![]() ![]() Here, we provide the first evidence that molecular chaperones can regulate conformational transitions in PrP. However, because neither the putative infectious nature of pure PrP Sc protein nor that of the in vitro converted PrP has been demonstrated, we refer to the in vitro converted material operationally as protease-resistant PrP (PrP-res). Together, these studies provide substantial evidence that in vitro converted, protease-resistant PrP is either authentic PrP Sc or has a very similar conformation. Last, this cell-free assay modeled accurately another in vivo TSE barrier, based on genetic polymorphisms in PrP, which render sheep either highly susceptible, moderately susceptible, or resistant to scrapie ( 22). Third, the known in vivo barriers to transmitting TSEs between different species were reflected well in the efficiencies of in vitro conversion ( 20, 21). Second, strain-specific PrP Sc protease digestion properties, specifically those associated with two mink TSE strains-hyper and drowsy-were precisely propagated from PrP Sc to radiolabeled PrP C in this assay ( 19). First, like experimental TSEs, in vitro conversion of PrP C to its protease-resistant form requires pre-existing PrP Sc ( 17– 22). This simple in vitro conversion reaction faithfully recapitulates several salient TSE features. In this altered state, PrP is aggregated and a specific portion of the molecule is highly resistant to proteolysis. To test for chaperone involvement, we used a cell-free assay, wherein metabolically labeled PrP C, purified from cultured cells in an acid-treated state, is converted to a conformational state characteristic of PrP Sc ( 17, 18). The goal of this study was to assess whether or not molecular chaperones, whose known functions are to alter the conformational states of proteins ( 14– 16), regulate the conversion of PrP C to PrP Sc. 13) and protein chaperones have been frequently speculated to be among them ( 7, 10– 12). None have been conclusively identified however, cellular osmolytes (sometimes called chemical chaperones ref. Genetic and inhibitor studies have suggested that other cellular factors may influence TSE pathogenesis or serve as regulators of disease ( 7– 12). However, the exact mechanism underlying conversion is not known. The conversion of PrP C to PrP Sc appears to involve direct interactions of PrP C with pre-existing PrP Sc ( 1, 2). Several lines of evidence show that PrP C is conformationally distinct from PrP Sc, although both molecules derive from the same primary sequence and have no detectable posttranslational differences ( 1, 2, 4– 6). Indeed, the “prion”, a term by which the agent is popularly known today, appears to be almost entirely proteinaceous: consisting primarily of PrP Sc ( 1, 2). Griffith ( 3) first proposed a “protein-only” model to explain the unconventional behavior of the infectious TSE agent. A central event in TSE pathogenesis is the accumulation in the nervous system of an abnormally folded version (PrP Sc) of a normal cellular protein, PrP C. ![]() The family of transmissible spongiform encephalopathies (TSEs) include scrapie in sheep, bovine spongiform encephalopathy or “mad cow disease” in cattle, and several rare human neuropathies: Creutzfeld–Jacob disease, fatal familial insomnia, Gertsmann–Straussler–Scheinker syndrome, and kuru ( 1, 2). 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. In contrast, chemical chaperones inhibited conversion. Both promoted it, but the reaction characteristics of conversions with the two chaperones were distinct. In its presence, only two, GroEL and Hsp104 (heat shock protein 104), significantly affected conversion. None affected conversion in the absence of pre-existing PrP Sc. 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 PrP C to its protease-resistant state. The “protein-only” hypothesis posits that PrP Sc is the infectious TSE agent that directly converts host-encoded PrP C to fresh PrP Sc, harming neurons and creating new agents of infection. PrP C is protease-sensitive, monomeric, detergent soluble, and primarily α-helical PrP Sc is protease-resistant, polymerized, detergent insoluble, and rich in β-sheet. A TSE hallmark is the conversion of the cellular protein PrP C to disease-associated PrP Sc (named for scrapie, the first known TSE). Transmissible spongiform encephalopathies (TSEs) are lethal, infectious disorders of the mammalian nervous system. ![]()
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