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

Abstract

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 alpha-helical; PrPSc is protease-resistant, polymerized, detergent insoluble, and rich in beta-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.

Figure 1

Effects of chaperones on cell-free conversion of [³⁵S]PrP^C to its protease-resistant form. (A) Conversions (as percent of total [³⁵S]PrP^C) obtained after 24 hr either with PrP^Sc or without PrP^Sc (100 ng), but with the indicated chaperones (each at 5 μM, with 5 mM ATP), by using the standard assay described. In indicated reaction (second from left), PrP^Sc was partially denatured with guanidinium hydrochloride (Gdn⋅HCl). Identical results were obtained, over a broad range of chaperone concentrations, with or without ATP (data not shown). (B) Conversions performed as in A, with the addition of untreated PrP^Sc. Mean values are from three to six experiments, with standard errors. Buffers for storing various chaperones differed slightly in salt and glycerol content, but none affected conversion (data not shown). (C) Concentration-dependent effects of chaperones in promoting conversion with untreated PrP^Sc. Other heat shock proteins were tested as in A. (D) SDS/PAGE phosphorimage of [³⁵S]PrP^C products from representative conversion reactions obtained with 3 ng [³⁵S]PrP^C and increasing amounts of PrP^Sc (3–1,000 ng). One-tenth of each reaction was left untreated (−PK); the remainder was digested with proteinase K (+PK). GroEL and GroES were at 1 μM. When indicated, PrP^Sc was partially denatured with Gdn⋅HCl. PrP^Sc fold represents the ratio of PrP^Sc/[³⁵S]PrP^C in the reaction. (E) ATP dependence of GroEL-mediated conversions. SDS/PAGE phosphorimages of representative conversion reactions obtained with untreated PrP^Sc and GroEL (WT and mutant D87K), with or without ATP. Both proteinase K-treated (+PK; Lower) and untreated samples (−PK, one-fifth sample; Upper) are shown. (F) [³⁵S]PrP^GPI- conversions with or without chaperones. Reactions contained either untreated PrP^Sc or Gdn⋅HCl-treated PrP^Sc, and a variant PrP missing the GPI anchor, [³⁵S]PrP^GPI-. [³⁵S]PrP^GPI- and [³⁵S]PrP^C preparations are compared (Right): UG, unglycosylated; MG, monoglycosylated; and DG, diglycosylated PrP species as indicated.

Figure 2

Time course of conversion with or without chaperone. (A) Appearance of PrP-res at 2, 6, 24, and 48 hr, in reactions treated with proteinase K and analyzed by quantitative phosphorimaging of SDS/PAGE. Mean values are from three independent measurements, with standard errors. (B) Pelletable [³⁵S]PrP determined by quantitative phosphorimaging of SDS/PAGE. At the indicated times, [³⁵S]PrP reaction products were centrifuged at 15,000 × g for 30 min at 22°C. After separating the supernatant fraction (S), the pelletable fraction (P) was resuspended in conversion buffer, and both fractions were prepared for SDS/PAGE. Mean values are from three independent experiments, with standard errors. (C) Protease-resistant [³⁵S]PrP in pellet (P) and supernatant (S) fractions quantified from SDS/PAGE phosphorimages of 24-hr reactions. Averages are of two independent experiments.

Figure 3

Combined effects of chaperones and partially denatured PrP^Sc on conversion. (A) Conversions obtained with partially denatured PrP^Sc (4 M urea pretreatment) with buffer alone, or with the indicated chaperones and control proteins (each at 5 μM). Mean values are from three to six independent measurements, with standard errors. (B) SDS/PAGE phosphorimage of representative conversion reactions obtained with untreated PrP^Sc (0) or PrP^Sc partially denatured in the presence of increasing urea concentrations (1–5 M), with or without chaperone (Hsp104 or GroEL, 3 μM). Only proteinase K-treated (+PK) samples are shown. (C) SDS/PAGE phosphorimage of representative conversion reactions obtained with Hsp104 (WT or mutant KT218), with or without ATP, and untreated or partially denatured PrP^Sc (4 M urea pretreatment). Only proteinase K-treated samples (+PK) are shown. (D) SDS/PAGE phosphorimage of representative conversion reactions obtained with partially denatured PrP^Sc (4 M urea pretreatment), with or without ATP, and with or without GroEL (WT or mutant D87K). Both proteinase K-treated (+PK; Lower) and untreated samples (−PK, one-fifth sample; Upper) are shown.

Figure 4

Conversion of [³⁵S]PrP^C in the presence of chemical chaperones. SDS/PAGE phosphorimages of representative conversion reactions obtained with partially denatured PrP^Sc (4 M urea pretreatment) in the presence of increasing concentrations of DMSO, glycerol, sucrose, or trehalose.

Figure 5

Model for chaperone-supervised PrP conversion. Conversion of [³⁵S]PrP^C to PrP-res in vitro requires pre-existing PrP^Sc (refs. –, and this study). Without chaperone, conversion is slow and inefficient likely because [³⁵S]PrP intermediates that productively associate with PrP^Sc are sparsely populated. The chaperone likely recognizes and binds near-native and nonnative intermediates derived from acid-treated [³⁵S]PrP^C, alters their conformation, and releases them in states that associate productively with PrP^Sc. Thereby, the chaperone facilitates the first step in conversion: specific binding of [³⁵S]PrP to PrP^Sc. In this stage, [³⁵S]PrP is pelletable, but remains protease-sensitive. A second slower step then follows, wherein PrP^Sc-bound [³⁵S]PrP undergoes a second conformational transition to form PrP-res, the converted state with protease digestion properties strikingly similar to PrP^Sc.