Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation - PubMed (original) (raw)

Evidence for protein X binding to a discontinuous epitope on the cellular prion protein during scrapie prion propagation

K Kaneko et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Studies on the transmission of human (Hu) prions to transgenic (Tg) mice suggested that another molecule provisionally designated protein X participates in the formation of nascent scrapie isoform of prion protein (PrPSc). We report the identification of the site at which protein X binds to the cellular isoform of PrP (PrPC) using scrapie-infected mouse (Mo) neuroblastoma cells transfected with chimeric Hu/MoPrP genes even though protein X has not yet been isolated. Substitution of a Hu residue at position 214 or 218 prevented PrPSc formation. The side chains of these residues protrude from the same surface of the C-terminal alpha-helix and form a discontinuous epitope with residues 167 and 171 in an adjacent loop. Substitution of a basic residue at positions 167, 171, or 218 also prevented PrPSc formation: at a mechanistic level, these mutant PrPs appear to act as "dominant negatives" by binding protein X and rendering it unavailable for prion propagation. Our findings seem to explain the protective effects of basic polymorphic residues in PrP of humans and sheep and suggest therapeutic and prophylactic approaches to prion diseases.

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Figures

Figure 1

Figure 1

Characterization of the binding site for protein X. Western blot analysis of each MHM2 chimeric construct expressed in ScN2a cells is shown. (A_–_C) Lanes: 1, MHM2 PrP; 2, MHMHu(A/B); 3, MHMHuA; 4, MHMHuB; and 5, untransfected control ScN2a cells. (D_–_F) Lanes: 6, MHM2 PrP; 7, MHM2(I214); 8, MHM2(E218,R219); 9, MHM2(E218); 10, MHM2(R219); 11, MHM2(I214,E218); and 12, untransfected control ScN2a cells. A and D demonstrate the expression of each chimeric MHM2 PrP construct: 40 μl of undigested cell lysates was applied to each lane and MHM2 PrP was detected by staining with α-PrP 3F4 mAb. B and E demonstrate the conversion of chimeric MHM2 PrPC into PrPSc and were stained with α-PrP 3F4 mAb. C and F show endogenous MoPrPSc as well as chimeric constructs detected with α-PrP RO73 rabbit antiserum. In B_–_C and E_–_F, 500 μl of cell lysate was digested with proteinase K (20 μg/ml) at 37°C for 1 h followed by centrifugation at 100,000 × g for 1 h and the loading of the resuspended pellet onto the gel.

Figure 2

Figure 2

Mutations at codons 214, 216, and 218 affect PrPSc formation. Western blot analysis of mutated MHM2 PrP constructs expressed in ScN2a cells. Films were exposed longer than other figures to detect faint signals. (A_–_C) Lane: 1, MHM2 PrP; 2, MHM2(I214,E218); 3, MHM2(E218); 4, MHM2(K218); 5, MHM2(I218); 6, MHM2(A218); 7, MHM2(W218); 8, MHM2(P218); 9, MHM2(R216); 10, MHM2(R216,E218); 11, MHM2(I214); 12, MHM2(K214); 13, MHM2(E214); 14, MHM2(A214); 15, MHM2(W214); 16, MHM2(P214); 17, MHM2(R171); 18, MHM2(N169); and 19, MHM2(R167). (D) Coexpression with MHM2 in the same orientation as in A_–_C. Samples were prepared and processed as described in the Fig. 1 legend.

Figure 3

Figure 3

The role of protein X in PrPSc formation and the influence of mutations in PrPC on the prion replication cycle. (A) NMR structure of SHa rPrP90–231. The color scheme is as follows: α-helices A (residues 144–157), B (172–193), and C (200–227) in pink; disulfide between Cys-179 and Cys-214 in yellow; hydrophobic cluster composed of residues 113–126 in red; loops in gray; residues 129–134 in green encompassing strand S1 and residues 159–165 in blue encompassing strand S2; the arrows span residues 129–131 and 161–163, as these show a closer resemblance to β-sheet (20). Structure of protein X binding site of SHa rPrP90–231 illustrating the proximity of the 165–171 loop, where residues Q168 and Q172 are depicted with a low density van der Waals rendering and helix C residues T215 and Q219 depicted with a high density van der Waals rendering. SHaPrP residues Q168, Q172, T215, and Q219 correspond to MoPrP residues Q167, Q171, T214, and Q218, respectively. The illustration was generated with the program UCSF

midasplus

. (B) Ordering experiments demonstrate that PrPC interacts with protein X prior to the creation of the PrPC/PrPSc complex. Two cycles are required for PrPSc formation. The left hand cycle shows protein X binding to PrPC (green) resulting in a heterologous complex that is then competent to interact with PrPSc (red). Upon conversion of PrPC to nascent PrPSc, protein X dissociates from the complex owing to its relative lack of affinity for PrPSc. Protein X is subsequently recycled. The right hand cycle depicts the interaction of PrPSc with the PrPC/protein X complex and the conversion of PrPC into nascent PrPSc. With time, the result is an exponential increase in PrPSc concentration as the template for conversion is recycled. (C) Type 1 inhibition (Table 2): mutant PrPC (blue) containing an amino acid substitution in the PrPC/protein X interface (e.g., E218 in MoPrP) interacts weakly with protein X. Dotted lines depict the failure of the mutant PrPC to interact with protein X and the subsequent inability to form the protein X/PrPC/PrPSc complex. Under these circumstances PrPSc formation either does not occur or proceeds slowly. (D) Type 2 inhibition: mutant PrPC (purple) containing an amino acid substitution in the protein X/PrPC interface (e.g., K218 in MoPrP) forms a very tight complex. PrPSc is able to bind to this protein X/PrPC complex but conversion of PrPC to PrPSc is prevented owing to the failure of the protein X/PrPC/PrPSc complex to release protein X. Dotted lines are shown for the steps in the replication cycle that are blocked. (E) Dominant-negative effect of tight binding mutants of PrPC. Mutant PrPC [e.g., K218 (purple)] successfully competes with wt PrPC (green) for binding to protein X. The protein X/PrPC(K218)/PrPSc complex is formed but conversion is inhibited as in D. (F) Type 3 inhibition: PrPC from a distinct species [e.g., SHa (gold)] is able to bind Mo protein X, but the Mo protein X/SHaPrPC/MoPrPSc complex is not competent for conversion. The result is that protein X is sequestered and scrapie prions are not replicated.

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