The long term objectives of this Project are: understanding of the molecular mechanisms of PrPC>PrPSc transformation, PrP interactions with non-nucleic acid and nucleic acid chaperones, elucidation of the role of these cellular components in PrPSc accumulation and disease progression. The major research tool of this Project is an in vitro tripartite PrP conversion model that has been assembled in our Company. This model is composed of proteinase K (pK) sensitive PrP (PrPSen) and a high molecular weight component of BCS that, in combination with RNA, generates a pK-resistant form of PrP (PrPRes). We propose to use this system to study molecular mechanisms of the PrPC conversion in the mouse. We will isolate active component of high molecular weight of BCS that participate in mouse PrPC conversion, characterize it and then elucidate the mode of its action. To achieve the Goals of this Proposal, the following Tasks will be accomplished: (1) Obtain cellular mouse prion protein (mPrPC) using eukaryotic expression systems, (2) Isolate active component(s) of a high molecular weight fraction of BCS that facilitate PrPC>PrPRes transition, (3) Purify this component(s), characterize it and determine its molecular structure and amino-acid composition, (4) Validate the ability of such component to generate corresponding mPrPRes isoforms from mPrPC in the presence of RQ 11 + 12 RNA. The success of this research should open the way for further understanding of the molecular mechanisms for TSE transmission and progression and cellular components that facilitate this process. Discovery and characterization of the high molecular weight component in BCS that chaperones PrPC>PrPRes transition should lead to the identification of corresponding counterparts in sheep, cow and humans and consequently, the development of corresponding diagnostics and specifically targeted therapeutic tools for TSE. Understanding of identified high molecular weight chaperone functions should suggest appropriate "rationally designed" strategies that could offer new opportunities for treating prion diseases and may lead to the creation of sensitive pre-symptomatic detection systems useful for human blood product and pre-mortem screening for livestock wasting diseases.
Thesaurus Terms: blood lipoprotein, molecular chaperone, prion, protein protein interaction, protein purification, technology /technique development apolipoprotein E, biotransformation, endopeptidase, molecular weight, protein degradation, protein folding, spongiform encephalopathy SDS polyacrylamide gel electrophoresis, gel mobility shift assay, high performance liquid chromatography, immunocytochemistry, polymerase chainreaction, two dimensional gel electrophoresis
