Chaperone-supervised conversion of prion protein to its protease-resistant form

Transmissible spongiform encephalopathies (TSEs) are lethal, infectious disorders of the mammalian nervous system. A TSE hallmark is the conversion of the cellular protein PrPC to disease-associated PrPSc (named for scrapie, the first known TSE). PrPC is protease-sensitive, monomeric, detergent soluble, and primarily α-helical; PrPSc is protease-resistant, polymerized, detergent insoluble, and rich in β-sheet. The “protein-only” hypothesis posits that PrPSc is the infectious TSE agent that directly converts host-encoded PrPC to fresh PrPSc, harming neurons and creating new agents of infection. To gain insight on the conformational transitions of PrP, we tested the ability of several protein chaperones, which supervise the conformational transitions of proteins in diverse ways, to affect conversion of PrPC to its protease-resistant state. None affected conversion in the absence of pre-existing PrPSc. In its presence, only two, GroEL and Hsp104 (heat shock protein 104), significantly affected conversion. Both promoted it, but the reaction characteristics of conversions with the two chaperones were distinct. In contrast, chemical chaperones inhibited conversion. Our findings provide new mechanistic insights into nature of PrP conversions, and provide a new set of tools for studying the process underlying TSE pathogenesis.

Ex vivo propagation of infectious sheep scrapie agent in heterologous epithelial cells expressing ovine prion protein

Transmissible spongiform encephalopathies, or prion diseases, are fatal degenerative disorders of the central nervous system that affect humans and animals. Prions are nonconventional infectious agents whose replication depends on the host prion protein (PrP). Transmission of prions to cultured cells has proved to be a particularly difficult task, and with a few exceptions, their experimental propagation relies on inoculation to laboratory animals. Here, we report on the development of a permanent cell line supporting propagation of natural sheep scrapie. This model was obtained by stable expression of a tetracycline-regulatable ovine PrP gene in a rabbit epithelial cell line. After exposure to scrapie agent, cultures were repeatedly found to accumulate high levels of abnormal PrP (PrPres). Cell extracts induced a scrapie-like disease in transgenic mice overexpressing ovine PrP. These cultures remained healthy and stably infected upon subpassaging. Such data show that (i) cultivated cells from a nonneuronal origin can efficiently replicate prions; and (ii) species barrier can be crossed ex vivo through the expression of a relevant PrP gene. This approach led to the ex vivo propagation of a natural transmissible spongiform encephalopathy agent (i.e., without previous experimental adaptation to rodents) and might be applied to human or bovine prions.