The applicant proposes to study the binding of HAP1 to other cellular proteins. The applicant identified HAP1 in the rat as a protein that binds to huntingtin, the protein that is abnormal in Huntington's disease. Huntington's disease is an autosomal dominant disease characterized by massive neuronal loss that is particularly severe in specific brain regions. The genetic defect is an expansion of a CAG trinucleotide repeat that produces a polyglutamine domain in an expressed protein named huntingtin. The applicant has documented that HAP1 binds to huntingtin in vivo and that there is increased binding of HAP1 to huntingtin that has an expanded polyglutamine domain. He has also found that HAP1 associates with accessory proteins for the microtubular motor proteins kinesin and dynein that participate in the anterograde and retrograde transport in the neuron. These accessory proteins are kinesin light chain and dynactin p150Glued. Also, HAP1 associates with various membranous organelles. Thus the applicant developed the ingenious and powerful hypothesis that HAP1 plays a role in targeting motor protein complexes to intracellular structures; also, that HAP1 may coordinate the two types of transport. The applicant further hypothesizes that mutant huntingtin may interact with HAP1 in such a way that the association of HAP1 with these other molecules is perturbed and thus intracellular trafficking is affected. Thus the applicant proposes three specific aims. The first is to clone and characterize homologues of human HAP1. In this aim, he proposes also to generate antibodies to human HAP1 protein, characterize the binding properties of human HAP1 to huntingtin, determine the regional distribution of human HAP1, and determine the subcellular localization of HAP1 in human and monkey brain and compare this to human huntingtin.
In Aim 2, he will investigate the association of HAP1 with microtubular motor binding proteins and intracellular organelles. In this aim, he will also determine which regions of HAP1 that bind to kinesin light chain, dynactin p150Glued, and intracellular organelles. He will also examine the effects of phosphorylation, calcium level, and other cellular factors on the association of HAP1 with huntingtin and to the proteins involved in intracellular transport. He will also identify the potential proteins that link HAP1 to intracellular vesicle membranes.
In Aim 3, he will study the effect of huntingtin on the association of HAP1 with motor binding proteins and organelles. In particular, he will study huntingtin with different numbers of glutamine repeats on the self-interaction of HAP1 and on HAP1's interactions with other molecules using a variety of techniques.
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