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 ubiquitinassociated; (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 A 2 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 AspSer-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.

Dimer Interface Organization is a Main Determinant of Intermonomeric Interactions and Correlates with Evolutionary Relationships of Retroviral and Retroviral-Like Ddi1 and Ddi2 Proteases

“…The relatively wider substrate cavity is supposed to be responsible for recognising secondary or tertiary structure-it permits the binding of smaller globular proteins [5,6,8]. Despite the special flap arrangement and larger substrate cavity compared with those in HIV-1 and other retroviral PRs, Ddi1-Lm PR exhibited substrate binding and catalytic activity [12]. A polypeptide substrate has been modelled to the substrate binding cavity of the homodimeric Ddi1-Sc PR, and the structure of the proposed complex indicated that the enzyme can bind a polypeptide, despite the lower number of closer enzyme-substrate contacts [5].…”

“…Despite the low sequence identity between the target and template structures, retroviral and retroviral-like PRs exhibit high structural similarity [5], which makes the homology modeling of retroviral-like PRs possible. Before the deposition of the first Ddi1-Lm crystal structure to the PDB in 2017 [8], model structures were previously prepared for Ddi1-like PR of L. major [5,12,13], and the PRs of Schistosoma mansoni and Hymenolepis microstoma [13]. Numerous eukaryotic organisms have been found to express homologs of retrovirus and retroelement PRs, which evolved from their ancestors and which were retained during their evolution [3,4].…”

“…Protease domain of Ddi1-Hs protein was found to undergo post-translational modification, ubiquitination sites were identified in the proximities of the active site (K192) and the dimer interface (K382) [68], but effect of these modifications of protease function has not been elucidated. Among the Ddi-like PRs, proteolytic activity of a recombinant protein was proved in vitro only for Ddi1-Lm [12]. Despite using various methods (e.g., screening an HEK293 cell line-derived peptide library or cleavage reactions by oligopeptide or protein substrates), neither the autoproteolytic activity (cis-activity) nor cleavage of any target protein (trans-activity) has been observed for purified Ddi2-Hs [6] and Ddi1-Sc [5,7] proteases.…”

Functional Study of the Retrotransposon-Derived Human PEG10 Protease

“…Our data imply low catalytic efficiency of PR PEG10 , but detailed comparison cannot be made due to the limited data on the catalytic efficiencies of retroviral-like proteases. To date, the proteolytic activity of a recombinant protein was proven in vitro only for Leishmania major Ddi1 [33] and Saccharomyces cerevisiae Ty1 retroviral-like proteases [30], while catalytic efficiency was determined only for the latter one. The structural characteristics of PR PEG10 indicate high similarity with other retroviral-like proteases; thus, the dimer stability of PR PEG10 is potentially lower than that of most retroviral proteases [31], and is more comparable to that of retroviral-like proteases.…”

Gene expression modulation and the molecular mechanisms involved in Nelfinavir resistance in Leishmania donovani axenic amastigotes

“…PIs block the active site of aspartyl protease, a HIV-1 enzyme known to be essential for the maturation of viral proteins, by mimicking target peptides (Deeks et al, 1997). Recently, it has been shown that a Ddi1-like protein from L. major is an active aspartyl proteinase and the biochemical analysis of the recombinant L. major Ddi1-like protein shows that it hydrolyses specific substrates in common with the A 2 aspartyl proteinase family, of which the HIV-1 protease is a member (Perteguer et al, 2012). In this regard, it has been shown that an orthologue of the yeast Ddi1-like protein in Leishmania is a potential target for the HIV-1 PIs including NFV (White et al, 2011).…”