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Results for “Protein Modification, Translational”

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PubMed2003

Detection of tyrosine phosphorylated peptides via skimmer collision-induced dissociation/ion trap mass spectrometry.

Phosphorylation of proteins is an important post-translational protein modification in cellular response to environmental change and occurs in both prokaryotes and eukaryotes. Identification of the amino acid on individual proteins that become phosphorylated in response to extracellular stimulus is essential for understanding the mechanisms involved in the intracellular signals that these modifications facilitate. Most protein kinases catalyze the phosphorylation of proteins on serine, threonine or tyrosine. Although tyrosine phosphorylation is often the least abundant of the three major phosphorylation sites, it is important owing to its role in signal pathways. Currently available methods for the identification of phosphorylation sites can often miss low levels of tyrosine phosphorylations. This paper describes a method for the identification of phosphotyrosine-containing peptides using electrospray ionization on an ion trap mass spectrometer. Skimmer-activated collision-induced dissociation (CID) was used to generate the phosphotyrosine immonium ion at m/z 216. This method is gentle enough that the protonated molecule of the intact peptide is still observed. In-trap CID was employed for the verification of the phosphotyrosine immonium ion. Using this technique, low levels of phosphotyrosine-containing peptides can be identified from peptide mixtures separated by nanoflow micro liquid chromatography/mass spectrometry.

Caseins↗

PubMed2005

Glycoproteomics of N-glycosylation by in-gel deglycosylation and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry mapping: application to congenital disorders of glycosylation.

A general strategy for the structural evaluation of N-glycosylation, a common post-translational protein modification, is presented. The methods for the release of N-linked glycans from the gel-separated proteins, their isolation, purification and matrix-assisted laser desorption/ionisation-mass spectrometry (MALDI-MS) analysis of their mixtures were optimised. Since many glycoproteins are available only at low quantities from sodium dodecyl sulphate-polyacrylamide gel electrophoresis or two-dimensional gels, high attention was paid to obtain N-glycan mixtures representing their actual composition in human plasma by in-gel deglycosylation. The relative sensitivity of solid MALDI matrices for MS analysis of acidic N-glycans was compared. The most favourable results for native acidic N-glycans were obtained with 2,4,6-trihydroxyacetophenone monohydrate/diammoniumcitrate as a matrix. This matrix provided good results for both neutral and acidic mixtures as well as for methylated N-glycans. In the second part of this paper the potential of such an optimised MS strategy alone or in combination with high pH anion-exchange chromatography profiling for the clinical diagnosis of congenital disorders of glycosylation is presented.

Carbohydrate Conformation↗

PubMed2006

Proteome analysis of B-cell maturation.

Proteins affected by anti-mIgM stimulation during B-cell maturation were identified using 2-DE-based proteomics. We investigated the proteome profiles of stimulated and nonstimulated Ramos B-cells at eight time points during 5 d and compared the obtained proteomic data to the corresponding data from DNA-microarray studies. Anti-mIgM stimulation of the cells resulted in significant differences (> or =twofold) in the protein abundance close to 100 proteins and differences in post-translational protein modifications. Forty-eight up- or down-regulated proteins were identified by mass spectrometric methods and database searches. The identities of a further nine proteins were revealed by comparing their positions to the known proteins in other lymphocyte 2-DE databases. Several of the proteins are directly related to the functional and morphological characteristics of B-cells, such as cytoskeleton rearrangement and intracellular signalling triggered by the crosslinking of B-cell receptors. In addition to proteins known to be involved in human B-cell maturation, we identified several proteins that were not previously linked to lymphocyte differentiation. The results provide deeper insights into the process of B-cell maturation and may lead to novel therapeutic strategies for immunodeficiencies. An interactive 2-DE reference map is available at http://bioinf.uta.fi/BcellProteome.

B-Lymphocytes↗

PubMedDate not supplied

Trans-acting factors involved in species-specificity and control of mouse ribosomal gene transcription.

Faithful and efficient transcription initiation at the mouse ribosomal gene promoter requires besides RNA polymerase I (pol I) four polypeptide trans-acting factors, termed TIF-IA, TIF-IB, TIF-IC, and mUBF. We have partially purified these proteins from cultured Ehrlich ascites cells and show that in the presence of TIF-IA and TIF-IB, pol I directs very low amounts of specific transcripts. Neither TIF-IC nor mUBF on their own significantly stimulate the efficiency of template utilization. However, both factors together strongly activate transcription. Interestingly, factor TIF-IB - the murine homologue of human SL1 - fails to program a human extract to transcribe the murine template, but requires its homologous RNA polymerase I. This finding implicates that not only some rDNA transcription factors but also pol I exhibits species-specific differences. The growth-related factor TIF-IA, on the other hand, stimulates both mouse and human rDNA transcription. This regulatory factor whose amount or activity fluctuates according to the proliferation rate of the cells, is functionally inactivated by antibodies against cdc2 protein kinase. This result together with the observation that transcription is stimulated by ATP-gamma S, an ATP analogue which is a substrate for protein kinases but not for protein phosphatases, strongly suggests that post-translational protein modification is involved in rDNA transcription regulation.

Adenosine Triphosphate↗

PubMed2006

Increase in HLA-G1 proteolytic shedding by tumor cells: a regulatory pathway controlled by NF-kappaB inducers.

HLA-G is expressed by tumors, in which it contributes to the evasion of immunosurveillance. NF-kappaB appears to be a candidate for regulating HLA-G expression, since it is considered to be a hallmark of cancer. We investigated the role of NF-kappaB in modulating HLA-G expression in HLA-G-positive tumor cells, JEG-3 (choriocarcinoma), FON (melanoma), and M8-HLA-G1 (HLAG1-transfected melanoma). The treatment of tumor cells with two NF-kappaB inducers, tumor necrosis factor-alpha and phorbol 12-myristate 13-acetate, decreased HLA-G1 cell surface expression but increased intracytoplasmic HLA-G proteins. Reduction in HLA-G1 cell surface expression is driven by NF-kappaB and involves a proteolytic shedding process dependent on metalloproteinase activity. In contrast, an increase in intracytoplasmic HLA-G proteins involves post-transcriptional mechanisms that are independent of NF-kappaB. These results, and the fact that soluble HLA-G1 reduces the cytotoxicity of the NKL cell line, lead us to propose a novel regulatory pathway for HLA-G expression by tumor cells that may have particular relevance in tumor escape.

Cell Line↗

PubMed2005

Using string kernel to predict signal peptide cleavage site based on subsite coupling model.

Owing to the importance of signal peptides for studying the molecular mechanisms of genetic diseases, reprogramming cells for gene therapy, and finding new drugs for healing a specific defect, it is in great demand to develop a fast and accurate method to identify the signal peptides. Introduction of the so-called {-3,-1, +1} coupling model (Chou, K. C.: Protein Engineering, 2001, 14-2, 75-79) has made it possible to take into account the coupling effect among some key subsites and hence can significantly enhance the prediction quality of peptide cleavage site. Based on the subsite coupling model, a kind of string kernels for protein sequence is introduced. Integrating the biologically relevant prior knowledge, the constructed string kernels can thus be used by any kernel-based method. A Support vector machines (SVM) is thus built to predict the cleavage site of signal peptides from the protein sequences. The current approach is compared with the classical weight matrix method. At small false positive ratios, our method outperforms the classical weight matrix method, indicating the current approach may at least serve as a powerful complemental tool to other existing methods for predicting the signal peptide cleavage site. The software that generated the results reported in this paper is available upon requirement, and will appear at http://www.pami.sjtu.edu.cn/wm.

Models, Genetic↗

PubMed1989

Protein changes during anterograde-to-retrograde conversion of axonally transported vesicles.

In the axon tip, cell biological mechanisms convert anterogradely transported membranous elements. To study the effects of these anterograde-to-retrograde (A-R) converting mechanisms on the electrophoretic behaviour of vesicle proteins, we compared the proteins of anterograde vesicles (before A-R conversion at the axon tip) with those of retrograde vesicles (after A-R conversion at the axon tip). The proteins in transported vesicles were pulse-labeled with [35S]methionine, and the radiolabeled vesicles were concentrated by ligating the axons-anterograde vesicles accumulate selectively on the proximal side of the ligature and retrograde vesicles accumulate on the distal side of the ligature. Analyses of vesicle proteins by polyacrylamide gel electrophoresis (SDS-PAGE) show that most of the anterograde proteins were also present in the retrograde vesicles. In addition to the conservation of these anterograde proteins in the retrograde vesicles, there were also many differences: some anterograde proteins were diminished in the retrograde vesicles, other anterograde proteins were absent from the retrograde vesicles, and the retrograde vesicles contained some new protein bands that were not present in the anterograde vesicles. These results indicate that A-R converting mechanisms modify membranous vesicle proteins in the axon tip. We propose that some of these post-translational protein modifications change the directional code on the vesicle surfaces, thereby converting anterograde membranous elements into retrograde membranous elements.

Animals↗

PubMed1989

Rift Valley fever virus M segment: phlebovirus expression strategy and protein glycosylation.

The M segment RNA of Rift Valley fever virus (RVFV) encodes four gene products: the two viral envelop glycoproteins G2 and G1, a glycosylated 78-kDa protein, and a nonglycosylated 14-kDa protein. These proteins are generated from a single open reading frame (ORF) by a strategy involving independent translational initiations at both the first and second in-phase ATG codons and co-translational processing of primary polyprotein products. The ORF encodes six sites for N-linked glycosylation: one present in the "preglycoprotein region" preceding the coding sequences of the mature envelop glycoproteins, and within the coding sequences of both the 78- and 14-kDa proteins; one site in the glycoprotein G2 coding region, also present in the 78-kDa protein; and four sites within glycoprotein G1. From analyses of RVFV proteins produced in cells infected with recombinant vaccinia viruses expressing various M segment regions, we show glycoprotein G2 was glycosylated at its single site and glycoprotein G1 at at least three sites. Both sites for N-linked glycosylation in the 78-kDa protein were occupied with glycan. This latter result indicated the preglycoprotein region glycosylation site was utilized in the 78-kDa protein, but this same site within the 14-kDa protein was not. Further analysis showed utilization of this glycosylation site, as well as proteolytic processing at the amino terminus of the mature glycoprotein G2, appeared to be determined by initiation codon usage. The two-site translational initiation expression strategy of this phlebovirus M segment and its role in the control of post-translational protein modification and processing are discussed.

Bunyaviridae↗

PubMed2006

Secretion and lysophospholipase D activity of autotaxin by adipocytes are controlled by N-glycosylation and signal peptidase.

Autotaxin (ATX) is a lysophospholipase D involved in synthesis of lysophosphatidic acid (LPA). ATX is secreted by adipocytes and is associated with adipogenesis and obesity-associated diabetes. Here we have studied the mechanisms involved in biosynthesis and secretion of ATX by mouse 3T3-F442A adipocytes. We found that inhibition of N-glycosylation with tunicamycin or by double point deletion of the amino-acids N53 and N410 of ATX inhibit its secretion. In addition, N-glycosidase treatment and point deletion of the amino-acid N410 inhibits the lysophospholipase D activity of ATX. Analysis of the amino-acid sequence of mouse ATX shows the presence of a N-terminal signal peptide. Treatment with the signal peptidase inhibitor globomycin inhibits ATX secretion by adipocytes. Transfection in Cos-7 cells of site-directed deleted ATX shows that ATX secretion is dependent on the hydrophobic core sequence of the signal peptide, not on the putative signal peptidase cleavage site sequence. Analysis of the amino-acid sequence of mouse ATX also reveals the presence of a putative cleavage site by the protein convertase furin. Treatment of adipocytes with the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone does not modified secretion or lysophospholipase D activity of ATX. Transfection in Cos-7 cells of site-directed deleted ATX shows that the furin recognition site is not required for secretion or lysophospholipase D activity of ATX. In conclusion, the present work demonstrates the crucial role of N-glycosylation in secretion and activity of ATX. The present work also confirms the crucial role signal peptidase in secretion of ATX by adipocytes.

Adipocytes↗

PubMed2004

Hepatic flavin-containing monooxygenase activity attenuated by cGMP-independent nitric oxide-mediated mRNA destabilization.

To identify the novel mechanism by which nitric oxide (NO) suppresses flavin-containing monooxygenase (FMO) activity in endotoxemic rat livers, NO-overproducing conditions were induced in primary cultured rat hepatocytes by treatment with a mixture (LCM) of lipopolysaccharide and proinflammatory cytokines (IL-1beta, TNF-alpha, and IFN-gamma), or by the addition of a pure NO donor, spermine-NONOate. mRNA levels of the major hepatic form, FMO1, decreased via a cGMP-independent destabilizing effect of NO rather than by decreased transcription. The decrease in the mRNA levels caused by LCM-induced inducible NO synthase (iNOS) was completely blocked by co-treatment with aminoguanidine, a selective iNOS inhibitor. Furthermore, spermine-NONOate, but not the cGMP analog, 8-bromo-cGMP, dose- and time-dependently attenuated FMO1 mRNA stability in actinomycin-D-pretreated cells, resulting in decreases in protein levels and biochemical activity. These results suggest that NO acts directly in a cGMP-independent mechanism by decreasing the half-life of FMO1 mRNA, thereby inducing impairment of FMO-related functions in endotoxemia.

Animals↗

PubMed2006

The methylation of the C-terminal region of hnRNPQ (NSAP1) is important for its nuclear localization.

Protein arginine methylation is an irreversible post-translational protein modification catalyzed by a family of at least nine different enzymes entitled PRMTs (protein arginine methyl transferases). Although PRMT1 is responsible for 85% of the protein methylation in human cells, its substrate spectrum has not yet been fully characterized nor are the functional consequences of methylation for the protein substrates well understood. Therefore, we set out to employ the yeast two-hybrid system in order to identify new substrate proteins for human PRMT1. We were able to identify nine different PRMT1 interacting proteins involved in different aspects of RNA metabolism, five of which had been previously described either as substrates for PRMT1 or as functionally associated with PRMT1. Among the four new identified possible protein substrates was hnRNPQ3 (NSAP1), a protein whose function has been implicated in diverse steps of mRNA maturation, including splicing, editing, and degradation. By in vitro methylation assays we were able to show that hnRNPQ3 is a substrate for PRMT1 and that its C-terminal RGG box domain is the sole target for methylation. By further studies with the inhibitor of methylation Adox we provide evidence that hnRNPQ1-3 are methylated in vivo. Finally, we demonstrate by immunofluorescence analysis of HeLa cells that the methylation of hnRNPQ is important for its nuclear localization, since Adox treatment causes its re-distribution from the nucleus to the cytoplasm.

Amino Acid Sequence↗

PubMed2004

The emerging role of poly(ADP-ribose) polymerase-1 in longevity.

In the present paper, the involvement of the family of poly(ADP-ribose) polymerases (PARPs), and especially of PARP-1, in mammalian longevity is reviewed. PARPs catalyse poly(ADP-ribosyl)ation, a covalent post-translational protein modification in eukaryotic cells. PARP-1 and PARP-2 are activated by DNA strand breaks, play a role in DNA base-excision repair (BER) and are survival factors for cells exposed to low doses of ionising radiation or alkylating agents. PARP-1 is the main catalyst of poly(ADP-ribosyl)ation in living cells under conditions of DNA breakage, accounting for about 90% of cellular poly(ADP-ribose). DNA-damage-induced poly(ADP-ribosyl)ation also functions as a negative regulator of DNA damage-induced genomic instability. Cellular poly(ADP-ribosyl)ation capacity in permeabilised mononuclear blood cells (MNC) is positively correlated with life span of mammalian species. Furthermore PARP-1 physically interacts with WRN, the protein deficient in Werner syndrome, a human progeroid disorder, and PARP-1 and WRN functionally cooperate in preventing carcinogenesis in vivo. Some of the other members of the PARP family have also been revealed as important regulators of cellular functions relating to ageing/longevity. In particular, tankyrase-1, tankyrase-2, PARP-2 as well as PARP-1 have been found in association with telomeric DNA and are able to poly(ADP-ribosyl)ate the telomere-binding proteins TRF-1 and TRF-2, thus blocking their DNA-binding activity and controlling telomere extension by telomerase.

Animals↗

PubMed2006

Control of protein expression through mRNA stability in calcium signalling.

Specific sequences (cis-acting elements) in the 3'-untranslated region (UTR) of RNA, together with stabilizing and destabilizing proteins (trans-acting factors), determine the mRNA stability, and consequently, the level of expression of several proteins. Such interactions were discovered initially for short-lived mRNAs encoding cytokines and early genes like c-jun and c-myc. However, they may also determine the fate of more stable mRNAs in a tissue and disease-dependent manner. The interactions between the cis-acting elements and the trans-acting factors may also be modulated by Ca(2+) either directly or via a control of the phosphorylation status of the trans-acting factors. We focus initially on the basic concepts in mRNA stability with the trans-acting factors AUF1 (destabilizing) and HuR (stabilizing). Sarco/endoplasmic reticulum Ca(2+) pumps, SERCA2a (cardiac and slow twitch muscles) and SERCA2b (most cells including smooth muscle cells), are pivotal in Ca(2+) mobilization during signal transduction. SERCA2a and SERCA2b proteins are encoded by relatively stable mRNAs that contain cis-acting stability determinants in their 3'-regions. We present several pathways where 3'-UTR mediated mRNA decay is key to Ca(2+) signalling: SERCA2a and beta-adrenergic receptors in heart failure, renin-angiotensin system, and parathyroid hormones. Other examples discussed include cytokines vascular endothelial growth factor, endothelin and endothelial nitric oxide synthase. Roles of Ca(2+) and Ca(2+)-binding proteins in mRNA stability are also discussed. We anticipate that these novel modes of control of protein expression will form an emerging area of research that may explore the central role of Ca(2+) in cell function during development and in disease.

3' Untranslated Regions↗

PubMed2005

Use of genetic immunization to raise antibodies recognizing toxin-related cell surface ADP-ribosyltransferases in native conformation.

ADP-ribosyltransferases (ARTs) transfer ADP-ribose from NAD to arginine, asparagine, or cysteine residues in target proteins. This post-translational protein modification is the mechanism by which cholera-toxin and other bacterial toxins cause pathology in human host cells. Molecular cloning has identified five toxin-related GPI-anchored cell surface ARTs in the mouse (ART1, ART2.1, ART2.2, ART3, and ART4) and three in the human (ART1, ART3, and ART4). ART2-which has sparked interest because of its ability to activate the cytolytic P2X7 purinergic receptor by ADP-ribosylation-is encoded by two functional gene copies in the mouse genome while the human genome carries two inactivated ART2 pseudogenes. We generated stable transfectants for FLAG-tagged versions of each of the functional human and mouse ARTs. Using genetic immunization we raised monoclonal antibodies that recognize the native human ARTs on the surface of living cells. Some of these mAbs recognize an epitope shared with the mouse ART orthologue but not with more distant ART paralogues. Screening of primary cells and established cell lines by FACS revealed expression of ART1 by monocytes, neutrophils and myeloid leukemia cell lines but not by cell lines derived from solid tumors. ART1 and ART4 have been assigned the designations: CD296, and CD297, respectively.

ADP Ribose Transferases↗

PubMed2006

STAT1 as a key modulator of cell death.

Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that mediate various biological responses, including cell proliferation, survival, apoptosis, and differentiation. Among the members of the STAT family, accumulating evidence now indicates an important role for STAT1 in various forms of cell death. Depending upon stimuli or cell types, STAT1 can modulate a broad spectrum of cell death, comprising both apoptotic and non-apoptotic pathways. STAT1-dependent regulation of cell death is largely dependent on a transcriptional mechanism such as the activation of death-promoting genes. However, non-transcriptional mechanisms such as STAT1 interaction with TRADD, p53, or HDAC have been implicated in the regulation of cell death by STAT1. Furthermore, STAT1 itself is also subject to complex forms of regulation such as post-translational protein modification, which can critically affect STAT1 signaling and STAT1-dependent cell death. Given the reports showing that dysregulation of STAT1 signaling is associated with various pathological conditions, including the development of cancer, a better understanding of the mechanism underlying STAT1 regulation of cell death may lead to successful strategies for targeting STAT1 in such pathological settings.

Apoptosis↗

PubMed2005

Mitosis: PARty time in the spindle.

Poly(ADP-ribose), a post-translational protein modification known to affect chromatin structure, has now been shown to regulate microtubule organization during mitosis. These findings alter conventional views of the mechanisms of spindle assembly and function.

Cell Cycle Proteins↗

PubMed2006

Membrane topology of the human seipin protein.

The Berardinelli-Seip congenital lipodystrophy type 2 (BSCL2) gene encodes an integral membrane protein, called seipin, of unknown function localized to the endoplasmic reticulum of eukaryotic cells. Seipin is associated with the heterogeneous genetic disease BSCL2, and mutations in an N-glycosylation motif links the protein to two other disorders, autosomal-dominant distal hereditary motor neuropathy type V and Silver syndrome. Here, we report a topological study of seipin using an in vitro topology mapping assay. Our results suggest that the predominant form of seipin is 462 residues long and has an N(cyt)-C(cyt) orientation with a long luminal loop between the two transmembrane helices.

Amino Acid Motifs↗

PubMed2006

The production of peroxynitrite by human spermatozoa may affect sperm motility through the formation of protein nitrotyrosine.

OBJECTIVE: To detect peroxynitrite and 3-nitrotyrosine production in human spermatozoa of asthenozoospermic infertile patients and normospermic donors, and evaluate any influence on kinetic sperm features. DESIGN: Basic study. SETTING: Andrology Unit, Dept of Internal Medicine and Biochemistry Institute, Polytechnic University of Marche, Italy. PATIENT(S): Sixty-nine infertile patients affected by idiopathic asthenozoospermia and 29 normal fertile donors. INTERVENTION(S): No therapeutic intervention was performed on patients. MAIN OUTCOME MEASURE(S): Production of peroxynitrite (ONOO-) and 3-nitrotyrosine by human spermatozoa; kinetic sperm cells parameters. RESULT(S): Normospermic fertile donors exhibited ONOO- concentrations significantly lower than those of asthenozoospermic infertile men (9.11 +/- 3.37 vs. 27.46 +/- 5.77 nmol/10(6) cells); confocal microscopy showed that ONOO- was more evident in spermatozoa of patients than in healthy donors. Moreover, a significant negative correlation was evident between ONOO- concentration and total sperm motility, curvilinear velocity (VCL), straight progressive velocity (VSL), and linearity coefficient. Finally, an increase was found in the nitration of the tyrosine residues in asthenozoospermic samples compared to controls. CONCLUSION(S): Spontaneous tyrosine nitration occurs in human spermatozoa. This post-translational protein modification is enhanced by an overproduction of peroxynitrite, which is more evident in asthenozoospermic infertile patients when compared with normospermic fertile donors. Motility parameters are negatively affected, suggesting that ONOO- may be involved in defective sperm function.

Adult↗