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Date of Award
Molecular Biology and Genetics
Myelin proteolipids (PLP and DM20) are encoded by a single gene and comprise >50% total protein mass in the CNS myelin. Proteolipid proteins play a role in survival and differentiation of oligodendrocytes and other CNS cell types, cell signaling, and myelin compaction. Mutations, duplications, and deletions of the PLP/DM20 gene produce dysmyelinating and demyelinating diseases. The first effort focused on developing a cell based proteolipid protein expression system to study the effects of these proteins on cell function. Wild type and mutant PLP and DM20 sequences were expressed as fusion proteins with several fluorescent protein tags in three human cell types. Transiently and stably transfected cells were examined for phenotypic changes in response to proteolipid protein expression. Consequently, a bank of stable cell lines was developed that expressed different levels of wild type and mutant proteolipids and mimicked the in-vivo responses to these proteins. These cells provided a homogeneous self-renewable system for studying proteolipid protein function. The second effort examined the role of the ubiquitin-proteasome system in proteolipid protein degradation using site directed mutagenesis and this cell based expression system. The degradation of wild type proteolipids, as well as proteins that carried mutations in the potential ubiquitination target residues was examined in the presence of a proteasomal inhibitor. Consequently, the PLP and DM20 proteins were identified as targets for the regulated degradation by the proteasome, and two lysine residues (K268 and K275) were shown to function as ubiquitin attachment sites. The third effort focused on identification and characterization of low molecular weight (LMW) proteolipid proteins that were preferentially expressed during apoptosis. Using site directed mutagenesis and a cell based expression system, these LMW proteolipids were shown to be produced by internal translation initiation from two AUG codons within the PLP/DM20 mRNAs (M205 and M234) in a process mediated by an internal ribosome entry site. The M205 protein contains a sequence that putatively displays a growth factor activity, while the M234 protein did not. The overlapping coding frames and the sequence of these proteins suggest that they function as components of a complex biological system.
Cypher, Maria A., "Novel synthesis and degradation pathways for the myelin proteolipid proteins" (2003). Wayne State University Dissertations. 3394.