Neurodegenerative diseases affect millions of people each year in ways that are emotionally and financially crippling. Restoration of nervous system function will require regeneration of neuron structure including axonal and dendritic processes. Microtubules form a cytoskeletal system known to contribute to the structural integrity of axons and to participate in the transport of materials within axons. In this study, the molecular structure and interactions of microtubule proteins associated with growing axons will be studied. The gene for one of these proteins - microtubule associated protein 1B - is likely to be the defective gene in the fatal human disease spinal muscular atrophy. An unusual processing mechanism that acts on microtubule-associated protein 1B (MAP1B) was recently identified. This mechanism produces two MAP1B subunits from a single polyprotein precursor. If the polyprotein is not processed, significant alterations in microtubule organization might occur due to microtubule cross-linking. The processing of MAP1B will be further characterized by identifying the protease that processes MAP1B. Polyprotein expressed in vitro will be used as the substrate to assay MAP1B specific protease activity. New evidence indicates that MAP1A is structurally related to MAP1B and that it is also derived from a polyprotein precursor containing two of its subunits, the MAP1A heavy chain and light chain 2. Antibodies that recognize epitopes near the carboxyl-terminus of the putative MAP1A polyprotein will be used in peptide-mapping experiments to determine if the MAP1A polyprotein is detectable in vivo as was recently suggested (Langkopf et al., In press). The primary sequence of light chain 3, a low molecular weight subunit common to MAP1A and MAP1B, will be determined by cloning and sequencing cDNA encoding this protein from a lambda gt 11 expression library. The antibody used to select these clones will be generated to a synthetic peptide whose sequence was obtained by Edman degradation of the N-terminus of light chain 3. The anti-light chain 3 antibody will also be used to determine if light chain 3 is part of the microtubule-binding domains of MAP1A and MAP1B. Finally, the distribution of light chain 1, light chain 2, and light chain 3 in brain will be determined by immunohistochemical methods. The localization of low molecular weight MAP1A and MAP1B subunits will be compared to the localization of the heavy chains. Differential localization of the subunits relative to their heavy chains may indicate that subunit composition regulates heavy chain function.
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