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ERAP1 is an aminopeptidase that is a component of antigen processing. To distinguish the role of ERAP1 from homologs ERAP2 and IRAP, I identified three specific ERAP1 inhibitors via a high-throughput screen. These compounds inhibit hydrolysis of a decamer peptide, and some inhibit ERAP1 in a cellular assay. These inhibitors enable dissection of ERAP1 mechanism. ERAP1 has been crystallized in two conformations: open and closed. I collected SAXS data on ERAP1 in the presence of various inhibitors. ERAP1 adopts an open conformation in solution, but some inhibitors stabilize the closed form. Compound 3 docks to a distal pocket 28Å from the active site zinc, while DG013 and DG014 bind to the active site. This distal pocket is an allosteric activation site, and allostery is mediated by stabilizing the closed state. I also identified an intermediate step in substrate binding where helix 4a becomes ordered while ERAP1 maintains an open conformation. Helix 4a then rotates and engages substrate when ERAP1 closes. The nonsynonymous SNP rs30187 at position 528 (Lys/Arg) subtly alters ERAP1 activity in vitro and correlates with disease incidence. Position 528 forms a conformation-dependent electrostatic interaction with Glu913 in the closed structure. The energetic contribution of this interaction is stronger for Lys528 than Arg528. Inhibitors that induce closing are more potent for Lys528 than Arg528. I propose a model where either helix 4a stabilization or allosteric site occupancy shift the conformational equilibrium towards a closed state, while substitution at position 528 alters the opening rate.

The increased understanding of underlying neurobiological processes and their connections to cognitive performance has opened new doors for novel therapeutic strategies that aim to prevent, and even reverse the effects of memory disorders, neurodegenerative diseases and other cognitive impairments. One of the new and exciting drug targets that has emerged as a result of this research is insulin-regulated aminopeptidase (IRAP). While the research is still in its early stages, inhibition of IRAP have promising therapeutic potential and several small molecule and peptidomimetic candidates that act as inhibitors of IRAP have been synthesized. Analysis of in vivo performed novel object recognition tests, in vitro cell viability and morphology assays were conducted in this study to further investigate the cognitive effects and other effects of an IRAP-inhibitor called HA08. Recorded novel object recognition test previously performed on rats were scored and analysed with statistical software. Primary cortical and hippocampal cells harvested from rat fetuses were treated with increasing concentrations of HA08 and oxytocin. The cell viability assays consisted of LDH assays to analyse HA08’s effect on the cells membrane integrity and MTT assays to analyse its effect on mitochondrial function. Immunocytochemistry was used to assess the difference in neurite length in cortical cells and the number of dendritic spines in hippocampal cells. No increases in recognition were found in HA08 treated rats. HA08 did not seem to affect the membrane integrity at concentrations used. Further research is needed to improve the accuracy of the results and to fully unlock the potential of IRAP-inhibitor HA08.

Plasmodium falciparum dipeptidyl aminopeptidase 1 (DPAP1) contributes to the degradation of hemoglobin by releasing dipeptides from globin oligopeptides in the food vacuole. The lack of success at DPAP1 gene disruption suggests that this exopeptidase is important for efficient growth during the erythrocytic asexual stage. DPAP1 is therefore an attractive target for the development of anti-malarial drugs that block the catabolism of hemoglobin. To guide the design of selective, potent DPAP1 inhibitors, it is necessary to characterize the substrate specificity of this enzyme along with its human homolog cathepsin C. Although native purification of DPAP1 is possible, the amount of purified enzyme obtained is insufficient for extensive biochemical characterization. To overcome this obstacle, a strategy was developed for the recombinant expression of soluble DPAP1 in the bacterium Escherichia coli and for its activation in vitro. The production of active recombinant DPAP1 presents three challenges: 1) expression of the protein in soluble form, 2) generation of the native N-terminus, and 3) cleavage of the pro-domain. Soluble expression of DPAP1 was achieved by fusing it to the C-terminus of maltose-binding protein (MBP). A linker sequence encoding a tobacco etch virus protease (TEVp) cleavage site was introduced between MBP and DPAP1 such that TEVp cleavage would generate the presumed native N-terminus of DPAP1. Incubation of the MBP-DPAP1 fusion with TEVp resulted in the release of free DPAP1which hydrolyzed the fluorogenic substrate proyly-arginyl-7-amido-4 methyl coumarin (Pro-Arg-AMC). Various proteases were tested for the ability to excise the pro-region. Treatment with both trypsin and papain removed the pro-region and increased DPAP1 activity two to three fold. When assayed with Pro-Arg-AMC, trypsin-treated DPAP1 had kinetic properties similar to native enzyme whereas papain-treated DPAP1 deviated from Michaelis-Menten kinetics. Using a combinational dipeptidyl substrate library, the substrate specificities of native and recombinant (trypsin-activated) DPAP1, as well as of human cathepsin C were profiled. We find that both DPAP1 and human cathepsin C accept a wide spectrum of amino acid side chains at the substrate P1 and P2 positions. Interestingly, several P2 residues show high selectivity for DPAP1 or cathepsin C. The collected data point to the feasibility of designing inhibitors that are specific for DPAP1 over cathepsin C. / Master of Science in Life Sciences

名古屋大学博士学位論文 学位の種類 : 博士(医学)(論文) 学位授与年月日:平成4年7月20日 淺田義正氏の博士論文として提出された

Die Hefe Candida albicans ist ein fakultativ humanpathogener Mikroorganismus, der insbesondere bei immungeschwächten Patienten schwere Erkrankungen der Haut und Schleimhäute sowie der inneren Organe hervorrufen kann. Seit langer Zeit wird eine Beteiligung des Hefepilzes an der Ätiopathogenese der Zahnkaries diskutiert, vor allem aufgrund der Säure-bildung, die zur Demineralisation der Zahnhartsubstanz beitragen kann. Hydrolytische Enzyme ermöglichen vermutlich die Gewebeinvasion von Candida albicans. In der vorliegenden Arbeit wurde ein sezerniertes peptidolytisches Enzym aus der Zellwand des Mikroorganismus isoliert, identifiziert und funktionell charakterisiert. Die mittels massenspektrometrischer Analyse der tryptischen Peptide und Datenbankrecherche ermittelte Primärstruktur und die Ergebnisse der funktionellen Charakterisierung ließen eine Identifi-zierung des peptidolytischen Enzyms als neutrale Arginin/Alanin/Leucin-spaltende Metallo-Aminopeptidase (CaApe2) zu, die durch den ORF CaO19.5197 (GenBank RefSeq XM 705313) kodiert wird. Mithilfe der Proteinanalytik wurde Serin-88 als N-terminale Aminosäure ermittelt. Die Aminosäuren 88 bis 954 des hypothetischen Genprodukts ergeben eine nominale Molekularmasse von 97,607 kDa. CaApe2 weist gleich hohe Ähnlichkeit mit den paralogen Genprodukten ScAap1 und ScApe2 auf, was eine Duplikation und Subfunktionalisierung des phylogenetischen Vorläufergens in Saccharomyces cerevisiae nahe legt. Die fehlende kollagenolytische Wirksamkeit von CaApe2 spricht gegen eine direkte Rolle des Enzyms in der Pathogenese der Dentinkaries von Candida albicans, schließt aber eine unterstützende Funktion nicht aus. Die Kollagendegradation durch aufgeschlossene Zellen und Kulturüberstand einer Flüssigkultur von Candida albicans wurde im sauren und neutralen Milieu mithilfe der Hydroxyprolin-Bestimmung untersucht. Dabei war keine Kollagenolyse mit Aktivitätsmaximum im neutralen Bereich nachweisbar. Im sauren pH-Bereich konnte eine deutliche Hydrolyse von säureunlöslichem Typ-I-Kollagen und auch von demineralisierter Dentinmatrix durch Kulturmedium gezeigt werden. Diese Kollagenolyse kann auf die bereits umfangreich charakterisierten sezernierten Aspartylproteinasen zurückgeführt werden. Die in der Literatur beschriebene Korrelation zwischen dem Ausmaß des Kariesbefalls und der Quantität der Besiedelung mit Candida albicans legt eine Beteiligung des Hefepilzes an der Kariogenese nahe. Auch die in der vorliegenden Arbeit gezeigte Fähigkeit von Candida albicans zur Dentinkollagendegradation unterstützt die Hypothese einer Kariogenität der Hefe. / The proteolytic potential of the pathogenic fungus Candida albicans was evaluated by the identification and functional characterization of a peptidolytic enzyme isolated from the cell wall of the microorganism. Determination of basic structural and kinetic data identified a neutral arginine/alanine/leucine-specific metallo-aminopeptidase of unknown function termed CaApe2 which is encoded by ORF CaO19.5197 (GenBank RefSeq XM_705313). Mass spectrometric tryptic peptide analysis and N-terminal protein sequencing revealed serine-88 to represent the N-terminus of CaApe2. The isolated CaApe2 protein shares equally high similarity with the gene products ScAap1 and ScApe2 suggesting duplication of a phylogenetically ancient precursor gene in Saccharomyces cerevisiae. The observed failure to cleave human type-I and type-IV collagen in vitro challenges a direct role secreted CaApe2 might play in the degradation of extracellular matrix components during host colonization, but does not exclude per se a contribution of the aminopeptidase to the pathogenicity of C. albicans.

Die Hefe Candida albicans ist ein fakultativ humanpathogener Mikroorganismus, der insbesondere bei immungeschwächten Patienten schwere Erkrankungen der Haut und Schleimhäute sowie der inneren Organe hervorrufen kann. Seit langer Zeit wird eine Beteiligung des Hefepilzes an der Ätiopathogenese der Zahnkaries diskutiert, vor allem aufgrund der Säure-bildung, die zur Demineralisation der Zahnhartsubstanz beitragen kann. Hydrolytische Enzyme ermöglichen vermutlich die Gewebeinvasion von Candida albicans. In der vorliegenden Arbeit wurde ein sezerniertes peptidolytisches Enzym aus der Zellwand des Mikroorganismus isoliert, identifiziert und funktionell charakterisiert. Die mittels massenspektrometrischer Analyse der tryptischen Peptide und Datenbankrecherche ermittelte Primärstruktur und die Ergebnisse der funktionellen Charakterisierung ließen eine Identifi-zierung des peptidolytischen Enzyms als neutrale Arginin/Alanin/Leucin-spaltende Metallo-Aminopeptidase (CaApe2) zu, die durch den ORF CaO19.5197 (GenBank RefSeq XM 705313) kodiert wird. Mithilfe der Proteinanalytik wurde Serin-88 als N-terminale Aminosäure ermittelt. Die Aminosäuren 88 bis 954 des hypothetischen Genprodukts ergeben eine nominale Molekularmasse von 97,607 kDa. CaApe2 weist gleich hohe Ähnlichkeit mit den paralogen Genprodukten ScAap1 und ScApe2 auf, was eine Duplikation und Subfunktionalisierung des phylogenetischen Vorläufergens in Saccharomyces cerevisiae nahe legt. Die fehlende kollagenolytische Wirksamkeit von CaApe2 spricht gegen eine direkte Rolle des Enzyms in der Pathogenese der Dentinkaries von Candida albicans, schließt aber eine unterstützende Funktion nicht aus. Die Kollagendegradation durch aufgeschlossene Zellen und Kulturüberstand einer Flüssigkultur von Candida albicans wurde im sauren und neutralen Milieu mithilfe der Hydroxyprolin-Bestimmung untersucht. Dabei war keine Kollagenolyse mit Aktivitätsmaximum im neutralen Bereich nachweisbar. Im sauren pH-Bereich konnte eine deutliche Hydrolyse von säureunlöslichem Typ-I-Kollagen und auch von demineralisierter Dentinmatrix durch Kulturmedium gezeigt werden. Diese Kollagenolyse kann auf die bereits umfangreich charakterisierten sezernierten Aspartylproteinasen zurückgeführt werden. Die in der Literatur beschriebene Korrelation zwischen dem Ausmaß des Kariesbefalls und der Quantität der Besiedelung mit Candida albicans legt eine Beteiligung des Hefepilzes an der Kariogenese nahe. Auch die in der vorliegenden Arbeit gezeigte Fähigkeit von Candida albicans zur Dentinkollagendegradation unterstützt die Hypothese einer Kariogenität der Hefe. / The proteolytic potential of the pathogenic fungus Candida albicans was evaluated by the identification and functional characterization of a peptidolytic enzyme isolated from the cell wall of the microorganism. Determination of basic structural and kinetic data identified a neutral arginine/alanine/leucine-specific metallo-aminopeptidase of unknown function termed CaApe2 which is encoded by ORF CaO19.5197 (GenBank RefSeq XM_705313). Mass spectrometric tryptic peptide analysis and N-terminal protein sequencing revealed serine-88 to represent the N-terminus of CaApe2. The isolated CaApe2 protein shares equally high similarity with the gene products ScAap1 and ScApe2 suggesting duplication of a phylogenetically ancient precursor gene in Saccharomyces cerevisiae. The observed failure to cleave human type-I and type-IV collagen in vitro challenges a direct role secreted CaApe2 might play in the degradation of extracellular matrix components during host colonization, but does not exclude per se a contribution of the aminopeptidase to the pathogenicity of C. albicans.

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Le premier acide aminé incorporé dans une chaîne polypeptidique naissante est toujours la méthionine. On identifie donc toujours ce premier résidu à la méthionine N-terminale. Cependant, les deux tiers des protéines accumulées à l'état stationnaire ne présentent plus leur méthionine initiatrice. Cet enlèvement résulte essentiellement d'une maturation protéolytique affectant chaque protéine. Ainsi, l'Excision de la Méthionine N-terminale (NME) concerne la majorité des protéines et ce dès que les premiers résidus émergent du ribosome. Ce mécanisme est retrouvé dans tous les compartiments cellulaires où une synthèse protéique a lieu : le cytoplasme, les plastes et les mitochondries. Les enzymes responsables du clivage de la méthionine initiatrice sont les METhionine AminoPeptidases (METAPs) ; les METAPs sont conservées dans le Règne vivant. Des études fonctionnelles de délétions géniques ont montré le caractère létal du maintien de la première méthionine dans tous les organismes. Il y a plus de dix ans, les METAPs ont été identifiées comme étant la cible de composés naturels ayant des effets anticellulaires. Aujourd'hui un nombre croissant d'études rapportent que la NME est une cible prometteuse pour le traitement de nombreuses pathologies. Néanmoins, les bases moléculaires qui expliquent le caractère essentiel de la NME restent très peu comprises, en particulier dans le cytoplasme des eucaryotes supérieurs. Grâce à un système inductible permettant de moduler finement la NME cytoplasmique dans la plante modèle Arabidopsis thaliana et différentes approches incluant des analyses protéomiques et métabolomiques, j'ai pu étudier les événements moléculaires précoces associés à l'inhibition de la NME cytoplasmique. J'ai également caractérisé la contribution relative des deux types de METAP cytoplasmiques au processus. Dans ce contexte, j'ai pu démontrer chez A. thaliana que la NME cytoplasmique agit sur deux voies de signalisation fréquemment dérégulées lors de conditions pathologiques : le statut des composés thiolés et la protéolyse. La diminution de la NME cytoplasmique induit une protéolyse accrue principalement via une augmentation du nombre de protéines destinées à une dégradation rapide. Ainsi, l'activité de la NME, en modulant la sensibilité de nombreuses protéines à subir la protéolyse, est un élément fondamental de la régulation de la demi-vie protéique. Finalement, mes résultats simialires obtenus également chez les Archées, levures et les lignées de cellules humaines suggèrent l'existence d'un mécanisme ubiquitaire associé à la NME.

Central infusion of the hexapeptide angiotensin IV (Ang IV) and its analogs have been demonstrated to markedly enhance memory retention and retrieval in rats using a range of learning and memory paradigms. This effect is mediated by the binding of the peptide to the specific binding site previously described as the AT4 receptor. The AT4 receptor has been isolated and identified as insulin-regulated aminopeptidase (IRAP), a type II transmembrane protein belonging to the M1 family of zinc-dependent aminopeptidases. Subsequently, AT4 receptor ligands, including Ang IV and its analogues and the unrelated peptide LVV-hemorphin-7, were demonstrated to be peptide inhibitors of IRAP. These findings suggest that AT4 ligands may exert their cognitive effects by inhibiting the catalytic activity of IRAP in the brain. Therefore, IRAP is an important target for the development of a new class of therapeutic agents for the treatment of memory loss. / To characterize IRAP at the molecular level and identify non-peptide inhibitors of IRAP for drug development, the aims of this study were to: 1) determine whether IRAP exists as a homodimer; 2) identify cysteine residue(s) involved in IRAP dimerization; 3) investigate the roles of the conserved residues of the HEXXH(X)18E Zn2+-binding motif and the GAMEN motif in substrate/inhibitor binding using site-directed mutagenesis; 4) use a molecular model of the catalytic domain of IRAP based on the crystal structure of a related M1 family metallopeptidase to: (i) identify key residues required for substrate/inhibitor binding; (ii) identify and characterize non-peptide IRAP inhibitors from a compound database by in silico virtual screening based on the homology model of IRAP. / Co-immunoprecipitation followed by Western blotting of IRAP under reducing and non-reducing conditions showed IRAP exists both as covalently- and non-covalently-bound homodimers. Serine scanning of cysteine residues potentially involved in forming inter-molecule disulfide-bonds was performed. Mutational analyses indicated that covalent homodimerization of IRAP is due to more than one cysteine residue. Limited trypsin digestion followed by co-immunoprecipitation suggests that non-covalent homodimerization of IRAP involves residues/regions within the last 130 amino acids of the protein. / The catalytic site of IRAP contains two consensus motifs, the H464EXXH468(X)18E487 Zn2+-binding motif and the G428AMEN432 motif. The role of conserved residues with these motifs was investigated using site-directed mutagenesis and pharmacological analyses. The conserved His and Glu residues of the Zn2+-binding motif were shown to be essential for IRAP catalytic activity. This was also observed for the Met and Glu residues of the GAMEN motif, while Asn mutant retained some catalytic activity. Residues important for substrate or inhibitor binding were identified as Gly, Ala and Asn. / A molecular model of the catalytic domain of IRAP based on the crystal structure of a homologous M1 metallopeptidase, leukotriene A4 hydrolase (LTA4H) was used to compare the catalytic sites of IRAP and LTA4H, and identified two amino acids at the putative substrate-binding pocket: Ala427 and Leu483 in IRAP, and the corresponding residues Tyr267 and Phe314 in LTA4H. A mutational analysis involving substitution of Ala427 and Leu483 with the corresponding residues revealed Ala427 and Leu483 characterize the enzyme S1 subsite, influencing the affinity and placement of substrates and peptide inhibitors in the catalytic site. / The molecular model of IRAP was also used for virtual screening of compound databases to identify novel non-peptide inhibitors. After two rounds of in silico screening, a family of compounds was identified and shown to be specific and competitive inhibitors of IRAP. Preliminary results suggest that one of these inhibitors, referred to as HFI 142, may possess memory-enhancing properties. The identification of non-peptide IRAP inhibitors will assist in pharmacological studies aimed at understanding the molecular mechanisms of IRAP aminopeptidase activity and physiological role of IRAP. In addition, the new inhibitors have the potential to form the basis for the development of a novel class of drugs useful for treating memory disorders.