Characterization of functional domains in GIMAP5 protein

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GIMAP (GTPase of the immune associated nucleotide binding protein) family of proteins was discovered following the identification of the gene responsible for the lymphopenic phenotype (lyp) in BB-DP (BioBreeding diabetes-prone) rats by positional cloning. Mature T lymphocytes undergo spontaneous death in the absence of functional GIMAP5 in these rats. It is presumed that the other members of this highly conserved GIMAP family may be involved in the survival of different hematopoietic cell types. However, the mechanisms by which GIMAP family regulates cell survival or other processes are not known. GIMAP family of proteins are characterized by a N-terminal GTPase domain followed by a coiled-coil domain and in certain members, a C-terminal membrane anchor that can associate with intracellular structures such as mitochondria or lysosomes. Research in my laboratory has been aimed at understanding the function of GIMAP5 in T lymphocytes. As the long-term survival of T lymphocytes in the quiescent state is necessary to maintain their numbers in circulation and in lymphoid organs, we hypothesized that GIMAP5 is required for the integration of these survival signals generated through the T cell antigen receptor (TCR) and the interleukin-7 receptor (IL-7R). Using primary T cells from control and GIMAP5 deficient rats and mice, we showed that: (i) TCR and IL-7 mediated signaling pathways are affected in the absence of functional GIMAP5; (ii) GIMAP5 deficiency impairs Ca2+ entry via plasma membrane channels due to the inability of their mitochondria to sequester Ca2+; (iii) GIMAP5 regulates lysosomal Ca2+. Based on our preliminary results, we hypothesize that GIMAP5 influences cellular Ca2+ homeostasis by regulating lysosomal Ca2+. Research Plan 1) Identification of GIMAP5 domains and the proteins interacting with GIMAP5: Using different mutant constructs of GIMAP5, we will identify the domains involved in the interaction with microtubules and the associated motor proteins. 2) Regulation of calcium homeostasis by GIMAP5: The endo-lysosomal system stores significant amounts of intracellular Ca2+, in addition to the ER and mitochondria. We will characterize the influence of GIMAP5 and the mutant constructs on the lysosomal Ca2+ compartment. 3) Relationship between lysosomal and mitochondrial Ca2+ stores: We posit that in the absence of GIMAP5, lysosomes may retain more Ca2+, accounting for elevated GPN-induced Ca2+ release. We propose that this elevated free-lysosomal Ca2+ store in cell lacking GIMAP5 may also be directly linked to mitochondria. We will follow mitochondrial Ca2+ uptake using Rhod2 and mitotracker as detailed by us. Significance: GIMAP5 plays an essential non-redundant role in keeping resting T cells alive. Hence, the proposed study will help in understanding the role of GIMAP5 in the survival of normal lymphocytes.