Peptides containing glutamine repeats as substrates for transglutaminase-catalyzed cross-linking: Relevance to diseases of the nervous system

Many proteins contain reiterated glutamine residues, but polyglutamine of excessive length may result in human disease by conferring new properties on the protein containing it. One established property of a glutamine residue, depending on the nature of the flanking residues, is its ability to act as an amine acceptor in a transglutaminase-catalyzed reaction and to make a glutamyl–lysine cross-link with a neighboring polypeptide. To learn whether glutamine repeats can act as amine acceptors, we have made peptides with variable lengths of polyglutamine flanked by the adjacent amino acid residues in the proteins associated with spinocerebellar ataxia type 1 (SCA1), Machado–Joseph disease (SCA3), or dentato-rubral pallido-luysian atrophy (DRPLA) or those residues adjacent to the preferred cross-linking site of involucrin, or solely by arginine residues. The polyglutamine was found to confer excellent substrate properties on any soluble peptide; under optimal conditions, virtually all the glutamine residues acted as amine acceptors in the reaction with glycine ethyl-ester, and lengthening the sequence of polyglutamine increased the reactivity of each glutamine residue. In the presence of transglutaminase, peptides containing polyglutamine formed insoluble aggregates with the proteins of brain extracts and these aggregates contained glutamyl–lysine cross-links. Repeated glutamine residues exposed on the surface of a neuronal protein should form cross-linked aggregates in the presence of any transglutaminase activated by the presence of Ca2+.

Covalent HLA-B27/peptide complex induced by specific recognition of an aziridine mimic of arginine.

The class I major histocompatibility complex (MHC) glycoprotein HLA-B27 binds short peptides containing arginine at peptide position 2 (P2). The HLA-B27/peptide complex is recognized by T cells both as part of the development of the repertoire of T cells in the cellular immune system and during activation of cytotoxic T cells. Based on the three-dimensional structure of HLA-B27, we have synthesized a ligand with an aziridine-containing side chain designed to mimic arginine and to bind covalently in the arginine-specific P2 pocket of HLA-B27. Using tryptic digestion followed by mass spectrometry and amino acid sequencing, the aziridine-containing ligand is shown to alkylate specifically cysteine 67 of HLA-B27. Neither free cysteine in solution nor an exposed cysteine on a class II MHC molecule can be alkylated, showing that specific recognition between the anchor side-chain pocket of an MHC class I protein and the designed ligand (propinquity) is necessary to induce the selective covalent reaction with the MHC class I molecule.

PR-39, a proline-rich antibacterial peptide that inhibits phagocyte NADPH oxidase activity by binding to Src homology 3 domains of p47 phox.

Reactive oxygen intermediates generated by the phagocyte NADPH oxidase are critically important components of host defense. However, these highly toxic oxidants can cause significant tissue injury during inflammation; thus, it is essential that their generation and inactivation are tightly regulated. We show here that an endogenous proline-arginine (PR)-rich antibacterial peptide, PR-39, inhibits NADPH oxidase activity by blocking assembly of this enzyme through interactions with Src homology 3 domains of a cytosolic component. This neutrophil-derived peptide inhibited oxygen-dependent microbicidal activity of neutrophils in whole cells and in a cell-free assay of NADPH oxidase. Both oxidase inhibitory and direct antimicrobial activities were defined within the amino-terminal 26 residues of PR-39. Oxidase inhibition was attributed to binding of PR-39 to the p47phox cytosolic oxidase component. Its effects involve both a polybasic amino-terminal segment and a proline-rich core region of PR-39 that binds to the p47phox Src homology 3 domains and, thereby, inhibits interaction with the small subunit of cytochrome b558, p22phox. These findings suggest that PR-39, which has been shown to be involved in tissue repair processes, is a multifunctional peptide that can regulate NADPH oxidase production of superoxide anion O2-. thus limiting excessive tissue damage during inflammation.

The chicken beta 2-microglobulin gene is located on a non-major histocompatibility complex microchromosome: a small, G+C-rich gene with X and Y boxes in the promoter.

beta 2-Microglobulin is an essential subunit of major histocompatibility complex (Mhc) class I molecules, which present antigenic peptides to T lymphocytes. We sequenced a number of cDNAs and two genomic clones corresponding to chicken beta 2-microglobulin. The chicken beta 2-microglobulin gene has a similar genomic organization but smaller introns and higher G+C content than mammalian beta 2-microglobulin genes. The promoter region is particularly G+C-rich and contains, in addition to interferon regulatory elements, potential S/W, X, and Y boxes that were originally described for mammalian class II but not class I alpha or beta 2-microglobulin genes. There is a single chicken beta 2-microglobulin gene that has little polymorphism in the coding region. Restriction fragment length polymorphisms from Mhc homozygous lines, Mhc congenic lines, and backcross families, as well as in situ hybridization, show that the beta 2-microglobulin gene is located on a microchromosome different from the one that contains the chicken Mhc. We propose that the structural similarities between the beta 2-microglobulin and Mhc genes in the chicken are due to their presence on microchromosomes and suggest that these features and the microchromosomes appeared by deletion of DNA in the lineage leading to the birds.

Specific interactions outside the proline-rich core of two classes of Src homology 3 ligands.

Two dodecapeptides belonging to distinct classes of Src homology 3 (SH3) ligands and selected from biased phage display libraries were used to investigate interactions between a specificity pocket in the Src SH3 domain and ligant residues flanking the proline-rich core. The solution structures of c-Src SH3 complexed with these peptides were solved by NMR. In addition to proline-rich, polyproline type II helix-forming core, the class I and II ligands each possesses a flanking sequence that occupies a large pocket between the RT and n-Src loops of the SH3 domain. Structural and mutational analyses illustrate how the two classes of SH3 ligands exploit a specificity pocket on the receptor differently to increase binding affinity and specificity.

The 94- to 97-kDa mouse macrophage membrane protein that recognizes oxidized low density lipoprotein and phosphatidylserine-rich liposomes is identical to macrosialin, the mouse homologue of human CD68.

We have previously reported the partial purification of a 94- to 97-kDa plasma membrane protein from mouse peritoneal macrophages that binds oxidatively modified low density lipoprotein (OxLDL) and phosphatidylserine-rich liposomes. We have now identified that protein as macrosialin, a previously cloned macrophage-restricted membrane protein in the lysosomal-associated membrane protein family (mouse homologue of human CD68). Early in the course of purification of the 94- to 97-kDa protein, a new OxLDL-binding band at 190-200 kDa appeared and copurified with the 94- to 97-kDa protein. The HPLC pattern of tryptic peptides from this higher molecular mass ligand-binding band closely matched that derived from the 94- to 97-kDa band. Specifically, the same three macrosialin-derived tryptic peptides (9, 9, and 15 residues) were present in the purified 94- to 97-kDa band and in the 190- to 200-kDa band and antisera raised against peptide sequences in macrosialin recognized both bands. An antiserum against macrosialin precipitated most of the 94- to 97-kDa OxLDL-binding material. We conclude that the binding of OxLDL to mouse macrophage membranes is in part attributable to macrosialin. Our previous studies show that OxLDL competes with oxidized red blood cells and with apoptotic thymocytes for binding to mouse peritoneal macrophages. Whether macrosialin plays a role in recognition of OxLDL and oxidatively damaged cells by intact macrophages remains uncertain.

A mammalian arginine/lysine transporter uses multiple promoters.

The mCAT-2 gene encodes a Na(+)-independent cationic amino acid (AA) transporter that is inducibly expressed in a tissue-specific manner in various physiological conditions. When mCAT-2 protein is expressed in Xenopus oocytes, the elicited AA transport properties are similar to the biochemically defined transport system y+. The mCAT-2 protein sequence is closely related to another cationic AA transporter (mCAT-1); these related proteins elicit virtually identical cationic AA transport in Xenopus oocytes. The two genes differ in their tissue expression and induction patterns. Here we report the presence of diverse 5' untranslated region (UTR) sequences in mCAT-2 transcripts. Sequence analysis of 22 independent mCAT-2 cDNA clones reveals that the cDNA sequences converge precisely 16 bp 5' of the initiator AUG codon. Moreover, analysis of genomic clones shows that the mCAT-2 gene 5'UTR exons are dispersed over 18 kb. Classical promoter and enhancer elements are present in appropriate positions 5' of the exons and their utilization results in regulated mCAT-2 mRNA accumulation in skeletal muscle and liver following partial hepatectomy. The isoform adjacent to the most distal promoter is found in all tissues and cell types previously shown to express mCAT-2, while the other 5' UTR isoforms are more tissue specific in their expression. Utilization of some or all of five putative promoters was documented in lymphoma cell clones, liver, and skeletal muscle. TATA-containing and (G+C)-rich TATA-less promoters appear to control mCAT-2 gene expression. The data indicate that the several distinct 5' mCAT-2 mRNA isoforms result from transcriptional initiation at distinct promoters and permit flexible transcriptional regulation of this cationic AA transporter gene.

Proline-rich sequences that bind to Src homology 3 domains with individual specificities.

To study the binding specificity of Src homology 3 (SH3) domains, we have screened a mouse embryonic expression library for peptide fragments that interact with them. Several clones were identified that express fragments of proteins which, through proline-rich binding sites, exhibit differential binding specificity to various SH3 domains. Src-SH3-specific binding uses a sequence of 7 aa of the consensus RPLPXXP, in which the N-terminal arginine is very important. The SH3 domains of the Src-related kinases Fyn, Lyn, and Hck bind to this sequence with the same affinity as that of the Src SH3. In contrast, a quite different proline-rich sequence from the Btk protein kinase binds to the Fyn, Lyn, and Hck SH3 domains, but not to the Src SH3. Specific binding of the Abl SH3 requires a longer, more proline-rich sequence but no arginine. One clone that binds to both Src and Abl SH3 domains through a common site exhibits reversed binding orientation, in that an arginine indispensable for binding to all tested SH3 domains occurs at the C terminus. Another clone contains overlapping yet distinct Src and Abl SH3 binding sites. Binding to the SH3 domains is mediated by a common PXXP amino acid sequence motif present on all ligands, and specificity comes about from other interactions, often ones involving arginine. The rules governing in vivo usage of particular sites by particular SH3 domains are not clear, but one binding orientation may be more specific than another.

TATPred:a Bayesian method for the identification of twin arginine translocation pathway signal sequences

The twin arginine translocation (TAT) system ferries folded proteins across the bacterial membrane. Proteins are directed into this system by the TAT signal peptide present at the amino terminus of the precursor protein, which contains the twin arginine residues that give the system its name. There are currently only two computational methods for the prediction of TAT translocated proteins from sequence. Both methods have limitations that make the creation of a new algorithm for TAT-translocated protein prediction desirable. We have developed TATPred, a new sequence-model method, based on a Nave-Bayesian network, for the prediction of TAT signal peptides. In this approach, a comprehensive range of models was tested to identify the most reliable and robust predictor. The best model comprised 12 residues: three residues prior to the twin arginines and the seven residues that follow them. We found a prediction sensitivity of 0.979 and a specificity of 0.942.

Novel inhibitors of the tRNA-dependent amidotransferase of "Helicobacter pylori": Peptides generated by phage display and dipeptide-like compounds

Cette thèse présente la découverte de nouveaux inhibiteurs de l’amidotransférase ARNt-dépendante (AdT), et résume les connaissances récentes sur la biosynthèse du Gln-ARNtGln et de l’Asn-ARNtAsn par la voie indirecte chez la bactérie Helicobacter pylori. Dans le cytoplasme des eucaryotes, vingt acides aminés sont liés à leur ARNt correspondant par vingt aminoacyl-ARNt synthétases (aaRSs). Ces enzymes sont très spécifiques, et leur fonction est importante pour le décodage correct du code génétique. Cependant, la plupart des bactéries, dont H. pylori, sont dépourvues d’asparaginyl-ARNt synthétase et/ou de glutaminyl-ARNt synthétase. Pour former le Gln-ARNtGln, H. pylori utilise une GluRS noncanonique nommée GluRS2 qui glutamyle spécifiquement l’ARNtGln ; ensuite, une AdT trimérique, la GatCAB corrige le Glu-ARNtGln mésapparié en le transamidant pour former le Gln-ARNtGln, qui lira correctement les codons glutamine pendant la biosynthèse des protéines sur les ribosomes. La formation de l’Asn-ARNtAsn est similaire à celle du Gln-ARNtGln, et utilise la même GatCAB et une AspRS non-discriminatrice. Depuis des années 2000, la GatCAB est considérée comme une cible prometteuse pour le développement de nouveaux antibiotiques, puisqu’elle est absente du cytoplasme de l’être humain, et qu’elle est encodée dans le génome de plusieurs bactéries pathogènes. Dans le chapitre 3, nous présentons la découverte par la technique du « phage display » de peptides cycliques riches en tryptophane et en proline, et qui inhibent l’activité de la GatCAB de H. pylori. Les peptides P10 (CMPVWKPDC) et P9 (CSAHNWPNC) inhibent cette enzyme de façon compétitive par rapport au substrat Glu-ARNtGln. Leur constante d’inhibition (Ki) est 126 μM pour P10, et 392 μM pour P9. Des modèles moléculaires ont montré qu’ils lient le site actif de la réaction de transmidation catalysée par la GatCAB, grâce à la formation d’une interaction π-π entre le résidu Trp de ces peptides et le résidu Tyr81 de la sous-unité GatB, comme fait le A76 3’-terminal de l’ARNt. Dans une autre étude concernant des petits composés contenant un groupe sulfone, et qui mimiquent l’intermédiaire de la réaction de transamidation, nous avons identifié des composés qui inhibent la GatCAB de H. pylori de façon compétitive par rapport au substrat Glu-ARNtGln. Cinq fois plus petits que les peptides cycliques mentionnés plus haut, ces composés inhibent l’activité de la GatCAB avec des Ki de 139 μM pour le composé 7, et de 214 μM pour le composé 4. Ces inhibiteurs de GatCAB pourraient être utiles pour des études mécanistiques, et pourraient être des molécules de base pour le développement de nouvelles classes d’antibiotiques contre des infections causées par H. pylori.

Gender effects in orientation on primary students’ performance on items rich in graphics

This study investigated the longitudinal performance of 378 students who completed mathematics items rich in graphics. Specifically, this study explored student performance across axis (e.g., numbers lines), opposed-position (e.g., line and column graphs) and circular (e.g., pie charts) items over a three-year period (ages 9-11 years). The results of the study revealed significant performance differences in the favour of boys on graphics items that were represented in horizontal and vertical displays. There were no gender differences on items that were represented in a circular manner.

Ghrelin gene-related peptides : multifunctional endocrine/autocrine modulators in health and disease

Ghrelin is a multi-functional peptide hormone which affects various processes including growth hormone and insulin release, appetite regulation, gut motility, metabolism and cancer cell proliferation. Ghrelin is produced in the stomach and in other normal and pathological cell types. It may act as an endocrine or autocrine/paracrine factor. The ghrelin gene encodes a precursor protein, preproghrelin, from which ghrelin and other potentially active peptides are derived by alternative mRNA splicing and/or proteolytic processing. The metabolic role of the peptide obestatin, derived from the preproghrelin C-terminal region, is controversial. However, it has direct effects on cancer cell proliferation. The regulation of ghrelin expression and the mechanisms through which the peptide products arise are unclear. We have recently re-examined the organisation of the ghrelin gene and identified several novel exons and transcripts. One transcript, which lacks the ghrelin-coding region of preproghrelin, contains the coding sequence of obestatin. Furthermore, we have identified an overlapping gene on the antisense strand of ghrelin, GHRLOS, which generates transcripts that may function as non-coding regulatory RNAs or code for novel, short bioactive peptides. The identification of these novel ghrelin-gene related transcripts and peptides raises critical questions regarding their physiological function and their role in obesity, diabetes and cancer.

Establishment of a mouse model of colitis and its use to evaluate the anti-inflammatory effects of two ghrelin peptides

Ghrelin is a gut-brain peptide hormone that induces appetite, stimulates the release of growth hormone, and has recently been shown to ameliorate inflammation. Recent studies have suggested that ghrelin may play a potential role in inflammation-related diseases such as inflammatory bowel diseases (IBD). A previous study with ghrelin in the TNBS mouse model of colitis demonstrated that ghrelin treatment decreased the clinical severity of colitis and inflammation and prevented the recurrence of disease. Ghrelin may be acting at the immunological and epithelial level as the ghrelin receptor (GHSR) is expressed by immune cells and intestinal epithelial cells. The current project investigated the effect of ghrelin in a different mouse model of colitis using dextran sodium sulphate (DSS) – a luminal toxin. Two molecular weight forms of DSS were used as they give differing effects (5kDa and 40kDa). Ghrelin treatment significantly improved clinical colitis scores (p=0.012) in the C57BL/6 mouse strain with colitis induced by 2% DSS (5kDa). Treatment with ghrelin suppressed colitis in the proximal colon as indicated by reduced accumulative histopathology scores (p=0.03). Whilst there was a trend toward reduced scores in the mid and distal colon in these mice this did not reach significance. Ghrelin did not affect histopathology scores in the 40kDa model. There was no significant effect on the number of regulatory T cells or TNF-α secretion from cultured lymph node cells from these mice. The discovery of C-terminal ghrelin peptides, for example, obestatin and the peptide derived from exon 4 deleted proghrelin (Δ4 preproghrelin peptide) have raised questions regarding their potential role in biological functions. The current project investigated the effect of Δ4 peptide in the DSS model of colitis however no significant suppression of colitis was observed. In vitro epithelial wound healing assays were also undertaken to determine the effect of ghrelin on intestinal epithelial cell migration. Ghrelin did not significantly improve wound healing in these assays. In conclusion, ghrelin treatment displays a mild anti-inflammatory effect in the 5kDa DSS model. The potential mechanisms behind this effect and the disparity between these results and those published previously will be discussed.