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Results for “Protein Modification, Translational”

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PubMed1998

Glycoaffinity chromatography and biological recognition.

The potential of bioaffinity chromatography as a tool for study of biological recognition mechanisms is gaining increasing recognition. Biochromatographic methods allow the separation of proteins according to both the structure of their polypeptidic chain and their post-translational modifications. Among the various post-translational modifications which proteins undergo, glycosylation has conducted to the development of original methods (glycotechnologies). This review discusses the applications of glycotechnologies in bioaffinity chromatography, and particularly the use of biochromatography to elucidate mechanisms involved in glycobiology.

Binding Sites↗
PubMed2006

Proteomics in clinical trials and practice: present uses and future promise.

The study of clinical proteomics is a promising new field that has the potential to have many applications, including the identification of biomarkers and monitoring of disease, especially in the field of oncology. Expression proteomics evaluates the cellular production of proteins encoded by a particular gene and exploits the differential expression and post-translational modifications of proteins between healthy and diseased states. These biomarkers may be applied towards early diagnosis, prognosis, and prediction of response to therapy. Functional proteomics seeks to decipher protein-protein interactions and biochemical pathways involved in disease biology and targeted by newer molecular therapeutics. Advanced spectrometry technologies and new protein array formats have improved these analyses and are now being applied prospectively in clinical trials. Further advancement of proteomics technology could usher in an era of personalized molecular medicine, where diseases are diagnosed at earlier stages and where therapies are more effective because they are tailored to the protein expression of a patient's malignancy.

Clinical Trials as Topic↗
PubMed2001

O-glycosylation of the mucin type.

While only about ten percent of the databank entries are defined as glycoproteins, it has been estimated recently that more than half of all proteins are glycoproteins. Mucin-type O-glycosylation is a widespread post-translational modification of proteins found in the entire animal kingdom, but also in higher plants. The structural complexity of the chains initiated by O-linked GalNAc exceeds that of N-linked chains by far. The process during which serine and threonine residues of proteins become modified is confined to the cis to trans Golgi compartments. The initiation of this process by peptidyl GalNAc-transferases is ruled by the sequence context of putative O-glycosylation sites, but also by epigenetic regulatory mechanisms, which can be mediated by enzyme competition. The cellular repertoir of glycosyltransferases with their distinct donor sugar and acceptor sugar specificities, their sequential action at highly-ordered surfaces, and their localizations in subcompartments of the Golgi finally determine the cell-specific O-glycosylation profile. Dramatic alterations of the glycosylation machinery are observed in cancer cells, resulting in aberrantly O-glycosylated proteins that expose previously masked peptide motifs and new antigenic targets. The functional aspects of O-linked glycans, which comprise among many others their potential role in sorting and secretion of glycoproteins, their influence on protein conformation, and their multifarious involvement in cell adhesion and immunological processes, appear as complex as their structures.

Animals↗
PubMed2005

Production of constitutively acetylated recombinant p53 from yeast and Escherichia coli by tethered catalysis.

Post-translational modification of proteins is a dynamic way of generating new protein-protein interaction interfaces that are critical for signaling networks in diverse cellular functions. Purified recombinant proteins frequently lack these signature modifications. Using the tumor suppressor p53 as the model protein, we present here a tethered catalysis approach for the production of acetylated p53 in vivo. P53 is a major tumor suppressor protein that protects the cell from various oncogenic stresses. Upon DNA damage, p53 is stabilized and activated by a plethora of post-translational modifications, including acetylation. Here, we show that constitutively acetylated p53 can be expressed and purified from both yeast and Escherichia coli. This method is highly suitable for studying protein-protein interactions in the conventional yeast two-hybrid screen that requires a constitutively acetylated state of p53. Furthermore, effective production and purification of acetylated p53 from E. coli supports future biochemical and structural characterization. The method described in this work can be applied to other proteins and modifications, and thus has widespread use in the fields of signal transduction and proteomic research.

Acetylation↗
PubMed2001

The role of mass spectrometry in proteome studies.

Mass spectrometry (MS) is an important tool in modern protein chemistry. In proteome analyses the expression of hundreds or thousands of proteins can be monitored at the same time. First, complex protein mixtures are separated by two-dimensional gel electrophoresis (2-DE) and then individual proteins are identified by using MS followed by database searches. Recent developments in this field have made it possible to do automated, high-throughput protein identification that is needed in proteome analyses. MS can also be used to characterize post-translational modifications in proteins and to study protein complexes. This review will introduce the current MS methods used in proteome studies, and discuss their advantages and disadvantages. New instrumental MS developments are also presented that are useful in these analyses.

Databases, Protein↗
PubMed2004

Protein modification by SUMO.

Small ubiquitin-related modifier (SUMO) family proteins function by becoming covalently attached to other proteins as post-translational modifications. SUMO modifies many proteins that participate in diverse cellular processes, including transcriptional regulation, nuclear transport, maintenance of genome integrity, and signal transduction. Reversible attachment of SUMO is controlled by an enzyme pathway that is analogous to the ubiquitin pathway. The functional consequences of SUMO attachment vary greatly from substrate to substrate, and in many cases are not understood at the molecular level. Frequently SUMO alters interactions of substrates with other proteins or with DNA, but SUMO can also act by blocking ubiquitin attachment sites. An unusual feature of SUMO modification is that, for most substrates, only a small fraction of the substrate is sumoylated at any given time. This review discusses our current understanding of how SUMO conjugation is controlled, as well as the roles of SUMO in a number of biological processes.

Amino Acid Sequence↗
PubMed1998

Polarized sorting of nicotinic acetylcholine receptors to the postsynaptic membrane in Torpedo electrocyte.

Several regulatory mechanisms contribute to the accumulation and maintenance of high concentrations of acetylcholine receptors (AChR) at the postsynaptic membrane of the neuromuscular junction, including compartmentalized gene transcription, targeting, clustering and anchoring to the cytoskeleton. The targeting of the AChR to the postsynaptic membrane is likely to involve a polarized sorting in the exocytic pathway. In this work, we used the electrocyte of Torpedo marmorata electric organ to study the intracellular trafficking of neosynthesized AChR and its delivery to the postsynaptic membrane. Gradient centrifugation and immunoisolation techniques have led to the isolation of two populations of post-Golgi transport vesicles (PGVs) enriched in proteins of either the innervated (AChR) or non-innervated (Na,K-ATPase) membrane domains of the cell. Immunolabelling of these vesicles at the EM level disclosed that very few PGVs contained both proteins. In AChR-enriched vesicles, high sialylation of AchR molecules, an expected post-translational modification of proteins exiting the trans-Golgi network, and the presence of a marker of the exocytic pathway (Rab6p), indicate that these vesicles are carriers engaged in the Golgi-to-plasma membrane transport. These data suggest that AChR and Na,K-ATPase are sorted intracellularly most likely within the trans-Golgi network. Furthermore, EM analysis and immunogold-labelling experiments provided in situ evidence that the AChR-containing PGVs are conveyed to the postsynaptic membrane, possibly by a microtubule-dependent transport mechanism. Our data therefore provide the first evidence that the targeting of receptors for neurotransmitters to synaptic sites could be contributed by intracellular sorting and polarized delivery in the exocytic pathway.

Animals↗
PubMed2004

A proteomic study of resistance to deoxycholate-induced apoptosis.

The development of apoptosis resistance appears to be an important factor in colon carcinogenesis. To gain an understanding of the molecular pathways altered during the development of apoptosis resistance, we selected three cell lines for resistance to induction of apoptosis by deoxycholate, an important etiologic agent in colon cancer. We then evaluated gene expression levels for 825 proteins in these resistant lines, compared with a parallel control line not subject to selection. Eighty-two proteins were identified as either over-expressed or under-expressed in at least two of the resistant lines, compared with the control. Thirty-five of the 82 proteins (43%) proved to have a known role in apoptosis. Of these 35 proteins, 21 were over-expressed and 14 were under-expressed. Of those that were over-expressed 18 of 21 (86%) are anti-apoptotic in some circumstances, of those that were under-expressed 11 of 14 (79%) are pro-apoptotic in some circumstances. This finding suggests that apoptosis resistance during selection among cultured cells, and possibly in the colon during progression to cancer, may arise by constitutive over-expression of multiple anti-apoptotic proteins and under-expression of multiple pro-apoptotic proteins. The major functional groups in which altered expression levels were found are post-translational modification (19 proteins), cell structure (cytoskeleton, microtubule, actin, etc.) (17 proteins), regulatory processes (11 proteins) and DNA repair and cell cycle checkpoint mechanisms (10 proteins). Our findings, overall, bear on mechanisms by which apoptosis resistance arises during progression to colon cancer and suggest potential targets for cancer treatment. In addition, assays of normal-appearing mucosa of colon cancer patients, for over- or under-expression of genes found to be altered in our resistant cell lines, may allow identification of early biomarkers of colon cancer risk.

Apoptosis↗
PubMed2001

Barley lipid transfer protein, LTP1, contains a new type of lipid-like post-translational modification.

In plants a group of proteins termed nonspecific lipid transfer proteins are found. These proteins bind and catalyze transfer of lipids in vitro, but their in vivo function is unknown. They have been suggested to be involved in different aspects of plant physiology and cell biology, including the formation of cutin and involvement in stress and pathogen responses, but there is yet no direct demonstration of an in vivo function. We have found and characterized a novel post-translational modification of the barley nonspecific lipid transfer protein, LTP1. The protein-modification bond is of a new type in which an aspartic acid in LTP1 is bound to the modification through what most likely is an ester bond. The chemical structure of the modification has been characterized by means of two-dimensional homo- and heteronuclear nuclear magnetic resonance spectroscopy as well as mass spectrometry and is found to be lipid-like in nature. The modification does not resemble any standard lipid post-translational modification but is similar to a compound with known antimicrobial activity.

Amino Acids↗
PubMed2003

Acetylation of the HIV-1 Tat protein: an in vitro study.

In the last few years, the understanding of lysine acetylation as a regulatory post-translational modification of proteins in cell signalling cascades has increased. It is now known that not only histones but also non-histone factors can serve as substrates of different acetyltransferase enzymes. Acetylated lysine residues in non-histone factors are often identified using radioactive labelling experiments and immunochemical analysis of synthetic peptides. In this study of the human immunodeficiency virus 1 (HIV-1) Tat protein, we demonstrate the benefits of matrix-assisted laser desorption/ionisation mass spectrometry, proteolytic digestion and Edman sequencing for the mapping of acetylation sites. We confirmed that the HIV-1 Tat protein is acetylated in vitro by the acetyltransferase p300 at a specific lysine residue at position 50 in its RNA binding region. Furthermore, we showed that the Tat cysteine-rich region is acetylated at multiple cysteine residues in the absence of enzyme. Since this non-enzymatic cysteine acetylation occurs independently from the surrounding peptide sequence, we consider the presence of cysteine residues in acetylated peptides an important factor for the interpretation of in vitro acetylation assays in general.

Acetylation↗
PubMed1995

Reversion of a human tumour cell line containing oncogenic p21ras is associated with a defect in the post-translational processing of the ras protein.

Correct post-translational modifications of the ras proteins are essential for their membrane localisation and functioning. The flat revertant cell lines 1aCB and 8b, derived from the human bladder carcinoma cell line EJ, contain the transforming gene V12Ha-ras and are resistant to retransformation by ras protein or DNA, but still do require the presence of ras for proliferation. Both revertant cell lines demonstrated reduced levels of membrane associated p21ras when compared to their parental EJ cell lines. This reduced level in 1aCB was reflected by an increase in nuclear associated p21ras, as seen by immunofluorescence of endogenous and introduced ras. In addition, 1aCB had a reduced ratio of ras in the detergent to aqueous phases after triton X114 partitioning, suggesting a defect in Step I processing of the p21ras in the cell line. This was not however due to defects in the Step I enzymes farnesyltransferase or carboxymethyltransferase whose activities were not reduced.

Alkyl and Aryl Transferases↗
PubMed2006

Investigation on glycosylation patterns of proteins from human liver cancer cell lines based on the multiplexed proteomics technology.

Glycosylation, a very important post-translational modification of proteins, is increasingly coming into notice. However, large-scale, throughput investigations on glycosylated proteins are few. We applied a sensitive and fast fluorescence-based multiplexed proteomics (MP) technology which included two-dimensional gel electrophoresis (2-DE) followed by the fluorescence staining of glycoprotein and mass spectrometry identification for the purpose of constructing glycoprotein databases of the typical human hepatocellular carcinoma cell lines including Hep3B cell line without metastasis and MHCC97H with highly metastatic potential as well as the control non-tumor Chang liver cell. 74+/-2 (n=3), 78+/-3 (n=3) and 72+/-5 (n=3) glycoprotein spots were detected on 2-DE gels from Chang liver, Hep3B and MHCC97H cell sample using this MP technique, respectively. In all, 80 glycoproteins from three cell lines were successfully identified via peptide mass profiling using MALDI-TOF-MS/MS and the identified glycoproteins were annotated to our databases. In addition, we also found the glycosylation pattern differences among these three cell lines. The protein glycosylation alteration would be have great significance for the diagnosis of HCC and prediction of its metastasis. This study described the construction of glycosylation patterns of proteins and glycoproteome databases of human liver cells by the novel technological platform. The glycoproteome databases also provide essential basis for following study.

Animals↗
PubMed2006

Protein arginine methylation in lymphocyte signaling.

Methylation of arginine is an additional option within the repertoire of post-translational modifications that proteins utilize for their communication with other partner proteins and nucleic acids, which ultimately contributes to cellular functions. Recent studies reveal that protein arginine methylation is more common and widespread than previously thought and that it is implicated in a number of key cellular processes including signal transduction. Two recent investigations have propelled this new world of protein modification into the immunological community by showing that TCR and CD28 signaling exploit this pathway. In contrast to other protein modifications utilized in intracellular signaling, arginine methylation seems to be long-lasting, raising interesting questions as to when, where and for what reason it can be utilized in the lymphocyte differentiation processes.

Animals↗
PubMed2005

Transcriptional profiling of a mouse model for Lafora disease reveals dysregulation of genes involved in the expression and modification of proteins.

Lafora's progressive myoclonus epilepsy (Lafora disease: LD) is caused by mutations in the EPM2A or NHLRC1 gene, but cellular mechanisms of the pathogenesis remain unclear. In an attempt to understand and elucidate the disease pathway, we have investigated the global gene expression profile in a mouse model for LD that developed a phenotype similar to that observed in human patients, including presence of Lafora bodies, neurodegeneration and profound neurological disturbances. We found 62 differentially expressed genes in the Epm2a knockout mice brains. These genes encode factors involved in protein catabolism, phosphatase, transcription factors, and molecules involved in protein translation, and homeostasis. The two largest functional groups of mRNAs that showed altered expression were predicted to be involved in post-translational modification of proteins and transcriptional regulation, suggesting that defects in protein activity and/or turnover may be the key trigger in the pathophysiology of LD. Furthermore we show that changes in gene expression are not limited to brain and are seen in other organs that develop Lafora bodies. Our study may provide valuable insights into the pathophysiology of LD and may aid in developing potential therapeutic targets.

Animals↗
PubMed2006

Selenoprotein synthesis: UGA does not end the story.

It is well established that the beneficial effects of the trace element selenium are mediated by its major biological product, the amino acid selenocysteine, present in the active site of selenoproteins. These fulfill different functions, as varied as oxidation-reduction of metabolites in bacteria, reduction of reactive oxygen species, control of the redox status of the cell or thyroid hormone maturation. This review will focus on the singularities of the selenocysteine biosynthesis pathway and its unique incorporation mechanism into eukaryal selenoproteins. Selenocysteine biosynthesis from serine is achieved on tRNA(Sec) and requires four proteins. As this amino acid is encoded by an in-frame UGA codon, otherwise signaling termination of translation, ribosomes must be told not to stop at this position in the mRNA. Several molecular partners acting in cis or in trans have been identified, but their knowledge has not enabled yet to firmly establish the molecular events underlying this mechanism. Data suggest that other, so far uncharacterized factors might exist. In this survey, we attempted to compile all the data available in the literature and to describe the latest developments in the field.

Models, Molecular↗
PubMed2006

Antibodies against nitric oxide damaged poly L-tyrosine and 3-nitrotyrosine levels in systemic lupus erythematosus.

Alterations in the amino acid structure or sequence can generate neo-epitopes from self-proteins causing autoaggressive immune attack. Reactive nitrogen species are an important factor that induces post-translational modification of proteins by cellular reduction and oxidation mechanism; cysteinyl-nitrosylation or tyrosine nitration leading to potentially pathogenic pathways. It was thought of interest to investigate the immunogenicity of nitrated poly L-tyrosine vis-á-vis its possible role in the induction of antibodies in systemic lupus erythematosus (SLE). Commercially available poly L-tyrosine was exposed to nitrating species and the damage was monitored by UV spectroscopy and alkaline gel electrophoresis. The results indicated the formation of 3-nitrotyrosine. Nitrated poly L-tyrosine induced higher titre antibodies as compared to the native form. Nitrated poly L-tyrosine was recognized by the autoantibodies present in the sera of patients suffering from SLE by enzyme immunoassays and band shift assay. The possible role of nitrated self-proteins has been discussed in the production of circulating anti-DNA antibodies in SLE.

Antibody Specificity↗
PubMed2003

Calmodulin-dependent and -independent activation of endothelial nitric-oxide synthase by heat shock protein 90.

Endothelial nitric oxide synthase (eNOS), which generates the endogenous vasodilator, nitric oxide (NO), is highly regulated by post-translational modifications and protein interactions. Heat shock protein 90 (HSP90) binds directly to eNOS, augmenting NO production. We have used purified proteins to characterize further the mechanism by which HSP90 increases eNOS activity at low (100 nm) and high (10 microm) Ca(2+) levels. In the presence of calmodulin (CaM), HSP90 increased eNOS activity dose dependently at both low and high Ca(2+) concentrations. This effect was abolished by the specific HSP90 inhibitor geldanamycin (GA) at both calcium concentrations. The EC(50) values of eNOS for both Ca(2+) and CaM were decreased in the presence of HSP90. HSP90 also significantly increased the rate of NADPH-dependent cytochrome c reduction by eNOS at both low and high Ca(2+) concentrations. HSP90 bound to eNOS in a dose-dependent manner, and the amount of bound HSP90 also increased with increasing Ca(2+)/CaM. At 100 nm Ca(2+), HSP90 promoted dose-dependent CaM binding to eNOS that was fully inhibitable by GA. At high calcium, HSP90 did not affect CaM binding to eNOS, but GA inhibited HSP90 binding to eNOS. At high Ca(2+), HSP90 caused the V(max) of eNOS for l-arginine to increase by 2-fold, but the K(m) of eNOS was unchanged. HSP90 bound preferentially to CaM-prebound eNOS and significantly increased both its NO synthesis and reductase activities. These data support that HSP90 promotes eNOS activity by two mechanisms: (i) a CaM-dependent mechanism operative at low Ca(2+) concentrations, characterized by an increase in the affinity of eNOS for CaM and (ii) a CaM-independent mechanism apparent at high Ca(2+) concentrations, characterized by stimulation of eNOS reductase activity without further change in CaM binding. These studies contribute to our understanding of eNOS activation by HSP90 and provide a basis for in vitro studies of other eNOS-interacting proteins.

Animals↗