Ubiquitin (Ub) is a small highly conserved multifunctional protein that is found in all eukaryotic cells as both the free molecule and covalently attached to other cellular proteins. Some Ub-protein conjugates are associated with the microtubule network in normal fibroblasts, with the cytoskeleton in bovine brain, with the senile plaques and neurofibrillary tangles (NFT) of Alzheimer's disease (AD), and with the inclusion bodies characteristic of other neurodegenerative diseases. It is possible that abnormal or defective ubiquitination of cytoskeletal proteins is involved in the pathology of AD and it is our long range goal to understand the cytoskeletal changes which occur in AD and the role ubiquitin plays in these events. As a first step, we will characterize normally ubiquitinated cytoskeletal proteins and the ubiquitinated proteins found in NFT. The cytoskeletal/NFT proteins will be isolated and the ubiquitinated species detected using mAbs to Ub already developed in this laboratory; the proteins will be purified directly from 2D gels. These proteins will be microsequenced and this information used in two attacks. 1) The structure, expression and regulation of these novel proteins will be studied using molecular biology approaches. Peptide sequence information will be used to clone and sequence the gene encoding the protein and this will provide probes to study expression and structural information for understanding function. Site directed mutagenesis will be used to alter the ubiquitin attachment sites to test the function of the Ub conjugate. Probes will be used to identify cells expressing these proteins and possible differences in normal and AD cells. 2) We will use protein chemistry and immunochemical approaches to study the proteins and their functions. Peptide specific antibodies will be developed and used in Western blots and immunoprecipitation studies to determine the dynamics of ubiquitin-protein conjugation in normal and pathological situations. The Ub conjugation site will be determined since differences in the ubiquitination pattern and sites of attachment could alter the function of Ub in these complexes and affect the stability of these proteins. Cellular localization of these proteins will be determined using indirect immunofluorescence and immunoelectron microscopy. Immunocytochemistry will be used to examine the distribution of these proteins in normal and AD brain. The identification and cellular localization of both the ubiquitinated and nonubiquitinated forms of these cytoskeletal proteins will provide insight into the structure and function of the normal cytoskeleton. The identification of specific ubiquitinated components in AD may provide new rationales for developing probes for diagnosis and therapeutic strategies.
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