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PubMed1994

The amino acid sequence of glutamate dehydrogenase from Pyrococcus furiosus, a hyperthermophilic archaebacterium.

The complete amino acid sequence of glutamate dehydrogenase from the archaebacterium Pyrococcus furiosus has been determined. The sequence was reconstructed by automated sequence analysis of peptides obtained after cleavage with cyanogen bromide, Asp-N endoproteinase, trypsin, or pepsin. The enzyme subunit is composed of 420 amino acid residues yielding a molecular mass of 47,122 D. In the recently determined primary structure of glutamate dehydrogenase from another thermophilic archaebacterium, Sulfolobus solfataricus, the presence of some methylated lysines was detected and the possible role of this posttranslational modification in enhancing the thermostability of the enzyme was discussed (Maras, B., Consalvi, V., Chiaraluce, R., Politi, L., De Rosa, M., Bossa, F., Scandurra, R., and Barra, D. (1992), Eur. J. Biochem. 203, 81-87). In the primary structure reported here, such posttranslational modification has not been found, indicating that the role of lysine methylation should be revisited. Comparison of the sequence of glutamate dehydrogenase from Pyrococcus furiosus with that of S. solfataricus shows a 43.7% similarity, thus indicating a common evolutionary pathway.

Amino Acid Sequence↗
PubMed2002

The Th1-specific costimulatory molecule, m150, is a posttranslational isoform of lysosome-associated membrane protein-1.

In an earlier report, we had shown a 150-kDa protein termed as M150, isolated from the surface of activated macrophages, to possess costimulatory activity for CD4(+) T cells. Significantly, this protein was found to specifically elicit Th1 responses. In this study, we characterize M150, which belongs to a unique subset of the lysosome-associated membrane protein-1 glycoprotein. Interestingly, the costimulatory activity of M150 depends on its posttranslational modification, which has a distinct glycosylation pattern restricted to macrophages. Furthermore, it has been demonstrated that in addition to stimulating Th1-specific responses, M150 is also capable of driving differentiation of naive CD4(+) T cells into the Th1 subset. This altered posttranslational modification of housekeeping protein appears to represent a novel pathway by which APCs can additionally regulate T cell responses.

Amino Acid Sequence↗
PubMed2006

Posttranslational nitrotyrosination of alpha-tubulin induces cell cycle arrest and inhibits proliferation of vascular smooth muscle cells.

Hyperproliferation of vascular smooth muscle cells is a hallmark of atherosclerosis and related vascular complications. Microtubules are important for many aspects of mammalian cell responses including growth, migration and signaling. alpha-Tubulin, a component of the microtubule cytoskeleton, is unique amongst cellular proteins in that it undergoes a reversible posttranslational modification whereby the C-terminal tyrosine residue is removed (Glu-tubulin) and re-added (Tyr-tubulin). Whereas the reversible detyrosination/tyrosination cycle of alpha-tubulin has been implicated in regulating various aspects of cell biology, the precise function of this posttranslational modification has remained poorly characterized. Herein, we provide evidence suggesting that alpha-tubulin detyrosination is a required event in the proliferation of vascular smooth muscle cells. Proliferation of rat aortic smooth muscle cells in response to serum was temporally associated with the detyrosination of alpha-tubulin, but not acetylation of alpha-tubulin; Glu-tubulin reached maximal levels between 12 and 18h following cell cycle initiation. Inclusion of 3-nitro-l-tyrosine (NO(2)Tyr) in the culture medium resulted in the selective nitrotyrosination of alpha-tubulin, that was paralleled by decreased elaboration of Glu-tubulin, decreased expression of cyclins A and E, decreased association of the microtubule plus-end binding protein EB1, and inhibited cell proliferation. Nitrotyrosination of alpha-tubulin did not induce necrotic or apoptotic death of rat aortic smooth muscle cells, but instead led to cell cycle arrest at the G(1)/S boundary coincident with decreased DNA synthesis. Collectively, these results suggest that the C-terminus of alpha-tubulin and its detyrosination are functionally important as a molecular switch that regulates cell cycle progression in vascular smooth muscle cells.

Animals↗
PubMed2006

MassSorter: a tool for administrating and analyzing data from mass spectrometry experiments on proteins with known amino acid sequences.

BACKGROUND: Proteomics is the study of the proteome, and is critical to the understanding of cellular processes. Two central and related tasks of proteomics are protein identification and protein characterization. Many small laboratories are interested in the characterization of a small number of proteins, e.g., how posttranslational modifications change under different conditions. RESULTS: We have developed a software tool called MassSorter for administrating and analyzing data from peptide mass fingerprinting experiments on proteins with known amino acid sequences. It is meant for small scale mass spectrometry laboratories that are interested in posttranslational modifications of known proteins. Several experiments can be compared simultaneously, and the matched and unmatched peak values are clearly indicated. The hits can be sorted according to m/z values (default) or according to the sequence of the protein. Filters defined by the user can mark autolytic protease peaks and other contaminating peaks (keratins, proteins co-migrating with the protein of interest, etc.). Unmatched peaks can be further analyzed for unexpected modifications by searches against a local version of the UniMod database. They can also be analyzed for unexpected cleavages, a highly useful feature for proteins that undergo maturation by proteolytic cleavage, creating new N- or C-terminals. Additional tools exist for visualization of the results, like sequence coverage, accuracy plots, different types of statistics, 3D models, etc. The program and a tutorial are freely available for academic users at http://www.bioinfo.no/software/massSorter. CONCLUSION: MassSorter has a number of useful features that can promote the analysis and administration of MS-data.

Algorithms↗
PubMed1997

P-selectin glycoprotein ligand-1 is essential for adhesion to P-selectin but not E-selectin in stably transfected hematopoietic cell lines.

P-selectin (CD62P) is a member of the selectin family of adhesion molecules involved in the regulation of leukocyte traffic. P-selectin glycoprotein ligand-1 (PSGL-1) is a mucin-like molecule that is thought to be a primary ligand for P-selectin. The interaction of P-selectin with PSGL-1 results in leukocyte rolling and recruitment of leukocytes to sites of inflammation and tissue injury. However, expression of PSGL-1 protein alone is insufficient for binding to P-selectin. Several posttranslational modifications of PSGL-1, including sialylation, sulfation, and fucosylation by alpha 1,3-fucosyltransferase(s) (FucT), are required for functional interaction with P-selectin. Recently, several groups have reported that PSGL-1 might also serve as a ligand for E-selectin. Differential posttranslational modifications of PSGL-1 may determine whether it can interact with either P- or E-selectin or both. To determine whether PSGL-1 is essential for adhesion to P- or E-selectin, we have constructed and analyzed a panel of stably transfected K562 cells. K562 cells express FucT-IV but not FucT-VII or PSGL-1, and do not bind to either E- or P-selectin. K562 cells transfected with PSGL-1 cDNA also did not bind to either P- or E-selectin. Binding to P-selectin occurred only when K562 cells were cotransfected with both FucT-VII and PSGL-1. In contrast, expression of FucT-VII alone was sufficient for E-selectin binding. These data demonstrate that expression of PSGL-1 is not required for adhesion of a stably transfected hematopoietic cell line to E-selectin, and suggest that FucT-IV alone cannot properly modify PSGL-1, expressed in transfected K562 cells, to bind P-selectin.

Carbohydrates↗
PubMed2004

Ubiquitin-dependent regulation of the synapse.

Posttranslational modification of cellular proteins by the covalent attachment of ubiquitin regulates protein stability, activity, and localization. Ubiquitination is rapid and reversible and is a potent mechanism for the spatial and temporal control of protein activity. By sculpting the molecular composition of the synapse, this versatile posttranslational modification shapes the pattern, activity, and plasticity of synaptic connections. Synaptic processes regulated by ubiquitination, as well as ubiquitination enzymes and their targets at the synapse, are being identified by genetic, biochemical, and electrophysiological analyses. This work provides tantalizing hints that neuronal activity collaborates with ubiquitination pathways to regulate the structure and function of synapses.

Animals↗
PubMed2004

Proteomic research: potential opportunities for clinical and physiological investigators.

Proteomics is the comprehensive and systematic study of proteins, which are functional molecules. Although proteins are products of gene expression, there are more proteins than genes due to the posttranslational modifications of proteins, making the study of proteins difficult. Protein expression is tissue specific, and its function is modulated by variety of factors, including other proteins, phosphates, sulfates, carbohydrates, and lipids, as well as other metabolites. Because of the dynamic nature of protein expression and posttranslational modifications, identification and quantification of proteins alone are not sufficient to understand functional changes. Emerging technologies will allow investigators to perform a combination of metabolic labeling and identification as well as quantification and measurement of the synthesis rates of a large number of proteins in a tissue. This offers the opportunity to better understand the regulation of tissue functions. Rapid advances in mass spectrometry, protein purification techniques, isotope labeling of proteins, and bioinformatics are likely to improve our understanding of physiological states and altered functions in diseased states. Such mechanistic information will improve the ability to perform early diagnosis of tumors and other diseases and develop prognostic indexes and novel therapies.

Endocrinology↗
PubMed1989

Biosynthesis of the secreted 24 K isoforms of prolactin.

PRL exists within the mammotroph population in a number of different molecular forms. Several of these forms are best described as isoforms, as they have the same mol wt (24 K), but differ in their net charges. In this study we have used in vitro translation assays to ascertain the number of 24 K translation products of normal pituitary messenger RNA (mRNA), and, finding only one, have used both in vitro translation assays and subcellular fractionation to determine the intracellular site of the posttranslational modification of this single translation product. Translation of mRNA from normal pituitary tissue or GH3 cells resulted in the apparent production of a number of pre-PRLs, but in only a single rough microsome-processed form of PRL, 24 K isoform 2. Longer term translation assays utilizing a variety of isotopes failed to show any evidence for rough microsomal posttranslational modification of isoform 2. Subcellular fractionation, using a discontinuous sucrose gradient, however, produced a membrane-bound large secretory granule fraction which, when isolated, contained essentially only isoform 2, and which had the capacity to convert isoform 2 to isoforms 3 and 3' by posttranslational phosphorylation.

Animals↗
PubMed1986

Peptide antibodies: new tools for cell biology.

The process of preparing antibodies against small peptide subsets of larger proteins is now a very routine and effective tool for cell biological investigations. Now that the identification of genes is commonplace, it is imperative to be able to identify, purify, and characterize the products of these genes. Antibodies against synthetic peptides will aid in discovering the elusive functions of these proteins. Over the past 5 years, peptide antibodies have contributed, and they will doubtless continue to contribute, to the identification of functional domains of proteins. Peptide antibodies provide a means for identifying functional domains conserved during the evolution of families of proteins, and for inhibiting specific functions of multifunctional proteins. Domain-specific antibodies have already increased the molecular resolution with which cell biologists can immunologically examine the function of cellular proteins. Finally, many proteins are now known to exist in subtly different forms, either as the products of separate genes or as the result of posttranslational modifications. Peptide antibodies allow molecular cell biologists, for the first time, to design antibodies for the specific assay of altered forms of a protein. Because they are amenable to specific immunolocalization of highly similar species, peptide antibodies can be considered to be subcellular probes of gene expression and posttranslational modification.

Antibodies↗
PubMed2000

Use of mass spectrometry to study signaling pathways.

Activation of cells by extracellular stimuli, such as growth factors, initiates a cascade of events involving posttranslational modifications, including phosphorylation, formation of protein complexes, and induction or repression of gene expression. Traditionally, genetic methods or specific biochemical assays have been used to identify molecules involved in signaling pathways. Lately, mass spectrometry, combined with elegant biochemical approaches, has become a powerful tool for identifying proteins and posttranslational modifications. With this protocol, we hope to bridge the gap between the biochemical and molecular aspects of signal transduction pathways and the mass spectrometric tools and techniques that are available to study them. We provide methods for large-scale cell culture and immunoprecipitation of tyrosine-phosphorylated proteins, silver staining of gels, trypsin digests, and protein identification by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry and nanoelectrospray tandem mass spectrometry. We discuss the special requirements for the identification of phosphorylation sites in proteins by mass spectrometry. We describe enrichment of phosphopeptides from unseparated peptide mixtures by immobilized metal affinity column (IMAC) and the use of phosphatases to identify phosphorylated peptides. We also discuss specialized methods, such as precursor ion scanning in the negative mode and direct sequencing of phosphopeptides in the positive mode. Our goal is to provide detailed methods in use today for proteomic applications in general and for receptor-mediated signaling pathways in particular.

Animals↗
PubMed1996

Axonemal tubulin polyglycylation probed with two monoclonal antibodies: widespread evolutionary distribution, appearance during spermatozoan maturation and possible function in motility.

Two monoclonal antibodies, AXO 49 and TAP 952, probed with carboxy-terminal peptides from Paramecium axonemal tubulin and with polyglycylated synthetic peptides, are found to recognize differently tubulin polyglycylation, the most recently identified posttranslational modification discovered in Paramecium axonemal tubulin. With these antibodies, we show that tubulin polyglycylation is widely distributed in organisms ranging from ciliated protozoa to mammals; it arose early in the course of evolution, but seems to be absent in primitive protozoa such as the Euglenozoa. Tubulin polyglycylation is the last posttranslational modification which takes place in the course of Drosophila spermatogenesis and its occurrence corresponds to the end of spermatozoan maturation. An involvement of polyglycylated tubulin in axoneme motility is suggested since AXO 49 and TAP 952 specifically inhibit the reactivated motility of sea urchin spermatozoa.

Amino Acid Sequence↗
PubMed2005

Chemical cytometry for monitoring metabolism of a Ras-mimicking substrate in single cells.

BACKGROUND: Chemical cytometry is an emerging technology that analyzes chemical contents of single cells by means of capillary electrophoresis or capillary chromatography. It has a potential to become an indispensable tool in analyses of heterogeneous cell populations such as those in tumors. Ras oncogenes are found in 30% of human cancers. To become fully functional products, oncogenic Ras proteins require at least three posttranslational modifications: farnesylation, endoproteolysis, and carboxyl-methylation. Therefore, enzymes that catalyze the three reactions, farnesyltransferase (FTase), endoprotease (EPase), and methyltransferase (MTase), are considered highly attractive therapeutic targets. In this work, we used chemical cytometry to study the metabolism of a pentapeptide substrate that can mimic Ras proteins with respect to their posttranslational modifications in solution. METHODS: Mouse mammary gland tumor cells (4T1) and mouse embryo fibroblasts (NIH3T3) were incubated with a fluorescently labeled pentapeptide substrate, 2',7'-difluorofluorescein-5-carboxyl-Gly-Cys-Val-Ilu-Ala. Cells were washed from the substrate and resuspended in phosphate buffered saline. Uptake of the substrate by the cells was monitored by laser scanning confocal microscopy. Single cells were injected into the capillary, lysed, and subjected to capillary electrophoresis. Fluorescent metabolic products were detected by laser-induced fluorescence and compared with products obtained by the conversion of the substrate by FTase, EPase, and MTase in solution. Co-sampling of single cells with the in-vitro products was used for such comparison. RESULTS: Confocal microscopy data showed that the substrate permeated the plasma membrane and clustered in the cytoplasm. Further capillary electrophoresis and chemical cytometry analyses showed that the substrate was converted into three fluorescently labeled products, two of which were secreted in the culture medium and one remained in the cells. The intracellular product was present at approximately 100,000 molecules per cell. The three metabolic products of the substrate were found to be different from the products of its processing by FTase, EPase, and MTase in solution. CONCLUSIONS: This is the first report of chemical cytometry in the context of Ras-signaling studies. The chemical cytometry method used in this work will find applications in the development of suitable peptide substrates for monitoring enzyme activities in single cells.

Alkyl and Aryl Transferases↗
PubMed2003

EH and UIM: endocytosis and more.

Exogenously and endogenously originated signals are propagated within the cell by functional and physical networks of proteins, leading to numerous biological outcomes. Many protein-protein interactions take place between binding domains and short peptide motifs. Frequently, these interactions are inducible by upstream signaling events, in which case one of the two binding surfaces may be created by a posttranslational modification. Here, we discuss two protein networks. One, the EH-network, is based on the Eps15 homology (EH) domain, which binds to peptides containing the sequence Asp-Pro-Phe (NPF). The other, which we define as the monoubiquitin (mUb) network, relies on monoubiquitination, which is emerging as an important posttranslational modification that regulates protein function. Both networks were initially implicated in the control of plasma membrane receptor endocytosis and in the regulation of intracellular trafficking routes. The ramifications of these two networks, however, appear to extend into many other aspects of cell physiology as well, such as transcriptional regulation, actin cytoskeleton remodeling, and DNA repair. The focus of this review is to integrate available knowledge of the EH- and mUb networks with predictions of genetic and physical interactions stemming from functional genomics approaches.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Oxidative modification of creatine kinase BB in Alzheimer's disease brain.

Creatine kinase (CK) BB, a member of the CK gene family, is a predominantly cytosolic CK isoform in the brain and plays a key role in regulation of the ATP level in neural cells. CK BB levels are reduced in brain regions affected by neurodegeneration in Alzheimer's disease (AD), Pick's disease, and Lewy body dementia, and this reduction is not a result of decreased mRNA levels. This study demonstrates that posttranslational modification of CK BB plays a role in the decrease of CK activity in AD brain. The specific CK BB activity and protein carbonyl content were determined in brain extracts of six AD and six age-matched control subjects. CK BB activity per microgram of immunoreactive CK BB protein was lower in AD than in control brain extracts, indicating the presence of inactive CK BB molecules. The analysis of specific protein carbonyl levels in CK BB, performed by two-dimensional fingerprinting of oxidatively modified proteins, identified CK BB as one of the targets of protein oxidation in the AD brain. The increase of protein carbonyl content in CK BB provides evidence that oxidative posttranslational modification of CK BB plays a role in the loss of CK BB activity in AD.

Aged↗
PubMedDate not supplied

Synthesis, modifications, and turnover of proteins during aging.

Slowing down of bulk protein synthesis is one of the most commonly observed biochemical changes during aging. The implications and consequences of slower rates of protein synthesis are manifold, including a decrease in the availability of enzymes for the maintenance, repair, and normal metabolic functioning of the cell, an inefficient removal of inactive, abnormal, and damaged macromolecules in the cell, the inefficiency of the intracellular and intercellular signalling pathways, and a decrease in the production and secretion of hormones, antibodies, neurotransmitters, and the components of the extracellular matrix. Age-related changes in the activity, specificity, and stability of a large number of proteins have been reported. However, the molecular mechanisms responsible for such alterations are still poorly understood. Studies on various components of the protein synthetic machinery have revealed a decline in the efficiency and accuracy of ribosomes, an increase in the levels of rRNA and tRNA, and a decrease in the amounts and activities of elongation factors. Because posttranslational modifications of proteins determine their activity and stability, alterations in the extent and level of various modifications such as phosphorylation, methylation, ADP-ribosylation, oxidation, glycation, and conformational changes during aging are being studied. Changes in the regulation of protein synthesis, posttranslational modifications, and protein turnover are crucial determinants of age-related decline in the maintenance, repair, and survival of the organism.

Adenosine Diphosphate Ribose↗
PubMed1992

Deletion studies to reveal the basis for size discrepancy in proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA), an essential component for DNA replication in eukaryotes, is a highly conserved nonhistone nuclear protein of 261 amino acids. The molecular weight of mammalian PCNA, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), differs notably from that predicted by the cDNA sequences, that is, 36,000 in comparison with 29,261 and 28,748 for human and rat PCNA, respectively. To investigate if this discrepancy is due to posttranslational modifications, we studied the PCNA protein synthesized by an in vitro transcription/translation system as well as the protein overproduced in bacteria. We found that both PCNA protein samples were indistinguishable from the authentic protein from the protein mobility in SDS-PAGE. The finding indicates that the size discrepancy is not due to the posttranslational modifications. Hence, the size discrepancy may be due to the protein sequence per se, namely a sequence-related anomaly in SDS-PAGE. Results from the analyses of a series of PCNA derivatives with various lengths of C- or N-terminal deletion indicate that the putative sequence is in the region of residues 128-150.

Amino Acid Sequence↗
PubMed1993

Primary structure of rabbit lens alpha-crystallins.

The primary structure and posttranslational modifications of rabbit lens alpha-crystallins were examined using electrospray ionization mass spectrometry to determine the molecular weights of the intact proteins and fast atom bombardment mass spectrometry to analyze proteolytic digests of the alpha A- and alpha B-crystallins. The previously determined primary structure of alpha A-crystallin was confirmed. Posttranslational modifications detected included one phosphorylation site and the presence of a truncated form minus the five C-terminal residues. The previously undetermined amino acid sequence of rabbit alpha B-crystallin was determined to be the same as the bovine alpha B-crystallin sequence except at three residues: Thr 40, Thr 132, and Pro 153. Rabbit alpha B-crystallin showed evidence of phosphorylation at the same three sites as bovine alpha B-crystallin. The molecular weights of the intact proteins indicated that any one molecule had a maximum of two phosphorylations. Also, there was a truncated form which did not include the five C-terminal residues.

Amino Acid Sequence↗
PubMed2005

Structural characteristics of hen egg ovalbumin expressed in yeast Pichia pastoris.

The recombinant ovalbumin (OVA) produced in yeast Pichia pastoris was purified from the culture medium by anion exchange chromatography, and its structural characteristics were compared with those of hen egg OVA, mainly from the point of view of posttranslational modification. The expressed OVA consisted of two molecular species immmunoreactive with antibody for hen egg OVA. The two molecular species, 45 and 47 kDa in molecular size, were thought to correspond to mono-glycosylated form and di-glycosylated form respectively. The non-glycosylated form was not produced in the system. The other posttranslational modifications (N-terminal acetylation and phosphorylation) observed in hen egg OVA were not detected in either of the molecular species. The two recombinant proteins displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as hen egg OVA. The melting temperature, Tm, which was determined from the thermal unfolding curve, was almost identical in the two recombinant proteins, despite the difference in glycosylation levels, while it decreased by about 2.5 degrees C as compared with that of hen egg OVA (77.3 degrees C). These data indicate that the additional glycosylation to Asn-311 in the recombinant protein does not affect protein conformation or thermostability.

Amino Acid Sequence↗