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One Amino Acid (one + amino_acid)
Selected Abstracts"Reverse degradomics", monitoring of proteolytic trimming by multi-CE and confocal detection of fluorescent substrates and reaction productsELECTROPHORESIS, Issue 13 2009Helene Piccard Abstract A platform for profiling of multiple proteolytic activities acting on one specific substrate, based on the use of a 96-channel capillary DNA sequencer with CE-LIF of labeled substrate peptides and reaction products is introduced. The approach consists of synthesis of a substrate peptide of interest, fluorescent labeling of the substrate, either aminoterminally by chemical coupling, or carboxyterminally by transglutaminase reaction, proteolysis by a biological mixture of proteases in the absence or presence of protease inhibitors, multi-channel analysis of substrate and reaction products, and data collection and processing. Intact substrate and reaction products, even when varying by only one amino acid, can be relatively semi-quantified in a high-throughput manner, yielding information on proteases acting in complex biological mixtures and without prepurification. Monitoring, classification and inhibition of multiple proteolytic activities are demonstrated on a model substrate, the aminoterminus of the mouse granulocyte chemotactic protein-2. In view of extensive processing of chemokines into various natural forms with different specific biological activities, and of the fragmentary knowledge of processing proteases, examples of processing by neutrophil degranulate, tumor cell culture fluids and plasma are provided. An example of selection and comparison of inhibitory mAbs illustrates that the platform is suitable for inhibitor screening. Whereas classical degradomics technologies analyze the substrate repertoire of one specific protease, here the complementary concept, namely the study of all proteases acting, in a biological context, on one specific substrate, is developed and tuned to identify key proteases and protease inhibitors for the processing of any biological substrate of interest. [source] Human haptoglobin structure and function , a molecular modelling studyFEBS JOURNAL, Issue 22 2008F. Polticelli Hemoglobin is the most prominent protein in blood, transporting O2 and facilitating reactive oxygen and nitrogen species detoxification. Hemoglobin metabolism leads to the release of extra-erythrocytic hemoglobin, with potentially severe consequences for health. Extra-erythrocytic hemoglobin is complexed to haptoglobin for clearance by tissue macrophages. The human gene for haptoglobin consists of three structural alleles: Hp1F, Hp1S and Hp2. The products of the Hp1F and Hp1S alleles differ by only one amino acid, whereas the Hp2 allele is the result of a fusion of the Hp1F and Hp1S alleles, is present only in humans and gives rise to a longer ,-chain. Haptoglobin consists of a dimer of ,,-chains covalently linked by a disulphide bond between the Cys15 residue of each ,-chain. However, the presence of the Hp1 and Hp2 alleles in humans gives rise to HPT1-1 dimers (covalently linked by Cys15 residues), HPT1-2 hetero-oligomers and HPT2-2 oligomers. In fact, the HPT2 variant displays two free Cys residues (Cys15 and Cys74) whose participation in intermolecular disulphide bonds gives rise to higher-order covalent multimers. Here, the complete modelling of both haptoglobin variants, together with their basic quaternary structure arrangements (i.e. HPT1 dimer and HPT2 trimer), is reported. The structural details of the models, which represent the first complete view of the molecular details of human haptoglobin variants, are discussed in relation to the known haptoglobin function(s). [source] Exquisite specificity and peptide epitope recognition promiscuity, properties shared by antibodies from sharks to humansJOURNAL OF MOLECULAR RECOGNITION, Issue 2 2001John J. Marchalonis Abstract This review considers definitions of the specificity of antibodies including the development of recent concepts of recognition polyspecificity and epitope promiscuity. Using sets of homologous and unrelated peptides derived from the sequences of immunoglobulin and T cell receptor chains we offer operational definitions of cross-reactivity by investigating correlations of either identities in amino acid sequence, or in hydrophobicity/hydrophilicity profiles with degree of binding in enzyme-linked immunosorbent assays. Polyreactivity, or polyspecificity, are terms used to denote binding of a monoclonal antibody or purified antibody preparation to large complex molecules that are structurally unrelated, such as thyroglobulin and DNA. As a first approximation, there is a linear correlation between degree of sequence identity or hydrophobicity/hydrophilicity and antigenic cross-binding. However, catastrophic interchanges of amino acids can occur where changing of one amino acid out of 16 in a synthetic peptide essentially eliminates binding to certain antibodies. An operational definition of epitope promiscuity for peptides is the case where two peptides show little or no identity in amino acid sequence but bind strongly to the same antibody as shown by either direct binding or competitive inhibition. Analysis of antibodies of humans and sharks, the two most divergent species in evolution to express antibodies and the combinatorial immune response, indicates that the capacity for both exquisite specificity and epitope recognition promiscuity are essential conserved features of individual vertebrate antibodies. Copyright © 2001 John Wiley & Sons, Ltd. [source] Fluorescent, internally quenched, peptides for exploring the pH-dependent substrate specificity of cathepsin BJOURNAL OF PEPTIDE SCIENCE, Issue 7 2006Paolo Ruzza Abstract Cathepsin B is a cysteine protease that in tumor tissues is localized in both acidic lysosomes and extracellular spaces. It can catalyze the cleavage of peptide bonds by two mechanisms: endoproteolytic attack with a pH optimum around 7.4, and attack from the C -terminus with a pH optimum at 4.5,5.5. In this work, seven fluorescent, internally quenched, decapeptides have been synthesized using the prototypical cathepsin B selective substrate Z-Phe-Arg-AMC as a lead, and used to identify the structural factors determining the susceptibility of peptides to hydrolysis at acidic and neutral pH values. Each peptide differs from the others in one amino acid (residue 6) and contains a highly fluorescent Nma group linked to the ,-amino function of the N -terminal Orn residue and a Dnp group linked to the side chain of the Lys8 residue acting as a quencher. Proteolytic cleavage was monitored by measuring the increase of fluorescence at 440 nm upon excitation at 340 nm, and the cleavage sites were determined by HPLC followed by ESI-MS analysis. Peptides containing Ala or Phe at position 6 are good substrates for the enzyme at both pH 5.0 and 7.4. By contrast, those containing Glu, Asp, Lys or Val are not cleaved at all by cathepsin B at pH 7.4, and are poorly hydrolyzed at pH 5.0. These findings provide new information for the rational design of cathepsin B-activated peptide-containing anticancer drugs. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd. [source] Distinct pharmacology of rat and human histamine H3 receptors: role of two amino acids in the third transmembrane domainBRITISH JOURNAL OF PHARMACOLOGY, Issue 7 2000X Ligneau Starting from the sequence of the human histamine H3 receptor (hH3R) cDNA, we have cloned the corresponding rat cDNA. Whereas the two deduced proteins show 93.5% overall homology and differ only by five amino acid residues at the level of the transmembrane domains (TMs), some ligands displayed distinct affinities. Thioperamide and ciproxifan were about 10 fold more potent at the rat than at the human receptor, whereas FUB 349 displayed a reverse preference. Histamine, (R),-methylhistamine, proxyfan or clobenpropit were nearly equipotent at H3 receptors of both species. The inverse discrimination patterns of ciproxifan and FUB 349 were partially changed by mutation of one amino acid (V122A), and fully abolished by mutation of two amino acids (A119T and V122A), in TM3 of the rH3R located in the vicinity of Asp114 purported to salt-link the ammonium group of histamine. Therefore, these two residues appear to be responsible for the distinct pharmacology of the H3R in the two species. British Journal of Pharmacology (2000) 131, 1247,1250; doi:10.1038/sj.bjp.0703712 [source] | ||||||||||