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PubMed1987

Human protein S cDNA encodes Phe-16 and Tyr 222 in consensus sequences for the post-translational processing.

Partial cDNAs coding for human protein S were isolated from a pUC9 human liver cDNA library. Together, the overlapping clones span a (partial) 5'-non-coding region, and the complete protein S coding and 3'-untranslated regions. The derived amino acid sequence deviates at five positions from two previously reported protein S sequences. Two of these differences (Phe instead of Leu at position -16 and Tyr instead of Asp at position 222) are found in regions that are important for the post-translational modification of protein S, the gamma-carboxylation of glutamic acid and the hydroxylation of asparagine, respectively.

Amino Acid Sequence↗
PubMed2001

Microbial transformations of steroids--XII. Progesterone hydroxylation profiles are modulated by post-translational modification of an electron transfer protein in Streptomyces roseochromogenes.

When Streptomyces roseochromogenes strain 10984 was incubated with exogenous progesterone for 25 h the major monohydroxylated metabolite, 16alpha-hydroxyprogesterone was produced in 3.6 fold excess to the minor metabolite 2beta,16alpha-dihydroxyprogesterone. In a reconstituted system containing highly purified progesterone 16alpha-hydroxylase cytochrome P-450, and electron transfer proteins ferredoxin-like redoxin (roseoredoxin) and redoxin reductase (roseoredoxin reductase), both metabolites were produced but in a 10:1 ratio. When S. roseochromogenes was pre-incubated for 8 h with 0.32 mM progesterone and the purified components of the hydroxylase system incubated as before, the ratio of 16alpha-hydroxyprogesterone to 2beta,16alpha-dihydroxyprogesterone produced decreased to 2.8:1, virtually identical to the ratio in whole cell transformations. Reconstitution assays containing all combinations of hydroxylase proteins purified from progesterone pre-incubated and control cells showed that the roseoredoxin was solely responsible for the observed changes in in vitro metabolite ratios. The fact that the lower 16alpha-hydroxyprogesterone to 2beta,16alpha-dihydroxyprogesterone ratio was also obtained when S. roseochromogenes was exposed to 0.335 mM cycloheximide for 8 h prior to the progesterone pre-incubation, pointed to post-translation modification of the roseoredoxin. Separation of two isoforms of roseoredoxin by isoelectric focusing supported this proposition.

Bacterial Proteins↗
PubMed1999

Regulation by phosphorylation of the zinc finger protein KRC that binds the kappaB motif and V(D)J recombination signal sequences.

The DNA binding protein KRC (forkappaB binding andrecognitioncomponent of the V(D)J recombination signal sequence) belongs to a family of large zinc finger proteins that bind to the kappaB motif and contains two widely separated DNA binding structures. In addition to the kappaB motif, KRC fusion proteins bind to the signal sequences of V(D)J recombination to form highly ordered complexes. Here, we report that KRC may be regulated by post-translational modifications. Specific protein kinases present in the nucleus of pre-B cells phosphorylated a KRC fusion protein at tyrosine and serine residues. Such protein modifications increased DNA binding, thereby providing a mechanism by which KRC responds to signal transduction pathways. KRC is a substrate of epidermal growth factor receptor kinase and P34cdc2 kinase in vitro. Our results suggest that activation of the KRC family of transcription factors may provide a mechanism by which oncogenic tyrosine kinases regulate genes with kappaB-controlled gene regulatory elements.

Amino Acid Sequence↗
PubMed2001

Post-translational modification of the hepatitis C virus core protein by tissue transglutaminase.

The hepatitis C virus (HCV) core protein is a structural protein that packages the viral genomic RNA. In this study, we demonstrate that a stable core protein dimer could be produced in liver cells. The production of this protein could be enhanced by calphostin C and serum deprivation. This protein was determined to be the core protein dimer because of its reactivity with the anti-core antibody, its similar electrophoretic mobility compared with that of the core protein dimer generated by cross-linking with glutaraldehyde, and its increase in size by a hemagglutinin tag fused to the core protein sequence. This core protein dimer was highly stable and resistant to SDS and beta-mercaptoethanol. The enzyme that mediated the formation of this stable core protein dimer was determined to be the tissue transglutaminase (tTG) because, first, tTG could be activated by calphostin C and serum deprivation; second, the formation of this dimer was suppressed by monodansylcadaverine, a tTG inhibitor; and third, the core protein could be cross-linked by tTG in vitro. Thus, the HCV core protein represents the first known viral structural protein substrate of tTG. The post-translational modification by tTG reduced the RNA binding activity of the core protein, raising the possibility that tTG may regulate the biological functions of the HCV core protein.

Cell Line↗
PubMed2005

Overview of molecular, cellular, and genetic neurotoxicology.

It has become increasingly evident that the field of neurotoxicology is not only rapidly growing but also rapidly evolving, especially over the last 20 years. As the number of drugs and environmental and bacterial/viral agents with potential neurotoxic properties has grown, the need for additional testing has increased. Only recently has the technology advanced to a level that neurotoxicologic studies can be performed without operating in a "black box." Examination of the effects of agents that are suspected of being toxic can occur on the molecular (protein-protein), cellular (biomarkers, neuronal function), and genetic (polymorphisms) level. Together, these areas help to elucidate the potential toxic profiles of unknown (and in some cases, known) agents. The area of proteomics is one of the fastest growing areas in science and particularly applicable to neurotoxicology. Lubec et al, provide a review of the potential and limitations of proteomics. Proteomics focuses on a more comprehensive view of cellular proteins and provides considerably more information about the effects of toxins on the CNS. Proteomics can be classified into three different focuses: post-translational modification, protein-expression profiling, and protein-network mapping. Together, these methods represent a more complete and powerful image of protein modifications following potential toxin exposure. Cellular neurotoxicology involves many cellular processes including alterations in cellular energy homeostasis, ion homeostasis, intracellular signaling function, and neurotransmitter release, uptake, and storage. The greatest hurdle in cellular neurotoxicology has been the discovery of appropriate biomarkers that are reliable, reproducible, and easy to obtain. There are biomarkers of exposure effect, and susceptibility. Finding the appropriate biomarker for a particular toxin is a daunting task. The appropriate biomarker for a particular toxin is a daunting task. The advantage to biomarker/toxin combinations is they can be detected and measured shortly following exposure and before overt neuroanatomic damage or lesions. Intervention at this point, shortly following exposure, may prevent or at least attenuate further damage to the individual. The use of peripheral biomarkers to assess toxin damage in the CNS has numerous advantages: time-course analysis may be performed, ethical concerns with the use of human subjects can partially be avoided, procedures to acquire samples are less invasive, and in general, peripheral studies are easier to perform. Genetic neurotoxicology comprises two focuses--toxin-induced alterations in genetic expression and genetic alterations that affect toxin metabolism, distribution, and clearance. These differences can be beneficial or toxic. Polymorphisms have been shown to result in altered metabolism of certain toxins (paraoxonase and paraoxon). Conversely, it is possible that some polymorphisms may be beneficial and help prevent the formation of a toxic by-product of an exogenous agent (resistance to ozone-induced lung inflammation). It has also become clear that interactions of potential toxins are not straightforward as interactions with DNA, causing mutations. There are numerous agents that cause epigenetic responses (cellular alterations that are not mutagenic or cytotoxic). This finding suggests that many agents that may originally have been thought of as nontoxic should be re-examined for potential "indirect" toxicity. With the advancement of the human genome project and the development of a human genome map, the effects of potential toxins on single or multiple genes can be identified. Although collectively, the field of neurotoxicology has recently come a long way, it still has a long way to go reach its full potential. As technology and methodology advances continue and cooperation with other disciplines such as neuroscience, biochemistry, neurophysiology, and molecular biology is improved, the mechanisms of toxin action will be further elucidated. With this increased understanding will come improved clinical interventions to prevent neuronal damage following exposure to a toxin.

Animals↗
PubMed2002

Detection of oxidant sensitive thiol proteins by fluorescence labeling and two-dimensional electrophoresis.

Oxidants can activate signaling pathways and modulate a variety of cellular activities. Their action at a molecular level involves the post-translational modification of protein thiols. We have developed a proteomic method to monitor the reduction and oxidation of protein thiols, and identify those thiol proteins most sensitive to oxidation. Cells were disrupted in the presence of N-ethylmaleimide to block the reduced thiol proteins and dithiothreitol was added to reduce the oxidized thiol proteins before labeling with 5-iodoacetamidofluorescein. Two-dimensional (2-D) electrophoresis was used to resolve the labeled samples. We applied the method to Jurkat T lymphocytes and examined the effect of diamide on the oxidized and reduced thiol protein profiles. A small percentage of protein thiols were already oxidized in untreated cells. Exposure of cells to 2 mM diamide for ten minutes led to a dramatic increase in thiol protein oxidation as seen in the oxidized thiol protein map. However, it was difficult to detect any change in the pattern of reduced thiol proteins. Separation of proteins by 2-D electrophoresis revealed approximately 200 thiol proteins that were oxidized by diamide treatment. This method will be valuable in elucidating redox signaling pathways.

Electrophoresis, Gel, Two-Dimensional↗
PubMed2006

Transcriptional dysregulation in Down syndrome: predictions for altered protein complex stoichiometries and post-translational modifications, and consequences for learning/behavior genes ELK, CREB, and the estrogen and glucocorticoid receptors.

The phenotype of Down syndrome, trisomy of chromosome 21, is hypothesized to be produced by the increased expression due to gene dosage of normal chromosome 21 genes. Chromosome 21 encodes a number of proteins that, based on experimental evidence or domain composition, are classed as transcription factors or their co-regulators. Other chromosome 21 proteins contribute to post-translational modification of transcription factors, including their phosphorylation, dephosphorylation and sumoylation. Several of these chromosome 21 proteins and the pathways in which they function have overlapping transcription factor specificities. Thus, altered stoichiometry in complexes and altered levels of activation of individual transcription factors may contribute to the Down syndrome phenotype by perturbation of downstream gene expression. Here we review recent data on four chromosome 21 proteins: NRIP1, GABPA, DYRK1A and SUMO3. We discuss the implications for activation of ELK, CREB, C/EBP alpha, beta estrogen and glucocorticoid receptors, and for expression of BDNF. Each of these proteins is relevant to learning, behavior and/or development and therefore perturbation of their activation may contribute to the Down syndrome phenotype.

Chromosome Mapping↗
PubMed1993

Identification of proteins encoded by avian reoviruses and evidence for post-translational modification.

Twelve avian reovirus strain 176 proteins, 10 structural (lambda 1, lambda 2, lambda 3, mu 1, mu 2/mu 2C, sigma 1, sigma 2, sigma 3, and sigma 4), and 2 nonstructural (mu NS and sigma NS) were identified and characterized by electrophoretic analysis of purified virions and infected cell lysates. Three of the identified proteins (mu 1, mu NS, and sigma 4) have not been previously described. In pulse-chase experiments, 10 of the proteins (lambda 1, lambda 2, lambda 3, mu 1, mu 2, mu NS, sigma 1, sigma 2, sigma NS, sigma 3) were shown to be primary translation products, whereas mu 2C was shown to be a post-translational cleavage product of mu 2.

Animals↗
PubMed2003

Decomposition of protein nitrosothiolsin matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry.

The S-nitrosylation of proteins is involved in the trafficking of nitric oxide (NO) in intra- and extracellular milieus. To establish a mass spectrometric method for identifying this post-translational modification of proteins, a synthetic peptide and transthyretin were S-nitrosylated in vitro and analyzed by electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The intact molecular ion species of nitrosylated compounds was identified in the ESI mass spectrum without elimination of the NO group. However, the labile nature of the S-NO bond was evident when the in-source fragmentation efficiently generated [M + H - 30](+) ions. The decomposition was prominent for multiply charged transthyretin ions with high charge states under ordinary ESI conditions, indicating that the application of minimum nozzle potentials was essential for delineating the stoichiometry of nitrosylation in proteins. With MALDI, the S-NO bond cleavage occurred during the ionization process, and the subsequent reduction generated [M + H - 29](+) ions.

Humans↗
PubMed2004

A cut above the rest: the regulatory function of plant proteases.

Proteolytic enzymes are intricately involved in many aspects of plant physiology and development. On the one hand, they are necessary for protein turnover. Degradation of damaged, misfolded and potentially harmful proteins provides free amino acids required for the synthesis of new proteins. Furthermore, the selective breakdown of regulatory proteins by the ubiquitin/proteasome pathway controls key aspects of plant growth, development, and defense. Proteases are, on the other hand, also responsible for the post-translational modification of proteins by limited proteolysis at highly specific sites. Limited proteolysis results in the maturation of enzymes, is necessary for protein assembly and subcellular targeting, and controls the activity of enzymes, regulatory proteins and peptides. Proteases are thus involved in all aspects of the plant life cycle ranging from the mobilization of storage proteins during seed germination to the initiation of cell death and senescence programs. This article reviews recent findings for the major catalytic classes, i.e. the serine, cysteine, aspartic, and metalloproteases, emphasizing the regulatory function of representative enzymes.

Aspartic Acid Endopeptidases↗
PubMed1995

Biomedical and biochemical applications of liquid chromatography-mass spectrometry.

This review centres on the application of various LC-MS and LC-MS-MS techniques to the study and solution of practical problems in biomedical research. For this purpose it covers a selection of publications in this area included in the MEDLINE database for the period 1991-mid-1994. As shown herein, LC-MS is increasingly gaining in importance in the biomedical field, especially after the revolution brought about by the introduction of the new liquid-phase atmospheric pressure ionization (API) techniques, such as electrospray (ES) and ionspray. The information in this database shows that thermospray (TS), which clearly dominated LC-MS coupling in the 1980s, is on a downward trend relative to the more modern API techniques which will certainly dominate this application field in the present decade. Studies on drug metabolism, therapeutic drug monitoring and pharmacology have been traditionally carried out by GC-MS. However, LC-MS has lately been replacing classical GC-MS techniques in many of these applications. For instance, LC-ES-MS has greatly facilitated the application of both qualitative and quantitative LC-MS methods to highly polar molecules. This is possible without the need to resort to elaborate sample handling and derivatization procedures for relatively high-molecular-mass compounds such as drug conjugates, biosynthetic and natural peptides and therapeutic proteins obtained by recombinant DNA technology, all of them formerly totally inaccessible to the standard GC-MS or LC-MS methods. With regard to studies on metabolism and biochemical phenomena of endogenous compounds, LC-ES-MS is also becoming very strong in the analysis of structural biopolymers such as peptides, proteins, glycoproteins and glycolipids, and also lower molecular mass compounds such as fatty acids, vitamins, steroids and nucleic acids. For example, structural verification of post-translational modifications in proteins can be efficiently obtained in the time frame of an LC run and suitable MS methods for the location of glycopeptide-containing fractions in proteolytic digests of glycoproteins have been developed. Interesting examples are also shown of the use of LC-MS in clinical studies and the determination of biological markers of disease.

Amino Acid Sequence↗
PubMed2022

Accessing isotopically labeled proteins containing genetically encoded phosphoserine for NMR with optimized expression conditions.

Phosphoserine (pSer) sites are primarily located within disordered protein regions, making it difficult to experimentally ascertain their effects on protein structure and function. Therefore, the production of 15N- (and 13C)-labeled proteins with site-specifically encoded pSer for NMR studies is essential to uncover molecular mechanisms of protein regulation by phosphorylation. While genetic code expansion technologies for the translational installation of pSer in Escherichia coli are well established and offer a powerful strategy to produce site-specifically phosphorylated proteins, methodologies to adapt them to minimal or isotope-enriched media have not been described. This shortcoming exists because pSer genetic code expansion expression hosts require the genomic ΔserB mutation, which increases pSer bioavailability but also imposes serine auxotrophy, preventing growth in minimal media used for isotopic labeling of recombinant proteins. Here, by testing different media supplements, we restored normal BL21(DE3) ΔserB growth in labeling media but subsequently observed an increase of phosphatase activity and mis-incorporation not typically seen in standard rich media. After rounds of optimization and adaption of a high-density culture protocol, we were able to obtain ≥10 mg/L homogenously labeled, phosphorylated superfolder GFP. To demonstrate the utility of this method, we also produced the intrinsically disordered serine/arginine-rich region of the SARS-CoV-2 Nucleocapsid protein labeled with 15N and pSer at the key site S188 and observed the resulting peak shift due to phosphorylation by 2D and 3D heteronuclear single quantum correlation analyses. We propose this cost-effective methodology will pave the way for more routine access to pSer-enriched proteins for 2D and 3D NMR analyses.

Humans↗
PubMed1994

Identification of proteins in polyacrylamide gels by mass spectrometric peptide mapping combined with database search.

Mass spectrometric peptide mapping of proteins separated by one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis has been investigated. The best results are obtained after blotting of the proteins onto polyvinylidene difluoride membranes followed by enzymatic digestion of the protein on the membrane. The peptide maps were investigated in terms of completeness and applicability for protein identification using a previously developed database search program as well as for the possibility for full characterization of covalent modifications in the proteins. The most complete peptide maps were obtained when the proteins were reduced and alkylated on the membrane prior to enzymatic digestion followed by separation of the resulting mixture by high performance liquid chromatography prior to mass spectrometric analysis. Such peptide maps cover up to 98% of the sequence and consequently may allow complete characterization of post-translational modifications in proteins for which the amino acid sequence is known. The fastest and most sensitive procedure to obtain peptide maps sufficient for protein identification was direct analysis of the extracted peptide mixture by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The use of external and internal calibration of MALDI spectra for database searches is evaluated as well as the possibility of including a post-calibration routine within the search program.

Alkylation↗
PubMed2003

Protein microarrays: molecular profiling technologies for clinical specimens.

Proteomics, the study of protein function within biologic systems, will further our understanding of cancer pathogenesis. Coupled with transcript profiling, proteomics can herald the advent of molecular therapy tailored to the individual patient's neoplasm. Protein microarrays, one emerging class of proteomic technologies, have broad applications for discovery and quantitative analysis. This technology is uniquely suited to gather information about the post-translational modifications of proteins reflecting the activity state of signal pathways and networks. Protein microarrays now make it feasible to conduct signal network profiling within cellular samples. Nevertheless, to be successful, design and use of protein microarrays must take into consideration enormous analytical challenges. A subclass of protein microarrays, Reverse Phase Arrays, created to meet these challenges, has been optimized for use with tissue specimens, and is now in use for the analysis of biopsy samples for clinical trial research.

Antibodies↗
PubMed1990

Secretion of sulfated and nonsulfated forms of parathyroid chromogranin A (secretory protein-I).

Chromogranin A (secretory protein-I) is an acidic, sulfated glycoprotein found in secretory granules of most endocrine cells but not in exocrine or epithelial cells. Parathyroid chromogranin A is sulfated on tyrosine residues, whereas adrenal chromogranin A appears to be sulfated mainly on oligosaccharide residues. Chromogranin B, on the other hand, is tyrosine-sulfated in the bovine adrenal whereas this protein is absent from the parathyroid. The role of this tissue- or species-specific sulfation of chromogranin is not known. Tyrosine sulfation is a common post-translational modification of proteins in the exocytotic pathway and has been suggested to play a role in the sorting or intracellular transport of secretory proteins. To test this, porcine parathyroid tissue slices were metabolically labeled with 35SO4 and [3H]Lys, and the tissue and incubation medium analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoprecipitation with chromogranin A-specific antiserum or by radioimmunoassay for parathormone. Secretion of total and 3H-labeled chromogranin A was about 3- and 7-fold higher, respectively, at 0.5 mM than at 3.0 mM Ca2+, and secretion of 35SO4-labeled chromogranin A was 67-fold higher. This indicates that either sulfated chromogranin A is directed primarily to the Ca2+-regulated pathway or that sulfation occurs following sorting to this pathway. Sodium chlorate (1-10 mM) inhibited sulfation in a dose-dependent manner by up to 95% but it had no effect on the onset or rate of chromogranin A secretion. These data indicate that regulated secretion of parathyroid chromogranin A does not require sulfation of tyrosine residues.

Animals↗
PubMed1989

Lipid modification of proteins and their membrane transport.

An effective method for artificial attachment of lipid anchors to water-soluble proteins has been developed. To this end, a protein molecule is modified in a system of reversed micelles by a water-insoluble reagent, e.g. fatty acid chloride. Fatty acylated proteins acquire an ability to translocate across lipid membranes and penetrate intact cells. This principle of imparting transmembrane properties to water-soluble proteins makes it possible to realize in vivo a direct transport of antibodies across the hemato-encephalic barrier into the brain and to develop a method for virus suppression by fatty acylated anti-viral antibodies capable of penetrating infected cells. The effect of a drastic increase in the biological activity of exogenous protein factors, e.g. Staphylococcus aureus enterotoxin A, as a result of their artificial fatty acylation has been discovered. The above-mentioned phenomena are discussed in relation to the in vivo data, indicating that post-translational modification of proteins by fatty acids and phospholipids is very widespread in nature and evidently plays an important role in protein transport and sorting. In this connection, lipid modification of proteins is regarded as a possible general step of protein transport in vivo.

Animals↗
PubMed2002

N-Terminal peptide labeling strategy for incorporation of isotopic tags: a method for the determination of site-specific absolute phosphorylation stoichiometry.

Determining the phosphorylation stoichiometry at specific sites in a phosphoprotein is a very challenging task. We describe here a novel mass spectrometry based method that is capable of measuring the absolute phosphorylation stoichiometry at specific sites without the need for specific internal standards, phospho-site antibodies or radioactivity. The method is based on a gentle chemical labeling strategy which specifically and differentially labels the N-terminus of all peptides in a sample with either a D(5)- or D(0)-propionyl group and measures the ratio of the abundance of the D(5)/D(0) peptide pairs simultaneously using mass spectrometry. Using matrix-assisted laser desorption/ionization (MALDI), the method can measure absolute stoichiometry to within at least 10% and can be applied to both in vitro and in vivo phosphorylated peptides and proteins. Furthermore, this method can potentially be applied to the quantitative study of other types of protein post-translational modifications, and the profiling of protein expression on the proteome level.

Amino Acid Sequence↗
PubMed2003

KaiB functions as an attenuator of KaiC phosphorylation in the cyanobacterial circadian clock system.

In the cyanobacterium Synechococcus elongatus PCC 7942, the KaiA, KaiB and KaiC proteins are essential for generation of circadian rhythms. We quantitatively analyzed the intracellular dynamics of these proteins and found a circadian rhythm in the membrane/cytosolic localization of KaiB, such that KaiB interacts with a KaiA-KaiC complex during the late subjective night. KaiB-KaiC binding is accompanied by a dramatic reduction in KaiC phosphorylation and followed by dissociation of the clock protein complex(es). KaiB attenuated KaiA-enhanced phosphorylation both in vitro and in vivo. Based on these results, we propose a novel role for KaiB in a regulatory link among subcellular localization, protein-protein interactions and post-translational modification of Kai proteins in the cyanobacterial clock system.

Bacterial Proteins↗