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At least 343 records · Page 19Linked to original sources
PubMed2006

Regulating the p53 pathway: in vitro hypotheses, in vivo veritas.

Mutations in TP53, the gene that encodes the tumour suppressor p53, are found in 50% of human cancers, and increased levels of its negative regulators MDM2 and MDM4 (also known as MDMX) downregulate p53 function in many of the rest. Understanding p53 regulation remains a crucial goal to design broadly applicable anticancer strategies based on this pathway. This Review of in vitro studies, human tumour data and recent mouse models shows that p53 post-translational modifications have modulatory roles, and MDM2 and MDM4 have more profound roles for regulating p53. Importantly, MDM4 emerges as an independent target for drug development, as its inactivation is crucial for full p53 activation.

Animals↗
PubMed1991

Post-translational modification of the tegument proteins (VP13 and VP14) of herpes simplex virus type 1 by glycosylation and phosphorylation.

VP13 and VP14, major tegument proteins of herpes simplex virus type 1 (HSV-1) and the products of the UL47 gene, have been shown by partial proteolytic mapping to have closely related protein sequences. These proteins are phosphorylated in virus-infected cells, but not in preparations of purified virus. They also contain O-linked oligosaccharide units which include beta-1,4-N-acetyl galactosamine residues, as demonstrated by the binding of Dolichos biflorus lectin. This modification was detected only in purified virus and appears to be restricted to VP13/14 and VP22, another HSV-1 tegument protein.

Electrophoresis, Polyacrylamide Gel↗
PubMed2006

Regulation of post-translational modifications of muskelin by protein kinase C.

Muskelin is a member of the kelch-repeat superfamily of proteins, identified as an intracellular protein involved in cell spreading responses to thombospondin-1. Muskelin is expressed by many adult tissues and has an evolutionarily conserved, multidomain architecture consisting of an amino-terminal discoidin-like domain, a central alpha-helical region and six kelch-repeats that are predicted to form a beta-propeller structure. We previous demonstrated that muskelin molecules undergo head-to-tail association, however the physiological, post-translational regulation of muskelin is not well understood. Here, we have examined the expression of muskelin during mouse embryonic development and report widespread expression that includes muscle tissues, multiple epithelia and the brain. In cultured skeletal myoblasts and vascular smooth muscle cells, muskelin exists as a complex set of isoelectric variants. Five potential sites for phosphorylation by protein kinase C (PKC), are conserved between vertebrate and Drosophila muskelins, therefore we examined the hypothesis that muskelin is regulated post-translationally by PKC activity. We demonstrate that PKC activation or inhibition regulates the profile of endogenous muskelin isoelectric variants and that muskelin is a substrate for PKCalphain vitro. Wild-type GFP-muskelin and a panel of alanine point mutations were used to test the sensitivity of self-association to PKC activation. Mutation of two of the sites, S324 and T515, partially inhibited the ability of muskelin to self-associate in cells and inhibited responsiveness to activated PKC. Interestingly, both sites are predicted to lie in surface-exposed loops on the same side of the beta-propeller, implicating a common binding interface.

Animals↗
PubMed2006

Proteomics studies of post-translational modifications in plants.

Post-translational modifications of proteins greatly increase protein complexity and dynamics, co-ordinating the intricate regulation of biological events. The global identification of post-translational modifications is a difficult task that is currently accelerated by advances in proteomics techniques. There has been significant development in sample preparation methods and mass spectrometry instrumentation. To reduce the complexity and to increase the amount of modified proteins available for analysis, proteins are usually subjected to prefractionation such as chromatographic purification and affinity enrichment. In this review, the post-translational modification studies in plants are summarized. The sample preparation strategies applied to each study are also described. These include affinity-based enrichment methods, immobilized metal affinity chromatography and immunoprecipitation used for phosphorylation and ubiquitination studies, respectively, and the phase partitioning approach for glycosylphosphatidylinositol modification studies.

Chromatography, Affinity↗
PubMed2002

Inhibitors of post-translational modifications of G-proteins as probes to study the pancreatic beta cell function: potential therapeutic implications.

It is well established that glucose-induced insulin secretion involves generation of intracellular second messengers. Using specific inhibitors of guanosine triphosphate [GTP] biosynthesis [e.g., mycophenolic acid; MPA], we have identified a permissive role for GTP in glucose-stimulated insulin secretion. While the exact site of action for GTP within the islet beta cell remains to be identified and defined, recent evidence from several laboratories, including our own, indicate that it could involve activation of GTP-binding proteins [G-proteins]. These studies have identified both trimeric and monomeric forms of G-proteins within the pancreatic beta cell. Recent data also indicate that these G-proteins, specifically the monomeric G-proteins and the gamma subunits of trimeric G-proteins undergo a series of posttranslational modifications at their C-terminal cysteine. Such modifications include, isoprenylation, carboxyl methylation and palmitoylation. These modification steps appear to be essential for translocation of these proteins to the membrane sites for interaction with their respective effector proteins. This review primarily focuses on recent findings that clearly support the viewpoint that these posttranslational modification steps not only play obligatory roles in fuel-induced insulin secretion, but also in cytokine-mediated apoptotic demise of the beta cell. In this review, we also attempted to describe those findings involving the use of specific inhibitors for each of these pathways, and it is our hope that these aspects of beta cell metabolism and function generate interest in development of therapeutic intervention modalities to states of perturbed insulin release.

Apoptosis↗
PubMed2002

Post-translational modification of bone morphogenetic protein-1 is required for secretion and stability of the protein.

Bone morphogenetic protein (BMP)-1 is a glycosylated metalloproteinase that is fundamental to the synthesis of a normal extracellular matrix because it cleaves type I procollagen, as well as other precursor proteins. Sequence analysis suggests that BMP-1 has six potential N-linked glycosylation sites (i.e. NXS/T) namely: Asn(91) (prodomain), Asn(142) (metalloproteinase domain), Asn(332) and Asn(363) (CUB1 domain), Asn(599) (CUB3 domain), and Asn(726) in the C-terminal-specific domain. In this study we showed that all these sites are N-glycosylated with complex-type oligosaccharides containing sialic acid, except Asn(726) presumably because proline occurs immediately C-terminal of threonine in the consensus sequence. Recombinant BMP-1 molecules lacking all glycosylation sites or the three CUB-specific sites were not secreted. BMP-1 lacking CUB glycosylation was translocated to the proteasome for degradation. BMP-1 molecules lacking individual glycosylation sites were efficiently secreted and exhibited full procollagen C-proteinase activity, but N332Q and N599Q exhibited a slower rate of cleavage. BMP-1 molecules lacking any one of the CUB-specific glycosylation sites were sensitive to thermal denaturation. The study showed that the glycosylation sites in the CUB domains of BMP-1 are important for secretion and stability of the molecule.

Amino Acid Substitution↗
PubMed2005

A genome-wide analysis of sumoylation-related biological processes and functions in human nucleus.

Protein sumoylation is an important reversible post-translational modification of proteins in the nucleus, and it orchestrates a variety of the cellular processes. Genome-wide analysis of functional abundance and distribution of Small Ubiquitin-related MOdifier (SUMO) substrates may shed a light on how sumoylation is involved in nuclear biological processes and functions. Two interesting questions about sumoylation have emerged: (1) how many SUMO substrates exist in mammalian proteomes, such as human and mouse, (2) and what are their functions and how are they involved in a variety of biological processes? To address these two questions,we present an in silico genome-scale analysis for SUMO substrates in human. Based on the pattern recognition and phylogenetic conservation, we retrieved a list of 2683 potential SUMO substrates conserved in both human and mouse. Then, by functional enrichment analysis, we surveyed the over-represented GO terms and functional domains of them against the whole human proteome. Besides the consistence between our analyses and in vivo or in vitro work, the in silico predicted candidates also point to several potential roles of sumoylation, e.g., perception of sound. These potential SUMO substrates in human are of great value for further in vivo or in vitro experimental analysis.

Animals↗
PubMed1994

Overview: protein palmitoylation in the nervous system: current views and unsolved problems.

Palmitoylation refers to a dynamic post-translational modification of proteins involving the covalent attachment of long-chain fatty acids to the side chains of cysteine, threonine or serine residues. In recent years, palmitoylation has been identified as a widespread modification of both viral and cellular proteins. Because of its dynamic nature, protein palmitoylation, like phosphorylation, appears to have a crucial role in the functioning of the nervous system. Several important questions regarding the post-translational acylation of cysteine residues in proteins are briefly discussed: (a) What are the molecular mechanisms involved in dynamic acylation? (b) What are the determinants of the fatty acid specificity and the structural requirements of the acceptor proteins? (c) What are the physiological signals regulating this type of protein modification, and (d) What is the biological role(s) of this reaction with respect to the functioning of specific nervous system proteins? We also present the current experimental obstacles that have to be overcome to fully understand the biology of this dynamic modification.

Amino Acid Sequence↗
PubMed2004

GPS: a novel group-based phosphorylation predicting and scoring method.

Protein phosphorylation is an important reversible post-translational modification of proteins, and it orchestrates a variety of cellular processes. Experimental identification of phosphorylation site is labor-intensive and often limited by the availability and optimization of enzymatic reaction. In silico prediction may facilitate the identification of potential phosphorylation sites with ease. Here we present a novel computational method named GPS: group-based phosphorylation site predicting and scoring platform. If two polypeptides differ by only two consecutive amino acids, in particular when the two different amino acids are a conserved pair, e.g., isoleucine (I) and valine (V), or serine (S) and threonine (T), we view these two polypeptides bearing similar 3D structures and biochemical properties. Based on this rationale, we formulated GPS that carries greater computational power with superior performance compared to two existing phosphorylation sites prediction systems, ScanSite 2.0 and PredPhospho. With database in public domain, GPS can predict substrate phosphorylation sites from 52 different protein kinase (PK) families while ScanSite 2.0 and PredPhospho offer at most 30 PK families. Using PKA as a model enzyme, we first compared prediction profiles from the GPS method with those from ScanSite 2.0 and PredPhospho. In addition, we chose an essential mitotic kinase Aurora-B as a model enzyme since ScanSite 2.0 and PredPhospho offer no prediction. However, GPS offers satisfactory sensitivity (94.44%) and specificity (97.14%). Finally, the accuracy of phosphorylation on MCAK predicted by GPS was validated by experimentation, in which six out of seven predicted potential phosphorylation sites on MCAK (Q91636) were experimentally verified. Taken together, we have generated a novel method to predict phosphorylation sites, which offers greater precision and computing power over ScanSite 2.0 and PredPhospho.

Algorithms↗
PubMed2005

Polycystic liver disease is a disorder of cotranslational protein processing.

Autosomal-dominant polycystic liver disease (PCLD) is a rare disorder that is characterized by the progressive development of fluid-filled biliary epithelial cysts in the liver. Positional cloning has identified two genes that are mutated in patients with polycystic liver disease, PRKCSH and SEC63, which encode the beta-subunit of glucosidase II and Sec63, respectively. Both proteins are components of the molecular machinery involved in the translocation, folding and quality control of newly synthesized glycoproteins in the endoplasmic reticulum. Most mutations are truncating and probably lead to a complete loss of the corresponding proteins and the defective processing of a key regulator of biliary cell growth. The finding that PCLD is caused by proteins involved in oligosaccharide processing was unexpected and implicates a new avenue for research into neocystogenesis, and might ultimately result in the identification of novel therapeutic drugs.

Calcium-Binding Proteins↗
PubMed2002

A micro-scale process for high-throughput expression of cDNAs in the yeast Saccharomyces cerevisiae.

Methods have been developed aimed at applying at high-throughput technology for expression of cloned cDNAs in yeast. Yeast is a eukaryotic host, which produces soluble recombinant proteins and is capable of introducing post-translational modifications of protein. It is, thus, an appropriate expression system both for the routine expression of various cDNAs or protein domains and for the expression of proteins, which are not correctly expressed in Escherichia coli. Here, we describe a standard system in Saccharomyces cerevisiae, based on a vector for intracellular protein expression, where the gene products are fused to specific peptide sequences (tags). These epitope tags, the N-terminal His(6) tag and the C-terminal StrepII tag, allow subsequent immunological identification and purification of the gene products by a two-step affinity chromatography. This method of dual-tagged recombinant protein purification eliminates contamination by degraded protein products. A miniaturization of the procedures for cloning, expression, and detection was performed to allow all steps to be carried out in 96-well microtiter plates. The system is, thus, suitable for automation. We were able to analyze the simultaneous protein expression of a large number of cDNA clones due to the highly parallel approach of protein production and purification. The microtiter plate technology format was extended to quantitative analysis. An ELISA-based assay was developed that detects StrepII-tagged proteins. The application of this high-throughput expression system for protein production will be a useful tool for functional and structural analyses of novel genes, identified by the Human Genome Project and other large-scale sequencing projects.

Base Sequence↗
PubMed2005

DNA-induced dimerization of poly(ADP-ribose) polymerase-1 triggers its activation.

In response to DNA strand breaks in the genome of higher eukaryotes, poly(ADP-ribose)polymerase 1 (PARP-1) catalyses the covalent attachment of ADP-ribose units from NAD(+) to various nuclear acceptor proteins including PARP-1 itself. This post-translational modification affecting proteins involved in chromatin architecture and in DNA repair plays a critical role in cell survival as well as in caspase-independent cell death. Although PARP-1 has been best-studied for its role in genome stability, several recent reports have demonstrated its role in the regulation of transcription. In this study, fluorescence spectroscopy and biochemical techniques are used to investigate the association of the amino-terminal DNA-binding domain of human PARP-1 (hPARP-1 DBD) with various DNA substrates, characterized by different DNA ends and sequence features (5'- or 3'-recessed end, double strands, telomeric repeats, and the palindromic sequence of a Not I restriction site). The correlation between the binding mode of hPARP-1 DBD to the DNA oligoduplexes and the enzymatic activation of hPARP-1 is analyzed. We show that hPARP-1 DBD binds a 5'-recessed DNA end cooperatively with a stoichiometry of two proteins per DNA molecule. In contrast, a 1:1 stoichiometry is found in the presence of a 3'-recessed end and double-strand DNA. A palindromic structure like the Not I restriction site is shown to induce protein dimerization and high enzymatic activation, suggesting that it can represent a recognition element for hPARP-1 in undamaged cells. Protein dimerization is found to be a requisite for high enzymatic activity. Taken together, our data allow further characterization of the features of hPARP-1 recognition in damaged cells and bring additional evidence that hPARP-1 may also play a role in undamaged cells.

Base Sequence↗
PubMed2002

The Sulfinator: predicting tyrosine sulfation sites in protein sequences.

UNLABELLED: Protein tyrosine sulfation is an important post-translational modification of proteins that go through the secretory pathway. No clear-cut acceptor motif can be defined that allows the prediction of tyrosine sulfation sites in polypeptide chains. The Sulfinator is a software tool that can be used to predict tyrosine sulfation sites in protein sequences with an overall accuracy of 98%. Four different Hidden Markov Models were constructed, each of them specialized to recognize sulfated tyrosine residues depending on their location within the sequence: near the N-terminus, near the C-terminus, in the center of a window with a size of at least 25 amino acids, as well as in windows containing several tyrosine residues. AVAILABILITY: The Sulfinator is accessible at (http://www.expasy.org/tools/sulfinator/). SUPPLEMENTARY INFORMATION: Sulfinator documentation is accessible at (http://www.expasy.org/tools/sulfinator/sulfinator-doc.html).

Amino Acid Sequence↗
PubMed2004

Hub1 is an essential ubiquitin-like protein without functioning as a typical modifier in fission yeast.

Hub1 exhibits 23% sequence identity to ubiquitin. However, Hub1 lacks the C-terminal Gly, which is essential for covalent attachment to target protein(s) of ubiquitin and other ubiquitin-like (UBL) modifiers. Instead, Hub1 proteins in all eukaryotes retain the di-Tyr just before a single variable residue at the C-terminus, so one intriguing question is whether Hub1 could be linked to substrate through the conserved Tyr or not. Here we studied Hub1 in Schizosaccharomyces pombe. Gene disruption experiment revealed that hub1+ is essential. Remarkably, the mutant cells harbouring Hub1 lacking the di-Tyr could grow similar to wild-type cells, indicating that the di-Tyr is dispensable for the essential function of Hub1. Moreover, we could not observe cleavage of Flag-tag fused with C-terminus of Hub1. It suggests that the processing for conjugation via conserved Tyr is not likely to occur in Hub1, and Hub1 is a novel class of the UBL protein family. Finally, we isolated a temperature-sensitive allele, hub1-1. This temperature sensitivity could be suppressed by overproduction of Rpb10 or Snu66, the former of which is one of the common subunits of the RNA polymerases and the other is the component of the spliceosome. We also observed that pre-mRNA splicing was impaired in hub1-1.

Alleles↗
PubMed2006

Structural and mechanistic features of protein O glycosylation linked to CD8+ T-cell apoptosis.

CD8+ T-cell apoptosis is essential for the contraction phase of the immune response, yet the initiating signals and precise pathways involved are unresolved. The ST3Gal-I sialyltransferase is a candidate mechanistic component and catalyzes sialic acid addition to core 1 O-glycans during protein O glycosylation. ST3Gal-I inactivation or enzymatic removal of its product renders CD8+ T cells, but not CD4+ T cells, susceptible to apoptosis by differential cross-linking of O-glycoproteins in the absence of interleukin-2 and T-cell receptor (TCR) signaling. This results in caspase activation, DNA fragmentation, and phosphatidylserine externalization prior to cell death. We further show that ST3Gal-I function is regulated by a posttranscriptional mechanism operating distal to Golgi core 2 O glycosylation and is invariably linked to CD8+ T-cell contraction following viral (lymphocytic choriomeningitis virus) infection and bacterial (staphylococcal enterotoxin B) antigen immunization. The mechanism does not involve the ST3Gal-I substrate CD43 or core 2 O-glycan induction and overcomes the ability of Bcl-2 to inhibit the contraction phase in vivo. Loss of ST3Gal-I function further reduces Bim-deficient CD8+ T-cell accumulation without diminishing apoptotic sensitivity. We propose that an endogenous lectin activates an apoptotic pathway constructed in CD8+ T cells following TCR stimulation and enables contraction upon attenuation of immune signaling.

Animals↗
PubMed2000

COS cells expression cloning of tyrosine-phosphorylated proteins by immunocytochemistry.

Tyrosine phosphorylation is an important post-translational modification of proteins, essential in many aspects of the cell economy, particularly in signal transduction pathways. Despite the importance of protein tyrosine phosphorylation, the approaches available for molecular cloning remain limited. We have developed a COS cell-based eukaryotic expression cloning procedure for phosphotyrosine-containing proteins by immunocytochemistry of cell monolayers. The approach takes advantage of the low basal levels of tyrosine phosphorylated, robust transient expression, availability of specific antibodies against tyrosine-phosphorylated residues, and rescue of episomal DNA after immunocytochemistry. The technique is validated by cloning the rat proto-oncogene c-fgr in its tyrosine-phosphorylated form out of a rat kidney cDNA library containing over 10(6) primary recombinants. This technique set the grounds for expression cloning of tyrosine-phosphorylated proteins in eukaryotic cells, and it is anticipated that further modifications and refinements will allow the identification of protein tyrosine phosphatase substrates.

Animals↗
PubMed1987

Interactions of aging and environmental agents: the gerontological perspective.

This article attempts to briefly review the current status of knowledge of biogerontology as a basis for a beginning set of discussions on the potential roles of environmental agents in the modulation of rates of aging in man. The article has tried to summarize "what the toxicologist should know about gerontology," whereas a companion review, by Dr. Jerry Williams, summarizes "what the gerontologist should know about toxicity." The review begins by defining some basic terminology, including introduction of the term "gerontogens" for those putative environmental agents that may in fact modulate the times of onset and/or rates of development of specific aspects of the senescent phenotype. That phenotype is then briefly described from the level of populations to the level of molecules, including an organ systems approach with emphasis on what is observed in humans. Among the important points that emerge from this description are: the variety of phenotypic alterations; the multiplicity of phenotypic alterations in any one aged individual; the special vulnerability in humans of the arterial system; the loss of proliferative homeostasis; the decline in the efficiency of enzyme adaptation; the decline in protein synthesis; the increasing evidence of post-translational modifications of proteins and DNA; the evidence for chromosomal instability (and, therefore, the potential importance of environmental clastogens). Arguments for a genetic basis for differential rates of aging are then developed, and a number of "genotropic" theories of aging are tabulated (i.e., those theories that invoke crucial roles for alterations of the genomes of somatic cells, whether changes in gene expression, gene structure, or both). It is concluded that no single theory is supported by a preponderance of evidence. Because aging appears to involve numerous processes and is highly polygenic in its determination, it is likely that a number of different mechanisms are responsible for the phenotypic alterations and that different individuals exhibit various patterns of aging based upon their specific inheritance and environmental experiences. At this early stage in the development of experimental approaches to the study of environmental-genetic interactions in the genesis of different aspects of senescence, the most vital issue involves the question of suitable model systems. Clearly, a number of different model systems will be required. A selection of these is tabulated based upon their amenability to a reasonable degree of genetic definition and genetic manipulation. Only by controlling the genotype can the environmental influences be properly assessed.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗