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At least 433 records · Page 24Linked to original sources
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↗
PubMed1998

DNA binding of activator protein-1 is increased in human mesangial cells cultured in high glucose concentrations.

Human mesangial cells (HMC) grown in high glucose environments synthesize excessive amounts of extracellular matrix proteins (ECM). The promoter regions of certain ECM genes contain TPA (phorbol ester)-responsive element (TRE) motifs that bind the transcription factor, activator protein-1 (AP-1), a complex of Jun and other phosphoproteins. AP-1 binding to the TRE promoter is regulated by the quantity, composition and post-translational modifications of proteins in the AP-1 complex. We report an increased binding of AP-1 to TRE oligonucleotides in HMC cultured chronically (5 days) in high glucose environments (30 mM d-glucose). This increased binding is not due to differences in the nuclear quantity of AP-1 proteins or in the composition of the AP-1 complex when compared to AP-1 proteins from cells grown in normal glucose (5 mM d-glucose). A 30 mM l-glucose environment also increased AP-1 binding, but to a degree less than d-glucose. The increased AP-1 binding was partly reversed by treatment of HMC with Calphostin C or Bisindolylmaleimide I suggesting a partial role of the protein kinase C (PKC) pathway in mediating AP-1 binding. AP-1 binding was unaffected by treatment of cells with the MEK inhibitor PD 98059. In addition, increased AP-1 binding persisted for at least 48 hours after media glucose concentrations were normalized. The level of Jun-NH2-terminal kinase (JNK) activity and the phosphorylation of the JNK kinase, SEK1, were unchanged by chronic high glucose concentrations. These studies suggest that in HMC cultured in chronic high glucose, post-translational modifications increase the binding of AP-1 to the TRE motif.

Binding Sites↗
PubMed1999

Protein Glycosylation in Heterogeneous Expression Systems.

Glycosylation the addition of sugar residues to peptide backbone is the most common post-translational modification of proteins in eukaryotic cells and has important functions in secretion antigenicity and metabolism of glycoproteins. Majority of proteins of pharmaceutical interest are glycoproteins. In recent years great advances have been made in understanding the versatile biological functions of carbohydrates in the accurate analysis of glycoprotein heterogeneity and in controlling the biosynthetic pathways of glycosylation. The aim of this mini review is to discuss general advantages and potential problems of different systems for expressing recombinant glycoproteins.

Journal Article↗
PubMed2003

Expression and enzymatic activity of small intestinal tissue transglutaminase in celiac disease.

OBJECTIVES: The molecular and functional properties of small intestinal tissue transglutaminase are largely unknown despite growing interest because of its role in celiac disease (CD). In this study, we aimed to evaluate tissue transglutaminase expression and enzymatic activity in bioptic fragments obtained from the duodenum of untreated individuals with CD and from control subjects. METHODS: Analysis of tissue transglutaminase mRNA expression was performed by reverse transcription-polymerase chain reaction (RT-PCR). The presence of the enzyme in bioptic fragments as well as in homogenates from CD patients and controls was revealed by immunohistochemistry and Western blot, respectively, using the antitissue transglutaminase CUB 7402 clone. To evaluate in situ transglutaminase activity, sections of bioptic fragments were incubated in the presence of 5 mmol/L CaCl(2) with 5-(biotinamido)pentylamine or, alternatively, with a biotinylated glutamine-containing hexapeptide (TVQQEL) and the biotinylated 31-43 A-gliadin-derived peptide. RESULTS: Tissue transglutaminase mRNA levels were 1.0-fold higher (p < 0.05) in CD patients than in controls. Immunohistochemistry and in situ demonstration of enzymatic activity in celiac mucosa clearly showed an increased expression of active tissue transglutaminase in the extracellular matrix of the subepithelial region and in the enterocytes. Staining of the biotinylated 31-43 A-gliadin peptide in the same area of tissue transglutaminase suggested the presence of lysine-donor substrates in intestinal mucosa. CONCLUSIONS: Tissue transglutaminase is more expressed and active in defined areas of the small intestinal mucosa from patients with CD. The presence in the celiac mucosa of proteins able to act as amine-donor substrates suggests that tissue transglutaminase-mediated post-translational modification of proteins cross-linked with gliadin peptides may represent a pathogenic mechanism of CD.

Adolescent↗
PubMed2006

Protein primary structure using orthogonal fragmentation techniques in Fourier transform mass spectrometry.

Proteomics analysis using tandem mass spectrometry requires informative backbone fragmentation of peptide ions. Collision-activated dissociation (CAD) of cations alone is not sufficiently informative to satisfy all requirements. Thus, there is a need to supplement CAD with a complementary fragmentation technique. Electron capture dissociation (ECD) is complementary to collisional excitation in terms of the cleavage of a different bond (N-Calpha versus C-N bond) and other properties. CAD-ECD combination improves protein identification and enables high-throughput de novo sequencing of peptides. ECD and its variants are also useful in mapping labile post-translational modifications in proteins and isomer differentiation; for example, distinguishing Ile from Leu, iso-Asp from Asp and even D- from L-amino acid residues.

Amino Acid Sequence↗
PubMed1979

Current status of age altered enzymes: alternative mechanisms.

The occurrence of inactive enzyme molecules in a variety of tissues and animal species has been shown to be of a general nature. The levels of inactive enzyme molecules found in old animals were produced by amino acid analogs in young animals. These levels have been shown to be initially detrimental but subsequently the young system shows recovery by efficiently disposing of the analog-modified proteins. In old animals this disposal is considerably less efficient. Evidence is presented which suggests that post-translational modifications of proteins are the main cause of enzyme inactivation in old animals. Amino acid substitutions and modifications involving charge differences apparently do not contribute significantly to this phenomenon.

Acetylation↗
PubMed2005

The physiology and pathophysiology of nitric oxide in the brain.

Nitric oxide (NO) is a molecule with pleiotropic effects in different tissues. NO is synthesized by NO synthases (NOS), a family with four major types: endothelial, neuronal, inducible and mitochondrial. They can be found in almost all the tissues and they can even co-exist in the same tissue. NO is a well-known vasorelaxant agent, but it works as a neurotransmitter when produced by neurons and is also involved in defense functions when it is produced by immune and glial cells. NO is thermodynamically unstable and tends to react with other molecules, resulting in the oxidation, nitrosylation or nitration of proteins, with the concomitant effects on many cellular mechanisms. NO intracellular signaling involves the activation of guanylate cyclase but it also interacts with MAPKs, apoptosis-related proteins, and mitochondrial respiratory chain or anti-proliferative molecules. It also plays a role in post-translational modification of proteins and protein degradation by the proteasome. However, under pathophysiological conditions NO has damaging effects. In disorders involving oxidative stress, such as Alzheimer's disease, stroke and Parkinson's disease, NO increases cell damage through the formation of highly reactive peroxynitrite. The paradox of beneficial and damaging effects of NO will be discussed in this review.

Animals↗
PubMed2001

Glycosyl transfer: a history of the concept's development and view of its major contributions to biochemistry.

Humans have fewer protein-coding genes than expected for all the inherent complexities of development. Supplementary factors include the post-translational modification of proteins by glycosylation. The latter term plus transglycosylation, glycosyltransferases, and 'to glycosylate' are used in biochemistry as though they always existed. Instead they have a history that can bring new insights in this science area to younger investigators. The present account describes five decades of findings and ideas on enzymic saccharide synthesis leading, finally to a rational theory that will surely continue to serve the biosciences well in the future.

Biochemistry↗
PubMed2004

Does looping and clustering in the nucleus regulate gene expression?

There has been considerable interest in the way that chromatin is spatially organised within the cell nucleus and how that may relate to gene expression and its control. New molecular techniques have identified looped chromatin domains at the mammalian beta-globin and the Drosophila hsp70 loci. Looped domains may insulate chromatin from the influence of neighbouring domains, and the bases of loops may also act to concentrate proteins locally within the nucleus. The spatial clustering of sequences from the Drosophila bithorax complex, located in trans, has also been demonstrated. An emerging theme is that bringing DNA and proteins together within a defined sub-region of the nuclear volume facilitates both the activation and the repression of gene expression. Nuclear compartments may also be involved in the post-translational modification of proteins by sumoylation and ubiquitylation.

Animals↗
PubMed1993

The human keratinocyte two-dimensional gel protein database: update 1993.

The master two-dimensional gel database of human keratinocytes currently lists 3038 cellular proteins (2127 isoelectric focusing, IEF; and 911 nonequilibrium pH gradient electrophoresis, NEPHGE) many of which correspond to post-translational modifications. 763 proteins have been identified (protein name, organelle components, etc.) and they are listed both in alphabetical order and with increasing SSP number, together with their M(r), pI, cellular localization and credit to the investigator(s) that aided in the identification. Furthermore we have listed 176 proteins that have been microsequenced so far and that are recorded in this database. We also include synthetic images depicting some interesting sets of proteins identified so far; these include components of hnRNP's, proteasomes or prosomes, ribosomes, as well as assorted organelle markers, GTP-binding proteins, calcium binding proteins, stress proteins, autoantigens, differentiation markers and psoriasis upregulated proteins. The aim of the comprehensive database is to gather, through a systematic study of keratinocytes, qualitative and quantitative information on proteins and their genes that may allow us to identify abnormal patterns of gene expression and ultimately to pinpoint signaling pathways and components affected in various skin diseases, cancer included.

Databases, Factual↗
PubMedDate not supplied

Carbamoylation of hemoglobin in uremic patients determined by antibody specific for homocitrulline (carbamoylated epsilon-N-lysine).

Polyclonal antisera to epsilon-amino-carbamoyl-lysine (homocitrulline) on guinea pig carbamoylated low density lipoprotein were used as probes to identify homocitrulline. These antisera detect homocitrulline in the immunogen and on other carbamoylated blood proteins including those from different species. In vivo carbamoylation of intracellular hemoglobin obtained from uremic patients and carbamoyl-proteins, carbamoylated in vitro, located on erythrocyte and lymphocyte cell surface membranes were detected. Solid phase, direct and competitive ELISAs for homocitrulline were used to analyze carbamoylated blood proteins. In uremic patients carbamoylation of protein by endogenous urea-derived cyanate occurs on the epsilon-amino group of lysine residues. Identification of carbamoylated proteins from uremic patients using an immunochemical probe of site specific antibody provides a way to monitor post-translational modification of proteins due to uremia and may give further insight for understanding the pathophysiology of end stage renal disease.

Animals↗
PubMed1998

Type II protein secretion by Pseudomonas aeruginosa: genetic suppression of a conditional mutation in the pilin-like component XcpT by the cytoplasmic component XcpR.

Pseudomonas aeruginosa exports a number of hydrolytic enzymes and toxins using the type II or general secretion pathway, found in a variety of Gram-negative bacteria and requiring the functions of at least 12 gene products (XcpP-Z and PilD/XcpA in P. aeruginosa). A number of these gene products are homologues of components of the type IV pilus biogenesis system, including four proteins, XcpT-W, which are highly similar to the pilin subunit in their size, localization and post-translational modifications. These proteins, in addition to the pilin subunit, are cleaved and methylated by the PilD/XcpA prepilin peptidase, but their interactions with other components of the export apparatus are unclear. Using a medium developed for the selection of export-proficient P. aeruginosa strains, we have isolated temperature-sensitive mutations in the xcpT gene and extragenic suppressors for one of the mutants. These suppressors fall into two classes, one that maps outside of the xcpP-Z gene cluster and may define additional cellular functions that are required for export, and a second that maps to the xcpR gene product and indicates a potential protein-protein interaction connecting two different cellular compartments and required for the assembly or function of the export apparatus.

Bacterial Outer Membrane Proteins↗
PubMed1982

Nonenzymatic galactosylation of human serum albumin. In vitro preparation.

Incubation of purified human serum albumin with D-[1-14C]galactose (5 mM) or D-[1-14C]glucose (5 mM) in vitro for 7 days under physiological conditions resulted in the time-dependent accumulation of radioactivity into trichloroacetic acid-precipitable material. Comparative studies indicated that the rate of sugar incorporation into albumin increased with increasing pH and temperature of incubation and followed a first order dependence with regard to monosaccharide and albumin concentrations. The extent of nonenzymatic galactosylation of human albumin was approximately 300% greater than the extent of nonenzymatic glucosylation under equivalent experimental conditions. Prolonged dialysis of the modified albumins against a large excess of the unlabeled monosaccharides failed to alter the amount of protein-bound radiolabeled carbohydrate, suggesting that the linkage between sugar and albumin is covalent in nature. The post-translational modification of proteins by nonenzymatic galactosylation may be of physiological significance in individuals with reduced galactokinase or galactose-1-phosphate uridyl transferase activities.

Carbon Radioisotopes↗
PubMed1995

The importance of the GTP-binding protein tissue transglutaminase in the regulation of cell cycle progression.

Tissue transglutaminase (tTgase) is a GTP-binding Ca(2+)-dependent enzyme which catalyses the post-translational modification of proteins via epsilon(gamma-glutamyl) lysine bridges. Recent evidence suggests that the GTP-binding activity of tTgase may be important in intracellular signaling thus explaining some of the diverse suggested roles for the enzyme. In the following work a malignant hamster fibrosarcoma (Met B) has been stably transfected with both the full length tTgase cDNA (wild type) and a mutant form of the cDNA whereby the active site cysteine (Cys 277) has been replaced by serine. Expression of this mutant cDNA leads to a protein with GTP binding activity which is deficient of protein crosslinking activity. When synchronised into S-phase and allowed to progress through the cell cycle tTgase transfected clones (both mutant and wild type), when compared to transfected controls, show a delayed progression from S-phase to G2/M when analysed by flow cytometry which appears to be elicited by the G-protein activity of the tTgase.

Animals↗
PubMed2002

Molecular characterisation of a xyloglucan oligosaccharide-acting alpha-D-xylosidase from nasturtium (Tropaeolum majus L.) cotyledons that resembles plant 'apoplastic' alpha-D-glucosidases.

We report the isolation, sequencing and analysis of the cDNA corresponding to an alpha-D-xylosidase involved in the mobilisation of xyloglucan from the cotyledons of germinated nasturtium (Tropaeolum majus L.) seeds. The translated open reading frame (2,808 bp including the stop codon), gave a polypeptide of 935 amino acids. It included the sequences of eleven peptides obtained by endo-proteinase digestion of the protein, and a putative hydrophobic signal sequence characteristic of a protein that is directed through the plasma membrane. The deduced molecular weight of the translated protein was appreciably higher than the molecular weight determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, suggesting post-translational modification. The protein sequence showed high homology (76.0% identity over 896 amino acids) with a putative alpha-xylosidase sequence from Arabidopsis thaliana and there was homology with several alpha-glucosidases, notably those associated with the plant cell apoplast. The enzyme is a member of Family 31 of the glycosyl hydrolases and it fits into Clade 1 of the phylogenic analysis of alpha-glucosidases. Although in vivo the nasturtium enzyme catalyses mobilisation of cell wall xyloglucan, the homology of its primary sequence with alpha-glucosidases prompted study of its action on a range of alpha-glucosides. It was active against several alpha-(1-->4)-and alpha-(1-->6)-linked substrates, the former being hydrolysed faster. The functional and evolutionary relationships between this alpha-D-xylosidase and plant "apoplastic" alpha-D-glucosidases are discussed.

Amino Acid Sequence↗
PubMed2002

Protein acylation in the cardiac muscle like cell line, H9c2.

Besides serving as oxidisable substrates, fatty acids (FA) are involved in co- and post-translational modification of proteins (protein acylation). Despite the high rate of fatty acid utilisation in the heart, information on protein acylation in cardiac muscle is scarce. To explore this subject in more detail, we used the H9c2 cell line as an experimental model. After incubation with 3H-palmitate or 3H-myristate, cells were lysed and proteins precipitated, followed by extensive delipidation. The delipidated proteins were subjected to SDS-PAGE and transferred to nitro-cellulose prior to autoradiography. In addition, TLC was used to separate the various lipid classes. The first aspect we addressed was the extent of protein acylation as a function of time, relative to fatty acid incorporation into various lipid classes. Cells were incubated for 30 min, 1 h and 2 h with 100 microCi palmitate (PA, 2.3 nmol) or 125 microCi myristate (MA, 2.5 nmol). Palmitoylation increased from 0.48 +/- 0.25 to 1.25 +/- 0.56 microCi/mg protein between 30 min to 2 h, while myristoylation increased from 0.25 +/- 0.12 to 0.77 +/- 0.36 microCi/mg protein. Furthermore, delipidated proteins subjected to autoradiography showed that a set of distinct proteins was labelled with 3H-palmitate. Incorporation into phospholipids (PL) increased from 40-60% of the total amount of radio-labelled PA or MA supplied between 30 min and 2 h. Only the FA pool differed between MA and PA, with a higher FA content present after incubations with MA. Second, we investigated palmitoylation and incorporation into cellular lipids as a function of the amount of PA applied. Palmitoylation showed saturation at high PA concentrations. The percentage incorporation of 3H-PA in the various lipids depended on the amount of PA added: a decline in the PL pool with a concomitant increase in the size of the diacylglycerol pool at high PA concentrations. Third, inhibition of palmitoylation by cerulenin and tunicamycin was investigated. While both were able to inhibit palmitoylation, cerulenin also inhibited the incorporation of PA into various lipid classes, indicating differences in inhibitory action.

Acylation↗