBHA, as previously observed for native HBHA. S-adenosylmethionine-dependent HBHA methyltransferase activity was detected in the cell-wall fractions of M. bovis bacillus Calmette-Guérin and of Mycobacterium smegmatis, a species that produces LBP but naturally lacks hbhA, suggesting that the same enzyme(s) methylate(s) both LBP and HBHA. This hypothesis was confirmed by the fact that HBHA produced by recombinant M. smegmatis was also methylated. These results show that mycobacteria use enzymatic methylation of lysines to ensure greater stability of their adhesins.

Adhesins, Bacterial↗

PubMed2005

O-GlcNAc level variations are associated with the development of skeletal muscle atrophy.

O-linked N-acetylglucosaminylation (O-GlcNAc) is a regulatory posttranslational modification of nucleocytoplasmic proteins, which consists of the attachment of N-acetylglucosamine to serine or threonine residues of a protein. This glycosylation is a ubiquitous posttranslational modification, which probably plays important roles in many aspects of protein function. Our laboratory has previously reported that, in skeletal muscle, proteins of the glycolytic pathway and energetic metabolism and contractile proteins were O-GlcNAc modified (Cieniewski-Bernard C, Bastide B, Lefebvre T, Lemoine J, Mounier Y, and Michalski JC. Mol Cell Proteomics 3: 577-585, 2004). O-GlcNAc has been recently demonstrated to play a role in modulating cellular function in response to nutrition and also in stress conditions. Therefore, we have investigated here the implication of the glycosylation/deglycosylation process in the development of atrophy in rat skeletal muscle after hindlimb unloading. The high O-GlcNAc level found in control soleus [compared with control extensor digitorum longus (EDL)] becomes lower in atrophied soleus. On the opposite side, the low rate of O-GlcNAc in control EDL reaches higher levels in EDL, not atrophied after hindlimb unloading. These variations in O-GlcNAc level are correlated with a variation of the O-GlcNAc process enzyme activities and could be associated with a differential expression of heat shock proteins. Our results suggest that O-GlcNAc variations could control the muscle protein homeostasis and be implicated in the regulation of muscular atrophy.

Acetylglucosamine↗

PubMed2005

What makes an autoantigen an autoantigen?

Multiple classes of proteins are modified to tailor them for specific physiological roles. The nature of these posttranslational modifications of proteins, as well as the relationships between them including those of the immune system proteins themselves, and immune system responses are reviewed. Aspects of protein posttranslational modification and their relationship to the pathogenesis of several autoimmune diseases and primary biliary cirrhosis are highlighted.

Animals↗

PubMed2000

Differential in vivo modifications of the HMGI(Y) nonhistone chromatin proteins modulate nucleosome and DNA interactions.

The HMGI(Y) family of "high mobility group" nonhistone proteins are architectural transcription factors whose overexpression is highly correlated with both cancerous transformation and increased malignancy and metastatic potential of tumors in vivo. Here we report on the types of posttranslational modifications found in vivo on the HMG-I and HMG-Y proteins isolated from two human breast epithelial cell lines, MCF-7 and MCF-7/PKC-alpha, that represent different stages of neoplastic progression. The MCF-7 cell line exhibits many characteristics of normal breast epithelial cells and does not form tumors when injected into nude mice, whereas the MCF-7/PKC-alpha cell line, a derivative of MCF-7 that expresses a transgene coding for the enzyme protein kinase C-alpha (PKC-alpha), is both malignant and highly metastatic. Using MALDI mass spectrometry, we show that the HMG-Y protein is more highly modified than the HMG-I protein in both the MCF-7 and the MCF-7/PKC-alpha cells. Significantly, the HMG-Y protein isolated from the highly metastatic MCF-7/PKC-alpha cells possesses a unique constellation of phosphorylations, methylations, and acetylations not found on the HMG-I protein isolated from either the MCF-7 or MCF-7/PKC-alpha cells. We further demonstrate that some of the same amino acid residues phosphorylated on recombinant HMGI(Y) proteins by purified PKC in vitro are also phosphorylated on the HMG-I(Y) proteins isolated from MCF-7/PKC-alpha cells, suggesting that PKC phosphorylates these proteins in vivo. Quantitative substrate binding analyses indicate that the biochemical modifications present on the HMG-I and HMG-Y proteins differentially influence the ability of these proteins to interact with both A.T-rich DNA substrates and nucleosome core particles in vitro, suggesting a similar modulation of such binding affinities in vivo. To our knowledge, this is the first demonstration of differences in the types of in vivo biochemical modifications found on the HMG-I and HMG-Y proteins in cells and also the first experimental evidence suggesting a possible linkage between such posttranslational modifications and the neoplastic potential of cells.

AT Rich Sequence↗

PubMed2005

A fluorescence resonance energy transfer-based assay to study SUMO modification in solution.

Analysis of posttranslational modifications with ubiquitin and ubiquitin-related proteins (Ubl) generally involves detection of the modified species by immunoblotting or autoradiography, techniques that are not easily applicable for kinetic, quantitative, or high-throughput assays. To circumvent these limitations for studies on ubiquitin-related proteins of the SUMO family, we have developed a fluorescence resonance energy transfer (FRET)-based assay system using yellow fluorescent protein (YFP)-tagged mature SUMO1 (amino acids 1-97) and cyan fluorescent protein (CFP)-tagged RanGAP1 (amino acids 400-589) as model substrates. Reactions are set up in 384-well microtiter plates and are followed online using a fluorescence microtiter plate reader. Applications may involve identification and analysis of SUMO-modifying enzymes and isopeptidases, comparison of enzyme and substrate mutants, and screens for small molecular weight inhibitors. The principal outline of the assay should be applicable to other Ubl conjugation systems as well.

Animals↗

PubMed2000

Heteronuclear NMR studies of the specificity of the post-translational modification of biotinyl domains by biotinyl protein ligase.

The lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes and the biotinyl domains of biotin-dependent enzymes have homologous structures, but the target lysine residue in each domain is correctly selected for posttranslational modification by lipoyl protein ligase and biotinyl protein ligase, respectively. We have applied two-dimensional heteronuclear NMR spectroscopy to investigate the interaction between the apo form of the biotinyl domain of the biotin carboxyl carrier protein of acetyl-CoA carboxylase and the biotinyl protein ligase (BPL) from Escherichia coli. Heteronuclear multiple quantum coherence NMR spectra of the 15N-labelled biotinyl domain were recorded in the presence and absence of the ligase and backbone amide 1H and 15N chemical shifts were evaluated. Small, but significant, changes in chemical shift were found in two regions, including the tight beta-turn that houses the lysine residue targetted for biotinylation, and the beta-strand 2 and the loop that precedes it in the domain. When compared with the three-dimensional structure, sequence alignments of other biotinyl and lipoyl domains, and mutagenesis data, these results give a clear indication of how the biotinyl domain is both recognised by BPL and distinguished from the structurally related lipoyl domain to ensure correct posttranslational modification.

Acetyl-CoA Carboxylase↗

PubMed2003

The human SUMF1 gene, required for posttranslational sulfatase modification, defines a new gene family which is conserved from pro- to eukaryotes.

Recently, the human C(alpha)-formylglycine (FGly)-generating enzyme (FGE), whose deficiency causes the autosomal-recessively transmitted lysosomal storage disease multiple sulfatase deficiency (MSD), has been identified. In sulfatases, FGE posttranslationally converts a cysteine residue to FGly, which is part of the catalytic site and is essential for sulfatase activity. FGE is encoded by the sulfatase modifying factor 1 (SUMF1) gene, which defines a new gene family comprising orthologs from prokaryotes to higher eukaryotes. The genomes of E. coli, S. cerevisiae and C. elegans lack SUMF1, indicating a phylogenetic gap and the existence of an alternative FGly-generating system. The genomes of vertebrates including mouse, man and pufferfish contain a sulfatase modifying factor 2 (SUMF2) gene encoding an FGE paralog of unknown function. SUMF2 evolved from a single exon SUMF1 gene as found in diptera prior to divergent intron acquisition. In several prokaryotic genomes, the SUMF1 gene is cotranscribed with genes encoding sulfatases which require FGly modification. The FGE protein contains a single domain that is made up of three highly conserved subdomains spaced by nonconserved sequences of variable lengths. The similarity among the eukaryotic FGE orthologs varies between 72% and 100% for the three subdomains and is highest for the C-terminal subdomain, which is a hotspot for mutations in MSD patients.

Alanine↗