IDENTIFICATION OF PTMS ON RIBOKINASE FROM LEISHMANIA MEXICANA

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Leishmania parasites are single cell protozoa that cause devastating diseases throughout much of the developing world and for which limited pharmacological therapies exist. Glucose is a nutrient of central importance for these parasites that is imported by permeases of the facilitative glucose transporter family. The Landfear laboratory has generated a glucose transporter null mutant in L. mexicana by targeted gene replacement, and this null mutant has strong biological phenotypes including the inability to survive and replicate within macrophages, the mammalian host cell that is invaded by the parasite. Analysis of wild type and null mutant parasites by 2-dimensional (2D) gel electrophoresis and mass spectrometry (MS) has identified over 40 proteins whose levels change significantly between these two lines. One such protein, ribokinase (RK), is upregulated ~7-fold in the null mutants, probably to replenish by an alternative route ribose-phosphate that cannot be made efficiently from glucose in the transport-deficient mutant. Western blots of 2D gels revealed that 4 new RK spots, of similar MW to wild type RK, appear in the null mutant. These results suggest that glucose starvation induces specific post-translational modifications (PTMs) on RK. To begin to examine the signal transduction pathway that results in these modifications, we propose to partner with PNNL to identify the PTMs that are induced on RK by glucose starvation. Initially, intact protein LC-MS will be performed on RK to identify the specific PTMs. Subsequently bottom-up analysis of RK peptides will be employed to identify the specific sites of modifications on RK. Although Leishmania parasites must respond to a variety of changes in environment during their life cycles, including pronounced changes in glucose availability, almost nothing is known at the molecular level about how they adapt to such environmental alterations. This focused project will begin to map in detail biochemical modifications in these pathogens that ensue from glucose limitation. The project will form the basis for ultimately assembling a biochemical pathway revealing how these parasites adapt to altering nutrient levels and thus maintain viability within their hosts during their life cycle.