The transmissible spongiform encephalopathies (TSEs) are a class of neurodegenerative diseases that are characterized by proteinaceous deposits in the brain. The deposits consist largely of an abnormal form of prion protein which is highly aggregated and resistant to degradation by proteases. The function of prion protein (PrP) is unknown and its normal form (PrPC) is sensitive to protease digestion. Some of the TSEs include scrapie in sheep, mice, and hamsters, bovine spongiform encephalopahty, and Creutzfeldt-Jakob disease in humans. Animals with scrapie accumulate a disease-specific form of PrP designated PrPSC. The identity of the infectious agent of the TSEs is unclear. No conventional agent or disease-specific nucleic acid has been found and treatments that destroy most normal viruses have no effect. Based on that information the infectious particle has been surmized to consist solely of protein.The "protein only" theory has been strengthened by the discovery of PrP and that preparations of PrPSc are greatly enriched for infectivity. Previously, the simplest system producing protease-resistant PrP was cell culture. Here is described a system that produces protease-resistant PrP in vitro. This system was used to study the mechanism of the conversion of PrPC to the protease-resistant form. A threshold concentration of aggregates of PrP S c was required for conversion. Guanidine-HCI solubilized PrPSC had no converting activity. The need for aggregates and a threshold concentration favors a seeded polymerization mechanism of conversion. The system was also used to study species barriers and different strains of scrapie. Different combinations of mouse and hamster PrPs were used in conversion reactions. The in vitro conversion results correlated with infectivity data on transmission. Scrapie strains with different PrPSc N-terminal protease cleavage sites were used with this system. These different cleavage sites were passed to newly-resistant PrPC depending on which strain was in the reaction. This system provided evidence that a "protein only" theory is compatible with the properties of TSEs.Thesis Supervisor: Dr. Peter T. Lansbury, Jr. Title:Associate Professor of Chemistry Chapter 1 CJD is characterized by dementia, ataxia (a loss of muscle coordination), and the formation of small holes in the brain known as spongiform degeneration.This spongiform degeneration is the result of a loss of neurons. CJD is often accompanied by abnormal proteinaceous deposits known as amyloid plaques.The onset of CJD can be months to years after infection, but once the disease presents itself a gradual deterioration of mental capabilities ensues. The deterioration ends in death usually within a year of onset. Heparin sulfates have been shown to offer some protection to mice inoculated with scrapie,7 but as of now there is no way to stop the deterioration and the disease is always fatal.GSS is a familial version of CJD with different pathology. All GSS cases are accompanied by the formation of amyloid plaques 2 and it i...

The transmissible spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by amylold formation in the brain. The major amylold protein is the prion protein (PrP). PrP and the (-amyloid protein of Mzhelmer disease share a similr sequence that, in both cases, may be respondble for the initiation of protein aggregation in Wivo. We report here that a peptide based on this sequence in PrP (PrP96-111M) forms amyloid fibrils. The existence of a kinetic barrier to amyloid formation by this peptide was demonstrated, suggesng that formation of an ordered nudeus Is the rate-determining step for aggregation. Seeding was demonstrated to occur with PrP96-111M amyloid fibrfls but not with amylold fibrils of a related peptide. This effect is consistent with the p al that the agegation of PrP, which characterizes TSE, involves a nucleation event analogous to the seeding of a crytlition.

In this study, we explored the application of a yeast three-hybrid (Y3H)-based compound/protein display system to scanning the proteome for targets of kinase inhibitors. Various known cyclin-dependent kinase (CDK) inhibitors, including purine and indenopyrazole analogs, were displayed in the form of methotrexate-based hybrid ligands and deployed in cDNA library or yeast cell array-based screening formats. For all inhibitors, known cell cycle CDKs as well as novel candidate CDK-like and/or CDK-unrelated kinase targets could be identified, many of which were independently confirmed using secondary enzyme assays and affinity chromatography. The Y3H system described here may prove generally useful in the discovery of candidate drug targets.