Rab proteins comprise the largest group of Ras-related GTPases in mammalian cells. Although several of these proteins are known to regulate specific vesicular trafficking pathways, many do not yet have an assigned function. Current studies have revealed that one such Rab protein, Rab24, exhibits properties distinct from other family members: For example, Rab24 has a very low intrinsic GTPase activity, is not efficiently modified by isoprenoid lipids and associates weakly with known Rab carrier proteins. When a putative dominant-negative (nucleotide-binding deficient) Rab24 mutant was expressed in cultured cells, it appeared to induce a striking array of autophagic vacuoles and nuclear inclusion bodies. Based on these preliminary observations, the following hypotheses have been developed: (a) Rab24 normally functions in the formation or maturation of autophagic vacuoles in mammalian cells. (b) Mutant forms of Rab24 trigger an autophagic cell death cascade that involves accumulation of degradative vacuoles and their ultimate fusion with the nucleus. The central objective of the proposed studies is to test these hypotheses. Toward this end, the biochemical properties of recombinant Rab24 will be defined, and the expression, nucleotide state, and localization of Rab24 will be studied in relation to autophagy in tumor cell models (Aim I). Genetic and biochemical strategies will be used to identify and characterize novel Rab24 effectors or carrier proteins (Aim 2). immunocytochemical and biochemical methods will be used to further define the nature of the vacuole-like structures induced by the Rab24 mutants (Aim 3). Domain-swapping and site-directed mutagenesis will be used to distinguish which structural features make Rab24 unique among the Rab family in being able to induce an autophagic response when expressed in the nucleotide-deficient form (Aim 4). Finally, novel cell-permeable fusion polypeptides will be used to test the possibility that synthetic peptides mimicking unique Rab24 sequence elements might be used to trigger autophagic death in transformed cells. The proposed studies will provide new information about the biological function of Rab24 and the poorly understood mechanisms that regulate cellular macroautophagy. Ultimately, these insights could reveal new targets for therapeutic intervention in malignant cells.
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