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Results for “Protein Modification, Translational”

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PubMed2004

Carbonylation of glycolytic proteins is a key response to drug-induced oxidative stress and apoptosis.

Recent work has highlighted the importance of protein post-translational modifications such as phosphorylation (enzymatic) and nitrosylation (nonenzymatic) in the early stages of apoptosis. In this study, we have investigated the levels of protein carbonylation, a nonenzymatic protein modification that occurs in conditions of cellular oxidative stress, during etopside-induced apoptosis of HL60 cells. Within 1 h of VP16 treatment, a number of proteins underwent carbonylation due to oxidative stress. This was inhibited by the antioxidant N-acetyl-L-cysteine. Among the proteins found to be carbonylated were glycolytic enzymes. Subsequently, we found that the rate of glycolysis was significantly reduced, probably due to a carbonylation mediated reduction in enzymatic activity of glycolytic enzymes. Our work demonstrates that protein carbonylation can be rapidly induced through cytotoxic drug treatment and may specifically inhibit the glycolytic pathway. Given the importance of glycolysis as a source of cellular ATP, this has severe implications for cell function.

Acetylcysteine↗

PubMed2006

Exercise affects energy metabolism and neural plasticity-related proteins in the hippocampus as revealed by proteomic analysis.

Studies were conducted to evaluate the effect of a brief voluntary exercise period on the expression pattern and post-translational modification of multiple protein classes in the rat hippocampus using proteomics. An analysis of 80 protein spots of relative high abundance on two-dimensional gels revealed that approximately 90% of the proteins identified were associated with energy metabolism and synaptic plasticity. Exercise up-regulated proteins involved in four aspects of energy metabolism, i.e. glycolysis, ATP synthesis, ATP transduction and glutamate turnover. Specifically, we found increases in fructose-bisphosphate aldolase C, phosphoglycerate kinase 1, mitochondrial ATP synthase, ubiquitous mitochondrial creatine kinase and glutamate dehydrogenase 1. Exercise also up-regulated specific synaptic-plasticity-related proteins, the cytoskeletal protein alpha-internexin and molecular chaperones (chaperonin-containing TCP-1, neuronal protein 22, heat shock 60-kDa protein 1 and heat shock protein 8). Western blot was used to confirm the direction and magnitude of change in ubiquitous mitochondrial creatine kinase, an enzyme essential for transducing mitochondrial-derived ATP to sites of high-energy demand such as the synapse. Protein phosphorylation visualized by Pro-Q Diamond fluorescent staining showed that neurofilament light polypeptide, glial fibrillary acidic protein, heat shock protein 8 and transcriptional activator protein pur-alpha were more intensely phosphorylated with exercise as compared with sedentary control levels. Our results, together with the fact that most of the proteins that we found to be up-regulated have been implicated in cognitive function, support a mechanism by which exercise uses processes of energy metabolism and synaptic plasticity to promote brain health.

Animals↗

PubMed2005

Effects of oxidative stress on the cardiac myocyte proteome: modifications to peroxiredoxins and small heat shock proteins.

Endogenous oxidative stress is a likely cause of cardiac myocyte death in vivo. We examined the early (0-2 h) changes in the proteome of isolated cardiac myocytes from neonatal rats exposed to H2O2 (0.1 mM), focussing on proteins with apparent molecular masses of between 20 and 30 kDa. Proteins were separated by two-dimensional gel electrophoresis (2DGE), located by silver-staining and identified by mass spectrometry. Incorporation of [35S]methionine or 32Pi was also studied. For selected proteins, transcript abundance was examined by reverse transcriptase-polymerase chain reaction. Of the 38 protein spots in the region, 23 were identified. Two families showed changes in 2DGE migration or abundance with H2O2 treatment: the peroxiredoxins and two small heat shock protein (Hsp) family members: heat shock 27 kDa protein 1 (Hsp25) and alphaB-crystallin. Peroxiredoxins shifted to lower pI values and this was probably attributable to 'over-oxidation' of active site Cys-residues. Hsp25 also shifted to lower pI values but this was attributable to phosphorylation. alphaB-crystallin migration was unchanged but its abundance decreased. Transcripts encoding peroxiredoxins 2 and 5 increased significantly. In addition, 10 further proteins were identified. For two (glutathione S-transferase pi, translationally-controlled tumour protein), we could not find any previous references indicating their occurrence in cardiac myocytes. We conclude that exposure of cardiac myocytes to oxidative stress causes post-translational modification in two protein families involved in cytoprotection. These changes may be potentially useful diagnostically. In the short term, oxidative stress causes few detectable changes in global protein abundance as assessed by silver-staining.

Amino Acid Sequence↗

PubMed1983

Acute adrenocorticotropic hormone stimulation of adrenal corticosteroidogenesis. Discovery of a rapidly induced protein.

Two-dimensional electrophoretic techniques were used to identify and characterize a protein that is not produced in quiescent isolated rat adrenal cells but is produced in response to acute stimulation by adrenocorticotropic hormone (ACTH) or dibutyryl cAMP. The molecular weight of this protein is 28,000 (sodium dodecyl sulfate electrophoresis), and its isoelectric point is 6.5 (isoelectric focusing). Mapping of proteolytic peptides suggests that this induced protein (i) is quite similar in primary structure to another protein (p), which is produced only in nonstimulated adrenal cells. The time course of formation of protein i and its ACTH dose response closely parallel the increase of corticosteroid production in stimulated cells. The possibility that protein i is produced in response to increased levels of some steroid of the glucocorticoid pathway is precluded by the observation that inhibition of corticosteroid synthesis by aminoglutethimide does not alter the rate of production of i. Addition of cycloheximide before ACTH, which prevents stimulation of corticosteroidogenesis, also prevents formation of protein i implying that the production of protein i depends on continuing protein synthesis. [35S/32S]Methionine pulse-chase experiments, i.e. addition of excess [32S] methionine and ACTH after prelabeling with [35S]methionine, show that protein i is not produced from pre-existing protein p or other pre-existing proteins even if protein synthesis (and increased steroid production) is not inhibited. These findings exclude post-translational modification as a mechanism for the production of i but are consistent with p and i being related by cotranslational modification. Addition of cycloheximide after stimulation causes the formation of protein i to cease, but the amount of the protein does not decrease with the same kinetics as the return of corticosteroid production to its unstimulated level. [35S/32S]Methionine pulse-chase experiments imply protein i, even under conditions of ongoing ACTH stimulation and protein synthesis, is degraded with approximately the same kinetics as after cycloheximide inhibition. The close concurrence under a wide variety of experimental conditions between the appearance of protein i and the increase in adrenal corticosteroid production, coupled with the fact that the former does not occur as a result of the latter, make protein i a likely candidate for the postulated corticosteroidogenic stimulatory protein (Ferguson, J.J. (1962) Biochim. Biophys. Acta 57, 616-617). The fact that stimulation of steroidogenesis may occur via co-translational modification of a regulatory protein is an intriguing possibility which readily explains both the observed rapid and protein synthesis-dependent stimulation and the lack of dependence of stimulation on transcription (Schulster, D. (1974) Mol. Cell. Endocr. 1, 55-64).

Adrenal Glands↗

PubMed2001

Post-translational modifications of the regulatory subunits of cAMP-dependent protein kinases during G1/S progression.

During the G1/S transition of the cell cycle variations in the labelling by 8-N3-[32P]cAMP of the protein kinase A regulatory subunits RI and RII, used as a probe to monitor post-translational modifications that may regulate cAMP binding, were observed in synchronized HeLa cells. A decrease in 8-N3-[32P]cAMP labelling of RI, RII and RII phosphorylated by the catalytic subunit of PKA was correlated with the increased percentage of cells in phases G1. An increase in 8-N3-[32P]cAMP incorporated into the 54-kDa RII subunit during progression from G1 to S was correlated with an increase in intracellular cAMP. A transient increase in Mn-SOD activity was detected in cells arrested at the G1/S transition using two different techniques, suggesting that oxidative modulation of regulatory subunits by free radicals may modify cAMP binding sites during the cell cycle. Decreased photoaffinity labelling by 8-N3-[32P]cAMP of RI, RII and autophosphorylated RII subunits was found to be an inherent characteristic of PKA in the G1/S transition.

Azides↗

PubMed2007

Automated mass correction and data interpretation for protein open-access liquid chromatography-mass spectrometry.

Characterization of recombinant protein purification fractions and final products by liquid chromatography-mass spectrometry (LC/MS) are requested more frequently each year. A protein open-access (OA) LC/MS system was developed in our laboratory to meet this demand. This paper compares the system that we originally implemented in our facilities in 2003 to the one now in use, and discusses, in more detail, recent enhancements that have improved its robustness, reliability, and data reporting capabilities. The system utilizes instruments equipped with reversed-phase chromatography and an orthogonal accelerated time-of-flight mass spectrometer fitted with an electrospray source. Sample analysis requests are accomplished using a simple form on a web-enabled laboratory information management system (LIMS). This distributed form is accessible from any intranet-connected company desktop computer. Automated data acquisition and processing are performed using a combination of in-house (OA-Self Service, OA-Monitor, and OA-Analysis Engine) and vendor-supplied programs (AutoLynx, and OpenLynx) located on acquisition computers and off-line processing workstations. Analysis results are then reported via the same web-based LIMS. Also presented are solutions to problems not addressed on commercially available, small-molecule OA-LC/MS systems. These include automated transforming of mass-to-charge (m/z) spectra to mass spectra and automated data interpretation that considers minor variants to the protein sequence-such as common post-translational modifications (PTMs). Currently, our protein OA-LC/MS platform runs on five LC/MS instruments located in three separate GlaxoSmithKline R&D sites in the US and UK. To date, more than 8000 protein OA-LC/MS samples have been analyzed. With these user friendly and highly automated OA systems in place, mass spectrometry plays a key role in assessing the quality of recombinant proteins, either produced at our facilities or bought from external sources, without dedicating extensive amounts of analyst resource.

Animals↗

PubMed2003

Tyrosine oxidation products: analysis and biological relevance.

Dityrosine is found in several proteins as a product of UV irradiation, gamma-irradiation, aging, exposure to oxygen free radicals, nitrogen dioxide, peroxynitrite, and lipid hydroperoxides. Interest of dityrosine in proteins is based on its potential as a specific marker for oxidatively damaged proteins and their selective proteolysis, hence it could be used as a marker for oxidative stress. Dityrosine is also the product of normal post-translational processes affecting specific structural proteins. Since post-translational modification of a given amino acid in a protein is equivalent to the substitution of that residue by an analogue, it has been proposed that the covalent modification of amino acids may serve as a "marking" step for protein degradation.

Animals↗

PubMed2002

Dynamic O-glycosylation of nuclear and cytosolic proteins: further characterization of the nucleocytoplasmic beta-N-acetylglucosaminidase, O-GlcNAcase.

beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and dynamic post-translational modification implicated in protein regulation that appears to be functionally more similar to phosphorylation than to classical glycosylation. There are nucleocytoplasmic enzymes for the attachment and removal of O-GlcNAc. Here, we further characterize the recently cloned beta-N-acetylglucosaminidase, O-GlcNAcase. Both recombinant and purified endogenous O-GlcNAcase rapidly release free GlcNAc from O-GlcNAc-modified peptide substrates. The recombinant enzyme functions as a monomer and has kinetic parameters (K(m) = 1.1 mm for paranitrophenyl-GlcNAc, k(cat) = 1 s(-1)) that are similar to those of lysosomal hexosaminidases. The endogenous O-GlcNAcase appears to be in a complex with other proteins and is predominantly localized to the cytosol. Overexpression of the enzyme in living cells results in decreased O-GlcNAc modification of nucleocytoplasmic proteins. Finally, we show that the enzyme is a substrate for caspase-3 but, surprisingly, the cleavage has no effect on in vitro O-GlcNAcase activity. These studies support the identification of this protein as an O-GlcNAcase and identify important interactions and modifications that may regulate the enzyme and O-GlcNAc cycling.

Acetylglucosaminidase↗

PubMed1980

Acetylation of histone-like proteins of adenovirus type 5.

We examined acetylation of the histone-like adenovirus core proteins VII and V and the precursor of the major core protein, pVII, by measuring the incorporation of [14C]acetate. Adenovirus proteins pVII and V appeared to be acetylated, whereas protein VII was not. Label incorporated into these viral proteins in the form of acetate was metabolically stable, and labeling was not enhanced by treatment with sodium butyrate, an inhibitor of histone deacetylases. Viral protein acetylation therefore differs from the reversible acetylation of histones that has been implicated in transient alterations of chromatin structure. Inhibition of protein synthesis in infected cells resulted in a proportional reduction in [14C]acetate uptake into pVII and V, suggesting that these proteins undergo acetylation during protein synthesis and not as a post-translational modification. Therefore, these viral proteins are probably acetylated amino-terminally, a characteristic shared by three of the five major histone classes.

Acetates↗

PubMed2006

S-Nitrosogluthathione reductase activity of amphioxus ADH3: insights into the nitric oxide metabolism.

Nitric oxide (NO) is a signalling molecule involved in many physiological functions. An important via of NO action is through the S-nitrosylation of proteins, a post-translational modification that regulates the activity of enzymes, protein-protein interactions and signal transduction pathways. Alcohol dehydrogenase class III (ADH3) recognises S-nitrosoglutathione (GSNO), the main reservoir of non-protein S-nitrosothiol, and functions as an effective GSNO reductase (GSNOR) and as a safeguard against nitrosative stress. To investigate the evolutionary conservation of this metabolic role, we have produced recombinant Branchiostoma floridae ADH3. Pure preparations of ADH3 showed 2-fold higher activity as GSNOR than as formaldehyde dehydrogenase, the previously assumed main role for ADH3. To correlate ADH3 expression in the gut with areas of NO production, we analysed the tissue distribution of the nitric oxide synthase (NOS) enzyme in amphioxus larvae. Immunostaining of the NOS enzyme revealed expression in the gut and in the dorsal region of the club-shaped gland. Co-localization in the gut supports the ADH3 and NOS joint contribution to the NO/SNO homeostasis.

Aldehyde Oxidoreductases↗

PubMed1980

Metabolism of individual proteins in exponentially growing Escherichia coli.

The metabolism of 184 individual proteins was examined in exponentially growing Escherichia coli. Cells were labeled with [3H]leucine and [35S]methionine using either a pulse-chase or a continuous labeling method. Proteins were fractionated by two-dimensional gel electrophoresis and their stabilities relative to total protein were determined from the 3H/35S ratios. Forty-seven proteins appeared to be unstable and were probably either degraded or modified to electrophoretically distinct forms. The apparent half-lives of these proteins calculated from the pulse-chase data varied from 2.0 to 23 h. Twenty-seven proteins appeared to be products of post-translational modifications. One hundred fourteen proteins appeared to be stable. The molar ratios of leucyl and methionyl residues in individual proteins and in total protein were calculated by comparing their 3H/35S ratios to that of a protein with known amino acid composition. These values varied from 1.7 to 12. The half-lives of the unstable proteins did not exhibit a correlation with protein molecular weights, isoelectric points, or leucine/methionine ratios. It may be significant, however, that 6 of the 10 most unstable proteins had low leucine/methionine ratios whereas only 11% of all proteins tested had similarly low ratios.

Bacterial Proteins↗

PubMed1994

ADP-ribosylation and gene expression.

Gene expression can be defined as the conversion of information existing in a molecule of DNA into a mature RNA or protein product and each step in the process, which requires the concerted action of several macromolecules for completion, may be perturbed by the post-translational modification of specific proteins with ADP-ribose. The participation of poly(ADP-ribose) in the regulation of transcription initiation was examined using cell-free systems for both ribosomal RNA and ribosomal proteins. The presence or absence of poly(ADP-ribose) polymerase did not influence the transcription process. Similarly, under conditions optimal for poly(ADP-ribose) polymerase activity, no change in transcription was observed. A direct contribution of poly(ADP-ribosyl)ation to gene transcription thus could not be detected. In contrast, the addition of 3-aminobenzamide to quiescent hepatoma cells treated with insulin inhibited the stimulation of rRNA synthesis. The high concentrations necessary for this effect suggest that a mono(ADP-ribosyl)ation event participates in the cellular action of insulin. A role in the signal transduction pathway leading to activation of rRNA gene expression has been proposed.

ADP Ribose Transferases↗

PubMed2006

Demonstration of lysosomal localization for the mammalian ependymin-related protein using classical approaches combined with a novel density shift method.

Most newly synthesized soluble lysosomal proteins are delivered to the lysosome via the mannose 6-phosphate (Man-6-P)-targeting pathway. The presence of the Man-6-P post-translational modification allows these proteins to be affinity-purified on immobilized Man-6-P receptors. This approach has formed the basis for a number of proteomic studies that identified multiple as yet uncharacterized Man-6-P glycoproteins that may represent new lysosomal proteins. Although the presence of Man-6-P is suggestive of lysosomal function, the subcellular localization of such candidates requires experimental verification. Here, we have investigated one such candidate, ependymin-related protein (EPDR). EPDR is a protein of unknown function with some sequence similarity to ependymin, a fish protein thought to play a role in memory consolidation and learning. Using classical subcellular fractionation on rat brain, EPDR co-distributes with lysosomal proteins, but there is significant overlap between lysosomal and mitochondrial markers. For more definitive localization, we have developed a novel approach based upon a selective buoyant density shift of the brain lysosomes in a mutant mouse lacking NPC2, a lysosomal protein involved in lipid transport. EPDR, in parallel with lysosomal markers, shows this density shift in gradient centrifugation experiments comparing mutant and wild type mice. This approach, combined with morphological analyses, demonstrates that EPDR resides in the lysosome. In addition, the lipidosis-induced density shift approach represents a valuable tool for identification and validation of both luminal and membrane lysosomal proteins that should be applicable to high throughput proteomic studies.

Animals↗

PubMed2001

p53 protein at the hub of cellular DNA damage response pathways through sequence-specific and non-sequence-specific DNA binding.

Our environment contains physical, chemical and pathological agents that challenge the integrity of our DNA. In addition to DNA repair, higher multicellular organisms have evolved multiple pathways of response to damage including programmed cell death-apoptosis. The p53 protein appears to sense multiple types of DNA damage and coordinate with multiple options for cellular response. The p53 protein activities depend upon its DNA binding. Specific p53 protein post-translational modifications are required for efficient sequence-specific binding and transcriptional activities. Non-sequence-specific DNA binding may involve a wide spectrum of p53 proteins and predominate as DNA damage is more severe or p53 protein is more highly induced. p53 protein is not strictly required for DNA damage sensing and repair. Rather, p53 protein may govern an apoptosis checkpoint through competition with DNA repair proteins for non-sequence-specific binding to exposed single-stranded regions in the DNA duplex. This model provides a framework for testing mechanisms of p53-mediated apoptosis dependent upon the p53 protein modification state, the level of p53 protein accumulation, the level of DNA damage and the capacity of the damaged cell to repair.

Animals↗

PubMed1995

A novel strategy to identify maternal and paternal inheritance in the mouse.

A novel strategy for identifying proteins which reveal maternal or paternal inheritance in the mouse is presented. Using two-dimensional electrophoresis we investigated protein expression patterns of adult liver and different embryonic and extraembryonic tissue in C57BL/6Crl and in DBA/2Crl mice, as well as in their reciprocal hybrids. We found three groups of protein spots which showed maternal or paternal inheritance of quantitative variations. These proteins were characterized by N-terminal or internal amino acid sequencing, by determination of the amino acid composition, by glycoprotein staining and RNA expression analysis. The three proteins identified were: alpha-enolase, cyclophilin and beta-group hemoglobins. The parental effects observed for alpha-enolase and cyclophilin were found to be due to parent-specific post-translational modifications of these proteins. For the beta-group hemoglobins our results suggested parental effects on the transcriptional level.

Amino Acid Isomerases↗

PubMed1990

Fatty acids on the A/Japan/305/57 influenza virus hemagglutinin have a role in membrane fusion.

The covalent attachment of fatty acid moieties to proteins is a widespread post-translational modification of viral and cell proteins yet the functional consequences of acylation are not well understood. We have determined that the A/Japan/305/57 influenza virus hemagglutinin (HA) contains three potential acylation sites at cysteine residues 211, 218 and 221 in the cytoplasmic domain of the molecule. Site-directed mutagenesis of one or more of these sites has no effect on biosynthesis, transport or receptor binding activity of the molecule; however, modification of any single site is sufficient to abolish completely or inhibit severely membrane fusion activity, a function essential for virus infectivity. We present a molecular model of the transmembrane and cytoplasmic domains of the HA to illustrate the potential orientation of these fatty acids and to provide a conceptual framework for further experimentation.

Acylation↗

PubMed2000

N-glycosylation and residue 96 are involved in the functional properties of UDP-glucuronosyltransferase enzymes.

The recent cloning of several human and monkey UDP-glucuronosyltransferase (UGT) 2B proteins has allowed the characterization of these steroid metabolic enzymes. However, relatively little is known about the structure-function relationship, and the potential post-translational modifications of these proteins. The mammalian UGT2B proteins contain at least one consensus asparagine-linked glycosylation site NX(S/T). Endoglycosidase H digestion of the human and monkey UGT2B proteins demonstrates that only UGT2B7, UGT2B15, UGT2B17, and UGT2B20 are glycosylated. Although UGT2B15 and UGT2B20 contain three and four potential glycosylation sites, respectively, site-directed mutagenesis revealed that both proteins are glycosylated at the same first site. In both proteins, abolishing glycosylation decreased glucuronidation activity; however, the K(m) values and the substrate specificities were not affected. Despite the similarities between UGT2B15 and UGT2B20, UGT2B20 is largely more labile than UGT2B15. Treating HK293 cells stably expressing UGT2B20 with cycloheximide for 2 h decreased the enzyme activity by more than 50%, whereas the activity of UGT2B15 remained unchanged after 24 h. The UGT2B20 protein is unique in having an isoleucine at position 96 instead of an arginine as found in all the other UGT2B enzymes. Changing the isoleucine in UGT2B20 to an arginine stabilized enzyme activity, while the reciprocal mutation in UGT2B15 R96I produced a more labile enzyme. Secondary structure predictions of UGT2B proteins revealed a putative alpha-helix in this region in all the human and monkey proteins. This alpha-helix is shortest in UGT2B20; however, the helix is lengthened in UGT2B20 I96R. Thus, it is apparent that the length of the putative alpha-helix between residues 84 and 100 is a determining factor in the stability of UGT2B enzyme activity. This study reveals the extent and importance of protein glycosylation on UGT2B enzyme activity and that the effect of residue 96 on UGT2B enzyme stability is correlated to the length of a putative alpha-helix.

Amino Acid Sequence↗

PubMed1989

Expression of the kinase domain of mouse protein kinase C in E. coli.

The kinase domain of mouse protein kinase C type alpha has been expressed at high levels in E. coli. The protein has been purified 500-fold taking advantage of the fact that highly expressed fusion proteins precipitate out in the bacterial cell and can be solubilized in 7 M urea. The purified protein can be detected with an antibody generated against a PKC alpha derived synthetic peptide. The purified kinase domain exhibits no measurable kinase activity in a protamin phosphorylation assay. This could be an indication that post-translational modifications of the protein kinase domain which do not happen in bacteria are a requirement for functional enzyme activity or that the regulatory domain of protein kinase C is indispensable for kinase function.

Animals↗