Serine proteases are a family of enzymes with a wide array of functions across both eukaryotes and prokaryotes. Here we have attempted to produce the serine proteases rat mast cell protease 2 and mouse mast cell protease 5 in a culture of HEK 293 cells; and mouse mast cell protease 4, platypus granzyme B-like protease and platypus hypothetical protease in a baculovirus expression system. Following production we wanted to analyse these serine proteases using a phage display assay and a battery of chromogenic substrates.

Serine proteases are a family of proteases that utilize an activated serine residue in the sub&not / strate-binding pocket to catalytically hydrolyze peptide bonds. Enzymes which belong to this family, with a diverse array of metabolic and regulatory functions, play critical roles in cell physiology and pathology. &lsquo / Clp&rsquo / s are a class of ATP dependent serine proteases which are composed of a protease (ClpP) and an ATPase (ClpA or ClpX) component. Their involvements in degrading proteins are especially implicated under stress conditions. In contrast to members of Bacteria and Eukarya, little is known about the energy-dependent proteolysis and there is no report on Clp family proteases in Archaea. In this study, for the fist time, a periplasmic Clp P-like (PSP) and a membrane bound serine protease (MSP) genes from thermophilic archaeon Thermoplasma volcanium GSS1 were cloned and expressed in E. coli. PCR amplifications at 55 &ordm / C yielded unique fragments of 971 and 1521bp, for PSP and MSP genes, respectively, which were ligated to p-Drive cloning vectors and introduced into E.coli TG1 competent cells. Recombinant clones were screened depending on blue/white colony selection. Putative recombinant plasmids were analyzed by restriction enzyme digestions. Serine protease activities of the three positive clones (E. coli TG-S1, E. coli TG-S4 and E. coli TG-M1) were determined spectrophotometrically by using chromogenic oligopeptide substrates. These results indicated that cloned PSP and MSP genes were successfully expressed in E. coli under the control of their own promoters. Heterologous expression of PSP gene was also attempted by adding 6xHis tag to the 5&acute / end of the PSP gene in pQE 30 expression vector. Competent E.coli TG1 cells were transformed by pQE expression constructs. Positive clones were detected on colony blots using Anti-His HRP conjugates and chromogenic DAB substrate. Plasmids of these colonies were analyzed by restriction digestions to select the true recombinants. Expression of the 6xHis-PSP fusion protein from the recombinant E. coli TG-pQE-S1.7 strain was confirmed by functional analysis and SDS-PAGE. An NCBI domain search and multiple sequence alignment using Clustal W 1.82 program indicated homologies between PSP and MSP of Tp. volcanium and various bacterial ATP dependent ClpPs. Signal peptide search using Signal P 3.0 server predicted a signal peptide sequence in MSP homologous to that of Gram (+) bacteria.

Scavenger receptors are a large family of structurally diverse molecules that have been implicated in a range of biological functions. In this work, a newly identified multi-domain scavenger receptor-serine protease IrSRP-1 from the tick Ixodes ricinus is characterized. IrSRP-1 is related to the serine protease Sp22D from the mosquito Anopheles gambiae. IrSRP-1 is expressed mainly in the tick gut but also in hemocytes, Malpighian tubules, tracheas and ovaries of fully fed females. This was confirmed with Western blots and immunohistological labeling with antibodies raised against recombinantly expressed IrSRP-1 trypsine-like domain. According to acquired qRT-PCR profiles relative expression of IrSRP-1 is strongly up-regulated during female feeding and remains unchanged in ticks experimentally injected with various microbes. Functional characterization by RNA interference revealed that lowering IrSRP1 expression leads to a higher mortality rate during tick female feeding.

No description available.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23433号 / 農博第2464号 / 新制||農||1086(附属図書館) / 学位論文||R3||N5348(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 谷 史人, 教授 橋本 渉 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

This year the American Cancer Society predicts that 565,650 individuals will lose their life as a result of their battle with cancer. Due to its established roles in cancer and extracellular presentation, the Epidermal Growth Factor Receptor (EGFR) is an excellent target for anti-cancer drugs. It has been determined that matriptase and prostasin serine proteases are proteolytic regulators of EGFR membrane presentation, and downstream signaling. Currently, there are several drugs that target EGFR, but research continues in order to further understand drug-resistant EGFR. In cancer cell lines that exhibit both EGFR signaling and these proteases, proteolytic cleavage may be a mechanism of resistance to drugs that target the EGFR extracellular domain (ECD). The specific aim of this project was to determine which protease was direct! y responsible for EGFR cleavage and establish the precise cleavage site within the EGFR ECD. DNA corresponding to amino acid residues 336-505 of the EGFR ECD was cloned into the p-GEX-6P-I vector and expressed as a GST-fusion protein in E.coli cells. This fusion protein was isolated and purified by affinity chromatography. Purified GSTEGFR BCD fusion protein was mixed with prostasin and matriptase and evaluated for cleavage. No cleavage was detected using this method. Trypsin serine protease was used to ensure the cleavability of the GST-EGFR ECD. The GST-EGFR ECD fusion protein was found to be inappropriate for determining matriptase or prostasin cleavage sites, which are now being pursued by other means.

Yes / The mammalian X and Y chromosomes originated from a pair of ordinary autosomes. Over the past ~180 million years, the X and Y have become highly differentiated and now only recombine with each other within a short pseudoautosomal region. While the X chromosome broadly preserved its gene content, the Y chromosome lost ~92% of the genes it once shared with the X chromosome. PRSSLY is a Y-linked gene identified in only a few mammalian species that was thought to be acquired, not ancestral. However, PRSSLY's presence in widely divergent species-bull and mouse-led us to further investigate its evolutionary history. We discovered that PRSSLY is broadly conserved across eutherians and has ancient origins. PRSSLY homologs are found in syntenic regions on the X chromosome in marsupials and on autosomes in more distant animals, including lizards, indicating that PRSSLY was present on the ancestral autosomes but was lost from the X and retained on the Y in eutherian mammals. We found that across eutheria, PRSSLY's expression is testis-specific, and, in mouse, it is most robustly expressed in post-meiotic germ cells. The closest paralog to PRSSLY is the autosomal gene PRSS55, which is expressed exclusively in testes, involved in sperm differentiation and migration, and essential for male fertility in mice. Outside of eutheria, in species where PRSSLY orthologs are not Y-linked, we find expression in a broader range of somatic tissues, suggesting that PRSSLY has adopted a germ-cell-specific function in eutherians. Finally, we generated Prssly mutant mice and found that they are fully fertile but produce offspring with a modest female-biased sex ratio compared to controls. PRSSLY appears to be the first example of a gene that derives from the mammalian ancestral sex chromosomes that was lost from the X and retained on the Y. Although the function of PRSSLY remains to be determined, it may influence the sex ratio by promoting the survival or propagation of Y-bearing sperm. / The group of D.C.P. is supported by the Howard Hughes Medical Institute, the Whitehead Institute, and philanthropic gifts from Brit and Alexander d’Arbeloff, Authur W. and Carol Tobin Brill, and Charles Ellis.

Doctor of Philosophy / Department of Biology / Lynn E. Hancock / The enterococci are gaining much notoriety as common nosocomial pathogens. One aspect of their pathogenesis, especially characteristic to infectious endocarditis and urinary tract infections, involves their ability to transition from the sessile state of existence to surface adherent structured communities called biofilms. Existence as biofilms, affords enterococci protection against a number of growth limiting challenges including antibiotic therapy and host immunity. In the current study a mechanistic role for two Fsr quorum-regulated extracellular proteases- gelatinase (GelE) and its cotranscribed serine protease (SprE), were explored in biofilm development of E. faecalis V583. Confocal imaging of biofilms suggested that GelE[superscript]– mutants were significantly reduced in biofilm biomass compared to V583, whereas the absence of SprE appeared to accelerate the progression of biofilm development. Culture supernatant and biofilm analysis confirmed that decreased biofilms observed in GelE[superscript]– mutants resulted from their inability to undergo autolysis and release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas SprE[superscript]– mutants produced significantly more eDNA as components of the biofilm matrix. The governing principle behind GelE mediated autolysis and eDNA release in E. faecalis V583 was demonstrated to be fratricide. GFP reporter assays of V583 populations confirmed that GBAP (gelatinase biosynthesis-activating pheromone encoded by fsrD) quorum non-responders (GelE[superscript]–SprE[superscript]–) were a minority subpopulation of prey cells susceptible to the targeted fratricidal action of the quorum responsive predatorial majority (GelE[superscript]+SprE[superscript]+). The killing action is dependent on GelE, and the GelE producer population is protected from self-destruction by the co-production of SprE as an immunity protein. Targeted gene inactivation and protein interaction studies demonstrate that extracellular proteases execute their characteristic effects following downstream interactions with the primary autolysin, AtlA. Finally, comparison of virulence effects of isogenic extracellular protease mutants (∆gelE, ∆sprE and ∆gelEsprE) relative to parental strain (V583) in a rabbit model of enterococcal endocarditis confirmed a critical role for GelE in the infection process. In conclusion, the data presented in this thesis are consistent with significant roles for GelE and SprE in biofilm mediated pathogenesis of enterococcal infections.

<p>Mast cells (MC) are potent inflammatory cells that are known primarily for their prominent role in IgE mediated allergies. However, they also provide beneficial functions to the host, e.g. in bacterial and parasitic defence. MCs react rapidly upon stimulation by releasing potent granule-stored mediators, and serine proteases of the chymase or tryptase families are such major granule constituents. </p><p>As a first step towards a better understanding of the biological function of these proteases, we have determined the extended cleavage specificities of four mammalian mast cell chymases, by utilizing a substrate phage display approach. The specificities of these enzymes have then been used to compare their functional characteristics.</p><p>The major mucosal MC chymase in mice, mMCP-1, was found to possess a strict preference in four amino acid positions of the peptide substrate. Using this sequence to search the mouse proteome for potential <i>in vivo</i> substrates led to the identification of several very interesting potential novel substrates. Some of them may explain the increased epithelial permeability provided by this enzyme.</p><p>Human MCs, express only one single α-chymase, and the rodent α-chymases have secondarily gained elastase-like primary cleavage specificity. However, rodents express additional chymases, the β-chymases, and rodent β-chymases may have adopted the function of the α-chymases. The cleavage specificities of the human chymase and two rodent β-chymases were therefore determined (rat rMCP-1 and mouse mMCP-4). N-terminal of the cleaved bond the three chymases showed similar preferences, but C-terminal the human chymase and mMCP-4 shared a high preference for acidic amino acids in the P2´ position and therefore seem to be functional homologues. The molecular interactions mediating the preference for acidic amino acids in position P2´ were further investigated. By site-directed mutagenesis of the human chymase, amino acids Arg143 and Lys192 were concluded to synergistically mediate this preference.</p><p>Our data show that chymases, of different MC subpopulations, display quite different extended cleavage specificities. However mouse do possess a MC chymase with almost identical cleavage specificity as the human MC chymase indicating a strong evolutionary pressure to maintain this enzyme specificity.</p>

Hepatitis C is a serious disease that affects about 200 million people worldwide. No anti-HCV vaccine or specific anti-viral drugs are available today. Non-structural protein 3 (NS3) of HCV is a bifunctional serine protease/helicase, and the protease has become a prime target in the search for anti-HCV drugs. In this work, the complete HCV NS3 gene has been cloned and expressed, and the protein has been purified using affinity chromatography. An assay for measuring the protease activity of full-length NS3 protease has been developed and used for inhibition studies. A series of peptide-based inhibitors of NS3 protease varying in length, the composition of the side-chain and the N- and C-terminal groups have been studied. Potent tetra-, penta- and hexapeptide inhibitors of the NS3 protease were discovered. Hexapeptides with an acyl sulfonamide C-terminal residue were the most potent inhibitors of the NS3 protease, having nanomolar Ki-values. The selectivity of the inhibitors was assessed using other serine and cysteine proteases. NS3 protease inhibitors with electrophilic C-terminal groups were non-selective while those comprising a C-terminal carboxylate or acyl sulfonamide group were selective. All inhibitors with a small hydrophobic P1 side-chain residue were non-selective for the NS3 protease, being good inhibitors of human leukocyte elastase. This result highlights the importance of the P1 residue for inhibitor selectivity, which stems from the major role of this residue in determining substrate specificity of serine proteases. Electrophilic inhibitors often cause slow-binding inhibition of serine and cysteine proteases. This was observed with other proteases used in our work but not with NS3 protease, which indicates that mechanism of inhibition of NS3 protease by electrophilic inhibitors may not involve formation of a covalent bond. The structure-activity relationships obtained in this work can be used for improvement of peptide-based inhibitors of HCV NS3 protease towards higher inhibitory potency and selectivity.