title: Ddi1-like protein from Leishmania major is an active aspartyl proteinase.
abstract: Eukaryotic cells respond to DNA damage by activating damage checkpoint pathways, which arrest cell cycle progression and induce gene expression. We isolated a full-length cDNA encoding a 49-kDa protein from Leishmania major, which exhibited significant deduced amino acid sequence homology with the annotated Leishmania sp. DNA damage-inducible (Ddi1-like) protein, as well as with the Ddi1 protein from Saccharomyces cerevisiae. In contrast to the previously described Ddi1 protein, the protein from L. major displays three domains: (1) an NH2-terminal ubiquitin like; (2) a COOH terminal ubiquitin-associated; (3) a retroviral aspartyl proteinase, containing the typical D[S/T]G signature. The function of the L. major Ddi1-like recombinant protein was investigated after expression in baculovirus/insect cells and biochemical analysis, revealing preferential substrate selectivity for aspartyl proteinase A₂ family substrates, with optimal activity in acidic conditions. The proteolytic activity was inhibited by aspartyl proteinase inhibitors. Molecular modeling of the retroviral domain of the Ddi1-like Leishmania protein revealed a dimer structure that contained a double Asp-Ser-Gly-Ala amino acid sequence motif, in an almost identical geometry to the exhibited by the homologous retroviral aspartyl protease domain of yeast Ddi1 protein. Our results indicate that the isolated Ddi1-like protein is a functional aspartyl proteinase in L. major, opening possibility to be considered as a potential target for novel antiparasitic drugs.
authors.affiliation: Servicio de Parasitología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid, Spain.
authors.forename: María J
authors.initials: MJ
authors.lastname: Perteguer
authors.forename: Paulino
authors.initials: P
authors.lastname: Gómez-Puertas
authors.forename: Carmen
authors.initials: C
authors.lastname: Cañavate
authors.forename: Francehuli
authors.initials: F
authors.lastname: Dagger
authors.forename: Teresa
authors.initials: T
authors.lastname: Gárate
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Valdivieso
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D057055:Aspartic Acid Proteases / Q000737:chemistry / Q000378:metabolism*; D019295:Computational Biology; D019281:Dimerization; D007700:Kinetics; D018320:Leishmania major / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D061987:Sf9 Cells; D013379:Substrate Specificity; D025801:Ubiquitin / Q000737:chemistry
chemical_list: C108125:DDI1 protein, S cerevisiae; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D057055:Aspartic Acid Proteases