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Results for “Protein Modification, Translational”

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PubMed2006

The impact of protein characterization in structural proteomics.

Protein characterization plays a role in two key aspects of structural proteomics. The first is the quality assessment of the produced protein preparations. Obtaining well diffracting crystals is one of the major bottlenecks in the structure-determination pipeline. Often, this is caused by the poor quality of the protein preparation used for crystallization trials. Hence, it is essential to perform an extensive quality assessment of the protein preparations prior to crystallization and to use the results in the evaluation of the process. Here, a protein-production and crystallization strategy is proposed with threshold values for protein purity (95%) and monodispersity (85%) below which a further optimization of the protein-production process is strongly recommended. The second aspect is the determination of protein characteristics such as domains, oligomeric state, post-translational modifications and protein-protein and protein-ligand interactions. In this paper, applications and new developments of protein-characterization methods using MS, fluorescence spectroscopy, static light scattering, analytical ultracentrifugation and small-angle X-ray scattering within the EC Structural Proteomics in Europe contract are described. Examples of the application of the various methods are given.

Crystallization↗

PubMed2005

Two separate motifs cooperate to target stathmin-related proteins to the Golgi complex.

The appropriate targeting of membrane-associated proteins involves a diversity of motifs including post-translational modifications and specific protein sequences. Phosphoproteins of the stathmin family are important regulators of microtubule dynamics, in particular in the developing and mature nervous system. Whereas stathmin is cytosolic, SCG10, SCLIP and the splice variants RB3/RB3'/RB3'' are associated with Golgi and vesicular membranes, through their palmitoylated N-terminal A domains. In order to identify essential motifs involved in this specific targeting, we examined the subcellular distribution of various subdomains derived from domain A of SCG10 fused with GFP. We show that the Golgi localization of SCG10 results from the cooperation of two motifs: a membrane-anchoring palmitoylation motif and a newly identified Golgi-specifying sequence. The latter displayed no targeting activity by itself, but retained a Golgi-specifying activity when associated with another membrane-anchoring palmitoylation motif derived from the protein GAP-43. We further identified critical residues for the specific Golgi targeting of domain A. Altogether, our results give new insight into the regulation of the subcellular localization of stathmin family proteins, an important feature of their physiological functions in differentiating and mature neural cells. More generally we provide new information on essential mechanisms of functional protein subcellular targeting.

Amino Acid Motifs↗

PubMed2005

MALDI QqTOF MS combined with off-line HPLC for characterization of protein primary structure and post-translational modifications.

The addition of off-line high-performance liquid chromatography to matrix-assisted laser desorption/ionization mass spectrometry greatly reduces congestion in the mass spectra, and also provides complete decoupling of the separation process from mass detection and measurement. This removes the time constraints inherent in on-line coupling, and so enables the detailed mass-spectrometric study of samples at later times. We describe here our use of this method to successfully characterize two "unknown" protein mixtures that were set as problems by the ABRF Proteomics Research Group (PRG) in the years 2003 and 2004.

Amino Acid Sequence↗

PubMed2005

Ubiquitinated annexin A2 is enriched in the cytoskeleton fraction.

Annexin A2 is a multifunctional protein and its cellular functions are regulated by post-translational modifications and ligand binding. When purified from porcine intestinal mucosa and transformed mouse Krebs II cells, SDS-PAGE revealed high-molecular-mass forms in addition to the 36 kDa protomer. These forms were identified as poly-/multi-ubiquitin conjugates of annexin A2, and ubiquitination represents a novel post-translational modification of this protein. Subcellular fractionation of mouse Krebs II cells revealed an enrichment of annexin A2-ubiquitin conjugates in the Triton X-100 resistant cytoskeleton fraction, suggesting that ubiquitinated annexin A2 may have a role associated with its function as an actin-binding protein.

Animals↗

PubMed2005

Multisite protein modification and intramolecular signaling.

Post-translational modification is a major mechanism by which protein function is regulated in eukaryotes. Instead of single-site action, many proteins such as histones, p53, RNA polymerase II, tubulin, Cdc25C and tyrosine kinases are modified at multiple sites by modifications like phosphorylation, acetylation, methylation, ubiquitination, sumoylation and citrullination. Multisite modification on a protein constitutes a complex regulatory program that resembles a dynamic 'molecular barcode' and transduces molecular information to and from signaling pathways. This program imparts effects through 'loss-of-function' and 'gain-of-function' mechanisms. Among the latter, covalent modifications specifically recruit a diverse array of modules, including the SH2 domain, 14-3-3, WW domain, Polo box, BRCT repeat, bromodomain, chromodomain, Tudor domain and motifs binding to ubiquitin and other protein modifiers. Such recruitments are often modulated by modifications occurred at neighboring and distant sites. Multisite modification thus coordinates intermolecular and intramolecular signaling for the qualitative and quantitative control of protein function in vivo.

Acetylation↗

PubMed2006

Effects of ligands on the stability of tissue transglutaminase: studies in vitro suggest possible modulation by ligands of protein turn-over in vivo.

Tissue transglutaminase catalyzes irreversible post-translational modification of specific protein substrates by either crosslinkage or incorporation of primary amines into glutamine residues, through glutamyl-amide isopeptide bonds. Modulation in vivo of these reactions (collectively called "transamidation") is brought about by both ligand dependent effects (chiefly, activation by calcium and inhibition by GTP) as well as by variation in enzyme tissue levels by transcriptional effects. Accumulating observations that the enzyme stability in vitro is greatly affected by interaction with ligands led us to postulate that also the turn-over in vivo might be modulated by ligands opening new scenarios on the regulation of the tissue transamidating activity. This proposal is consistent with data obtained in in vitro cell culture systems and has important implications for the expression of activity in vivo.

Animals↗

PubMed2005

Accumulation of the hydroxyl free radical markers meta-, ortho-tyrosine and DOPA in cataractous lenses is accompanied by a lower protein and phenylalanine content of the water-soluble phase.

Post-translational modifications of lens proteins play a crucial role in the formation of cataract during ageing. The aim of our study was to analyze protein composition of the cataractous lenses by electrophoretic and high-performance liquid chromatographic (HPLC) methods. Samples were obtained after extracapsular cataract surgery performed by phacoemulsification technique from cataract patients with type 2 diabetes mellitus (DM CAT, n = 22) and cataract patients without diabetes (non-DM CAT, n = 20), while non-diabetic non-cataractous lenses obtained from cadaver eyes served as controls (CONTR, n = 17). Lens fragments were derived from the surgical medium by centrifugation. Samples were homogenized in a buffered medium containing protease inhibitor. Soluble and insoluble protein fractions were separated by centrifugation. The electrophoretic studies were performed according to Laemmli on equal amounts of proteins and were followed by silver intensification. Oxidized amino acid and Phe content of the samples were also analyzed by HPLC following acid hydrolysis of proteins. Our results showed that soluble proteins represented a significantly lower portion of the total protein content in cataractous lenses in comparison with the control group (CONTR, 71.25%; non-DM CAT, 32.00%; DM CAT, 33.15%; p < 0.05 vs CONTR for both). Among the proteins, the crystallin-like proteins with low-molecular weight can be found both in the soluble and insoluble fractions, and high-molecular weight aggregates were found mainly in the total homogenates. In our HPLC analysis, oxidatively modified derivatives of phenylalanine were detected in cataractous samples. We found higher levels of m-Tyr, o-Tyr and DOPA in the total homogenates of cataractous samples compared to the supernatants. In all three groups, the median Phe/protein ratio of the total homogenates was also higher than that of the supernatants (total homogenates vs supernatants, in the CONTR group 1102 vs 633 micromol/g, in the DM CAT group 1187 vs 382 micromol/g and in the non-DM CAT group 967 vs 252 micromol/g; p < 0.05 for all). In our study we found that oxidized amino acids accumulate in cataractous lenses, regardless of the origin of the cataract. The accumulation of the oxidized amino acids probably results from oxidation of Phe residues of the non-water soluble lens proteins. We found the presence of high-molecular weight protein aggregates in cataractous total homogenates, and a decrease of protein concentration in the water-soluble phase of cataractous lenses. The oxidation of lens proteins and the oxidative modification of Phe residues in key positions may lead to an altered interaction between protein and water molecules and thus contribute to lens opacification.

Aged↗

PubMed2002

Transmembrane topology of AgrB, the protein involved in the post-translational modification of AgrD in Staphylococcus aureus.

The accessory gene regulator (agr) of Staphylococcus aureus is the central regulatory system that controls the gene expression for a large set of virulence factors. This global regulatory locus consists of two transcripts: RNAII and RNAIII. RNAII encodes four genes (agrA, B, C, and D) whose gene products assemble a quorum sensing system. RNAIII is the effector of the Agr response. Both the agrB and agrD genes are essential for the production of the autoinducing peptide, which functions as a signal for the quorum sensing system. In this study, we demonstrated the transmembrane nature of AgrB protein in S. aureus. A transmembrane topology model of AgrB was proposed based on AgrB-PhoA fusion analyses in Escherichia coli. Two hydrophilic regions with several highly conserved positively charged amino acid residues among various AgrBs were found to be located in the cytoplasmic membrane as suggested by PhoA-AgrB fusion studies. However, this finding is inconsistent with the putative transmembrane profile of AgrB by computer analysis. Furthermore, we detected an intermediate peptide of processed AgrD from S. aureus cells expressing AgrB and a 6 histidine-tagged AgrD. These results provide direct evidence that AgrB is involved in the proteolytic processing of AgrD. We speculate that AgrB is a novel protein with proteolytic enzyme activity and a transporter facilitating the export of the processed AgrD peptide.

Amino Acid Sequence↗

PubMed1998

Novel inhibitors against the transglutaminase-catalysed crosslinking of lens proteins.

Post-translational modifications by transglutaminase may contribute to the remodeling of cellular architecture in the development of lens fiber cells, and there is evidence that the enzyme may also play a role in cataract formation. It catalyses hydrolytic deamidations as well as amide exchanges on select glutamine side chains at endo positions in a small subset of proteins of the lens. N epsilon(gamma-glutamyl)lysine crosslinks, the characteristic hallmarks of transglutaminase activity, were identified in polymers isolated from human cataract. Following up on our earlier studies relating to the inhibition of protein crosslinking by the Ca(2+)-activated transglutaminase in the lens, we have now examined the effects of 2-[(2-oxopropyl)thio]-imidazolium derivatives, recently described as active site-directed inhibitors for this family of enzymes. First, we have shown that the compounds at concentrations of 1-2 microM were effective in blocking the transamidating activities of partially purified lens transglutaminase. Then we focused on their efficacy in preventing the formation of the ca. 55 kDa beta crystallin dimers in the whole lens tissue. The production of these dimers, crosslinked by N epsilon(gamma-glutamyl)lysine isopeptide bridges, is an early sign of transglutaminase action in rabbit lens, and it can be readily documented by the SDS-PAGE analysis of proteins remaining in the soluble phase after brief exposure of the homogenate to Ca2+. The new compounds proved to be potent inhibitors of transglutaminase also in this preparation, preventing the crosslinking event at ca. 1 microM concentration. Moreover, even when applied at a 1,000-fold greater concentration (2 mM), they did not interfere with the action of calpain which, similarly to the activation of the transglutaminase system, is triggered by the addition of Ca2+. The high selectivity of the new compounds for differentially blocking only the transglutaminase and not the calpain of the lens, is all the more remarkable because these two enzymes share several mechanistic and structural similarities.

Animals↗

PubMed1999

Food plant-delivered cholera toxin B subunit for vaccination and immunotolerization.

Developments in recombinant DNA technology have enabled molecular biologists to introduce a variety of novel genes into plant species for specific purposes. From crop improvement to vaccine antigen and antibody production, plants are attractive bioreactors for production of recombinant proteins, as their eukaryotic nature often permits appropriate post-translational modification of recombinant proteins to retain native biological activity. The autotrophic growth of plants requires only soil minerals, water, nitrogen, sunlight energy and carbon dioxide for the synthesis of constituent proteins. Furthermore, production of biologically active proteins in food plants provides the advantage of direct delivery through consumption of edible transformed plant tissues. The production of cholera toxin B subunit in potato plants and applications for prevention of infectious and autoimmune disease are explained in this contribution.

Animals↗

PubMed2001

Protein kinase C isoforms in human erythrocytes.

Erythrocytes are the most abundant cells in blood and carry out the vital function of oxygen transport. These cells lack nuclei and do not synthesise new proteins. Their cellular responses are modulated entirely by post-translational modifications in existing proteins. Phosphorylation mediated by protein kinase C (PKC) plays a significant role in red cell physiology. To date PKC alpha and zeta are the only isoforms reported to be expressed in erythrocytes. Upon activation they influence cytoskeletal integrity and erythrocyte functions. In this study we report for the first time the presence of PKC iota and PKC mu in addition to PKC alpha and zeta in human erythrocytes. All isoforms were present only in the cytosolic fraction. PKC alpha was the only isoform that translocated to the membrane upon stimulation with phorbol myristate acetate (PMA). It could thus mediate several of the reported PMA-regulated membrane modifications. Investigations on alterations in PMA-mediated phosphorylation of erythrocyte skeletal components in disorders such as chronic myeloid leukaemia can now focus on PKC alpha, which is the only isoform in erythrocytes, which translocates to the membrane on stimulation of the cells.

Enzyme Activation↗

PubMed2000

DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts.

The cell cycle checkpoint mechanisms ensure the order of cell cycle events to preserve genomic integrity. Among these, the DNA-replication and DNA-damage checkpoints prevent chromosome segregation when DNA replication is inhibited or DNA is damaged. Recent studies have identified an outline of the regulatory networks for both of these controls, which apparently operate in all eukaryotes. In addition, it appears that these checkpoints have two arrest points, one is just before entry into mitosis and the other is prior to chromosome separation. The former point requires the central cell-cycle regulator Cdc2 kinase, whereas the latter involves several key regulators and substrates of the ubiquitin ligase called the anaphase promoting complex. Linkages between these cell-cycle regulators and several key checkpoint proteins are beginning to emerge. Recent findings on post-translational modifications and protein-protein interactions of the checkpoint proteins provide new insights into the checkpoint responses, although the functional significance of these biochemical properties often remains unclear. We have reviewed the molecular mechanisms acting at the DNA-replication and DNA-damage checkpoints in the fission yeast Schizosaccharomyces pombe, and the modifications of these controls during the meiotic cell cycle. We have made comparisons with the controls in fission yeast and other organisms, mainly the distantly related budding yeast.

Animals↗

PubMed1997

Cloning and characterization of porcine enamelin mRNAs.

Dental enamel forms by matrix-mediated biomineralization. The components of the developing enamel matrix are generally specific for that matrix. The primary structures of three enamel proteins-amelogenin, tuftelin, and sheathlin (ameloblastin/amelin)-have been derived from cDNA sequences. Here we report the cloning and characterization of mRNA encoding a fourth enamel protein: enamelin. The longest porcine enamelin cDNA clone has 3907 nucleotides, exclusive of the poly(A) tail. The primary structure of the secreted protein is 1104 amino acids in length. Without post-translational modifications, the secreted protein has an isotope-averaged molecular mass of 124.3 kDa and an isoelectric point of 6.5. Polymerase chain-reaction phenotyping of enamelin cDNA suggests that porcine enamelin transcripts are not alternatively spliced and use a single polyadenylation/cleavage site. Immunohistochemical and Western blot analyses with an affinity-purified antipeptide antibody specific for the enamelin carboxyl terminus demonstrate that enamelin is synthesized and secreted by secretory-phase ameloblasts. The parent protein is a 186-kDa glycoprotein that concentrates along the secretory face of the ameloblast Tomes' process. Intact enamelin and proteolytic cleavage products containing its carboxyl terminus are limited to the most superficial layer of the developing enamel matrix, while other enamelin cleavage products are observed in deeper enamel.

Amino Acid Sequence↗

PubMed2001

Small heat shock proteins (sHSPs) as potential drug targets.

Small heat shock proteins (sHSPs) belong to a family of 12- to 43-kDa proteins that are ubiquitous and are largely conserved in amino acid sequence among all organisms. The principal heat-shock proteins that have chaperone activity (that is, they protect newly made proteins from misfolding) belong to five conserved classes: HSP100, HSP90, HSP70, HSP60 and the small heat-shock proteins (sHSPs). The sHSPs (which include alpha crystallin) can form large multimeric structures and have a wide range of cellular functions, including endowing cells with thermotolerance in vivo and being able to act as molecular chaperones in vitro; sHSPs do this by forming stable complexes with folding -or unfolding--intermediates of their protein substrates, probably the molten globule. This paper includes: a brief survey of the chaperone family, the small heat shock protein superfamily, transcription of sHSPs, sequence comparisons and structural models of small heat shock proteins--structural elements as potential drug targets, sHSPs as chaperone-like proteins, alpha crystallin chaperone-like activity, conformational diseases--the role of alpha crystallin small heat shock protein superfamily proteins, post-translational modification and useful pharmacological agents. Functionality of small heat shock proteins--targets and diseases where pharmacologically active agents are of importance, alpha crystallin--small heat shock proteins and prion diseases: specific targets for diagnostic tests and drug development, details of some specific small heat shock proteins as drug targets, structural and functional implications for treatment.

Animals↗

PubMed1996

In vitro glycosylation of proteins: an enzymatic approach.

The glycosylation pathway is the most important post-translational modification of a protein and is moreover a highly specific process. The majority of proteins of pharmaceutical interest are glycoproteins. Therefore, it is necessary to identify the composition, the structure, the function and the biosynthesis of the glycoproteins. The present knowledge is described here. In addition, the performed studies about structure-function relationship of the glycoproteins have shown that the oligosaccharide part of a glycoprotein confers important and specific biological roles. Thus, the modification of the structure of the glycan chains can lead to a modification of the activity of the glycoprotein. This phenomenon is encountered at the time of the production of recombinant glycoprotein in a heterologous system. Indeed, the glycosylation profile of a protein is specific to both the host cell and the culture conditions of this cell. Thus, the advantages and the drawbacks of the different host cells used for the glycosylation engineering are presented. In this way, the identification of the different specific enzymes glycosyltransferases and glycosidases involved in the glycosylation pathway is now necessary to improve the production of recombinant glycoprotein. The structure and the characteristics of these enzymes, and more particularly the oligosaccharyltransferase and the galactosyltransferase, are also described.

Carbohydrate Conformation↗

PubMed2000

Heteronuclear NMR studies of the specificity of the post-translational modification of biotinyl domains by biotinyl protein ligase.

The lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes and the biotinyl domains of biotin-dependent enzymes have homologous structures, but the target lysine residue in each domain is correctly selected for posttranslational modification by lipoyl protein ligase and biotinyl protein ligase, respectively. We have applied two-dimensional heteronuclear NMR spectroscopy to investigate the interaction between the apo form of the biotinyl domain of the biotin carboxyl carrier protein of acetyl-CoA carboxylase and the biotinyl protein ligase (BPL) from Escherichia coli. Heteronuclear multiple quantum coherence NMR spectra of the 15N-labelled biotinyl domain were recorded in the presence and absence of the ligase and backbone amide 1H and 15N chemical shifts were evaluated. Small, but significant, changes in chemical shift were found in two regions, including the tight beta-turn that houses the lysine residue targetted for biotinylation, and the beta-strand 2 and the loop that precedes it in the domain. When compared with the three-dimensional structure, sequence alignments of other biotinyl and lipoyl domains, and mutagenesis data, these results give a clear indication of how the biotinyl domain is both recognised by BPL and distinguished from the structurally related lipoyl domain to ensure correct posttranslational modification.

Acetyl-CoA Carboxylase↗

PubMed2001

Histone deacetylase 1 phosphorylation promotes enzymatic activity and complex formation.

Accessibility of the genome to DNA-binding transcription factors is regulated by proteins that control the acetylation of amino-terminal lysine residues on nucleosomal histones. Specifically, histone deacetylase (HDAC) proteins repress transcription by deacetylating histones. To date, the only known regulatory mechanism of HDAC1 function is via interaction with associated proteins. Although the control of HDAC1 function by protein interaction and recruitment is well precedented, we were interested in exploring HDAC1 regulation by post-translational modification. Human HDAC1 protein was analyzed by ion trap mass spectrometry, and two phosphorylated serine residues, Ser(421) and Ser(423), were unambiguously identified. Loss of phosphorylation at Ser(421) and Ser(423) due to mutation to alanine or disruption of the casein kinase 2 consensus sequence directing phosphorylation reduced the enzymatic activity and complex formation of HDAC1. Deletion of the highly charged carboxyl-terminal region of HDAC1 also decreased its deacetylase activity and protein associations, revealing its requirement in maintaining HDAC1 function. Our results reinforce the importance of protein associations in modulating HDAC1 function and provide the first step toward characterizing the role of post-translational modifications in regulating HDAC activity in vivo.

Alanine↗

PubMed2005

Post-translational disulfide modifications in cell signaling--role of inter-protein, intra-protein, S-glutathionyl, and S-cysteaminyl disulfide modifications in signal transmission.

Cell signaling entails a host of post-translational modifications of effector-proteins. These modifications control signal transmission by regulating the activity, localization or half-life of the effector-protein. Prominent oxidative modifications induced by cell-signaling reactive oxygen species (ROS) are cysteinyl modifications such as S-nitrosylation, sulfenic acid and disulfide formation. Disulfides protect protein sulfhydryls against oxidative destruction and simultaneously influence cell signaling by engaging redox-regulatory sulfhydryls in effector-proteins. The types of disulfides implicated in signaling span (1) protein S-glutathionylation, e.g. as a novel mode of Ras activation through S-glutathionylation at Cys-118 in response to a hydrogen-peroxide burst, (2) intra-protein disulfides, e.g. in the regulation of the stability of the protein phosphatase Cdc25C by hydrogen-peroxide, (3) inter-protein disulfides, e.g. in the hydrogen peroxide-mediated inactivation of receptor protein-tyrosine phosphatase alpha (RPTPalpha) by dimerization and (4) protein S-cysteaminylation by cystamine. Cystamine is a byproduct of pantetheinase-catalyzed pantothenic acid recycling from pantetheine for biosynthesis of Coenzyme A (CoA), a ubiquitous and metabolically indispensable cofactor. Cystamine inactivates protein kinase C-epsilon (PKCepsilon), gamma-glutamylcysteine synthetase and tissue transglutaminase by S-cysteaminylation-triggered mechanisms. The importance of protein S-cysteaminylation in signal transmission in vivo is evident from the ability of cystamine administration to rescue the intestinal inflammatory-response deficit of pantetheinase knockout mice. These mice lack the predominant epithelial pantetheinase isoform and have sharply reduced levels of cystamine/cysteamine in epithelial tissues. In addition, intraperitoneal administration of cystamine significantly delays neurodegenerative pathogenesis in a Huntington's disease mouse model. Thus, cystamine may serve as a prototype for the development of novel therapeutics that target effector-proteins regulated by S-cysteaminylation.

Animals↗