Submitted to: Protein and Peptide Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 15, 2007
Publication Date: February 1, 2008
Citation: Zukas, A.A., Bruederle, C.E., Carter, J.M. 2008. Sonication Induced Intermediate in Prion Protein Conversion. Protein and Peptide Letters. 53(2):206-211 Interpretive Summary: Prion diseases, such as scrapie and “mad cow” disease in cattle are related to changes in a brain protein known as PrPC. It is believed that a healthy PrPC adopts certain intermediate shapes before becoming an abnormal prion PrPSc, but very little is known about those shapes. We studied shapes of PrPC. We found that some shapes of PrPC were more susceptible to misfolding than others. We looked at the environmental factors that might change the shape of PrPC and cause prion diseases.
Technical Abstract: In vivo conversion of prion protein (PrPC) to its abnormal pathogenic isoform (PrPSc) is associated with conformational transition of alpha-helices and unstructured regions to beta-sheets. Protein misfolding cyclic amplification (PMCA) is thought to mimics this conversion in vitro. PMCA involves sonication and incubation of PrPC with trace amounts of PrPSc. The technique produces large amounts of protease-resistant (PrPres) isoform at the expense of PrPC, but very little is known about the mechanism. We have observed that hamster PrPC undergoes structural changes on exposure to heat or sonication, in absence of seeding with added PrPSc. The concentration of PrPC in its native conformation decreases as a function of sonication time. However, by Western after denaturing SDS-PAGE, the concentration of PrPC does not change. This suggests the presence of a third stable PrPC conformer, which may be intermediate in the conversion of PrPC to PrPSc. If this conformer is seeded with a small amount of PrPSc, then it undergoes a significant conversion to PrPres even in the absence of additional sonication. The PCMA reaction is not limited by the supply of PrPC. Instead conversion slows over time. Eventually it comes to a stop, even though nearly half the starting amount of PrPC remains. This suggests PCMA requires interaction between PrPC and other cofactors present in brain homogenate. The cofactors appear to be the limiting reagents that are consumed in the conversion process and not recycled. Addition of fresh brain homogenate restores the conversion. These findings provide a new set of tools for studying the cofactor dependent conversion process and understanding pathogenesis of transmissible spongiform encephalopathies (TSE).