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

Abstract

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.

Contributions of the EBNA1 Protein of Epstein-Barr Virus Toward B-Cell Immortalization and Lymphomagenesis

“…Studies from several groups, including ours, indicated that EBNA1's AT-hooks were essential to recruit ORC to DS (Sears, Ujihara et al 2004;Norseen, Thomae et al 2008). This conclusion has been reiterated by our observations that chimeric proteins in which ATH1 and ATH2 were substituted by cellular AT-hook proteins support licensed replication from DS (Sears, Kolman et al 2003;Kelly, Singh et al 2011). Lieberman and co-workers have demonstrated that EBNA1 uses its AT-hooks to recruit the ORC complex to DS via a Gquadruplex RNA intermediate, underscoring a critical role for these domains in oriPreplication (Norseen, Thomae et al 2008).…”

“…EBNA1 mutants in which either ATH1 or ATH2 is deleted retain the capacity to bind mitotic chromosomes and support the segregation of oriP-plasmids (Sears, Ujihara et al 2004). Further, both ATH1 and ATH2 can be substituted by cellular AT-hook proteins, and the resulting chimeras associate with mitotic chromosomes and support segregation of oriP-plasmids (Hung, Kang et al 2001;Sears, Kolman et al 2003;Kelly, Singh et al 2011). Because both ATH1 and ATH2 function equivalently to support oriP-plasmid segregation, but only ATH2 associates with EBP2/p40, it is likely that EBP2 does not mediate the partitioning of oriP-plasmids or EBV genomes.…”

Nuclear DNA replication and repair in parasites of the genusLeishmania: Exploiting differences to develop innovative therapeutic approaches

“…The multi-subunit ORC complex in budding and fission yeast recognizes AT-rich DNA sequences (Chuang & Kelly, 1999;Kelly et al, 2011) (Table 1). AT-rich sequences have been associated with DNA replication, repair and transcription in many eukaryotes and are recognized by AT-hook motifs.…”

“…These motifs can bind to the minor groove of AT-rich DNA, thus inducing a conformational change or recruiting other proteins (Kelly et al, 2011). In fission yeast, the Orc4 subunit contains nine AT-hooks, whereas S. cerevisiae Orc2 contains only one AT-hook (Chuang & Kelly, 1999;Kelly et al, 2011). HMGA1a is a classic AT-hook protein.…”

“…Its three AT-hook domains are essential to form transcription enhanceosomes that are involved in the recruitment of DNA replication components to origins (Dragan et al, 2008;Norseen et al, 2008). Mammalian viruses also use AThook proteins to recruit ORC to the viral origin of replication (Kelly et al, 2011).…”