Molecular and Cellular Characterization of an AT-Hook Protein from Leishmania

AT-rich DNA, and the proteins that bind it (AT-hook proteins), modulate chromosome structure and function in most eukaryotes. Unlike other trypanosomatids, the genome of Leishmania species is unusually GC-rich, and the regulation of Leishmania chromosome structure, replication, partitioning is not fully understood. Because AT-hook proteins modulate these functions in other eukaryotes, we examined whether AT-hook proteins are encoded in the Leishmania genome, to test their potential functions. Several Leishmania ORFs predicted to be AT-hook proteins were identified using in silico approaches based on sequences shared between eukaryotic AT-hook proteins. We have used biochemical, molecular and cellular techniques to characterize the L. amazonensis ortholog of the L. major protein LmjF06.0720, a potential AT-hook protein that is highly conserved in Leishmania species. Using a novel fusion between the AT-hook domain encoded by LmjF06.0720 and a herpesviral protein, we have demonstrated that LmjF06.0720 functions as an AT-hook protein in mammalian cells. Further, as observed for mammalian and viral AT-hook proteins, the AT-hook domains of LmjF06.0720 bind specific regions of condensed mammalian metaphase chromosomes, and support the licensed replication of DNA in mammalian cells. LmjF06.0720 is nuclear in Leishmania , and this localization is disrupted upon exposure to drugs that displace AT-hook proteins from AT-rich DNA. Coincidentally, these drugs dramatically alter the cellular physiology of Leishmania promastigotes. Finally, we have devised a novel peptido-mimetic agent derived from the sequence of LmjF06.0720 that blocks the proliferation of Leishmania promastigotes, and lowers amastigote parasitic burden in infected macrophages. Our results indicate that AT-hook proteins are critical for the normal biology of Leishmania . In addition, we have described a simple technique to examine the function of Leishmania chromatin-binding proteins in a eukaryotic context amenable to studying chromosome structure and function. Lastly, we demonstrate the therapeutic potential of compounds directed against AT-hook proteins in Leishmania .