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Identifying Critical Sites for PrPC-PrPSc Interaction Through Dominant-Negative Inhibition Arla M.A. Mistica Senior Honors Project Undergraduate Research Day April 21, 2012 Transmissible Spongiform Encephalopathy Prion Diseases Creutzfeldt-Jakob disease (Humans) Bovine Spongiform Encephalopathy (BSE; Cattle) Scrapie (Sheep) Chronic Wasting Disease (Cervids) Source: Acquired, Familial & Sporadic Universally fatal and there is no treatment What is a prion? Prion: Abnormally-folded Prion Protein (PrP) C PrP : normal cellular isoform Sc PrP : Infectious isoform Enigma: Despite its lack of genetic material High Infectivity Various Strains Jacquemot et al., J Virol 2005 Daggett’s Model (Spiral model) Replication of Prion (PrPSc) Endogenous PrPC Interaction between PrPC and PrPSc Spontaneous generation of PrPSc Conversion of mutant PrPC to PrPSc Inoculation of PrPSc Conversion of PrPC to PrPSc Accumulation of PrPSc The conversion of PrPC to PrPSc is still not completely understood. PrPSc-PrPC or PrPSc-PrPSc Interaction • Structures of PrPSc is stable in Oligomers PrPSc-PrPSc interaction • Strain-specific structures are inherited with High fidelity PrPC-PrPSc interaction Investigation into PrP-PrP interactions are important Dominant-Negative Inhibition • When a conversion-incompetent PrP (incPrP) co-exists with a conversion-competent PrP, the former inhibit the conversion of the latter. • Efficiencies of the inhibition varies between incPrPs • Due to variation in Binding affinity of incPrPs to PrPSc? PrPC PrPC PrPSC PrPSC PrPSC incPrP Hypothesis If efficiency of dominant-negative inhibition depends on binding affinity of inPrP to PrPSc, the region necessary for PrPC-PrPSc interaction can be identified by making a series of Internal-deletion mutants and evaluating their inhibitory effects. (3F4)MoPrP MoPrP Δ159 Δ159-163 Δ159-167 Δ159-171 Δ159-175 -YRYPNQVYYRPVDQYSNQNNFVHD-YRYPN VYYRPVDQYSNQNNFVHD-YRYPN PVDQYSNQNNFVHD-YRYPN YSNQNNFVHD-YRYPN NNFVHD-YRYPN HD-- --- --- ------------------------------ --------------------------------------------------------------- -------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------ Methods • Create Internal-deletion PrPs (ΔPrPs) by site-directed mutagenesis • Amplify the plasmids encoding ΔPrPs in competent cells and purify the plasmids • Transfect the plasmids to scrapie-infected cultured cells (Co-express conversion-competent PrP with ΔPrP). • Harvest, SDS-PAGE & immunoblot to evaluate dominant-negative inhibition Transfection to cultured cells Expression of mutant PrPs Results Results • ∆PrPs showed insolubility to detergent & mildly proteaseresistant unlike wild-type PrP 4% Sarcosyl Non-infected Sup Pellet 4H11 (Endogenous Wild-type PrP) Chymotrypsin concentration (µg/ml) 0 1 ∆159 2 4 8 ∆159 ∆159-175 ∆159 ∆159-175 ∆159 ∆159-175 ∆159 ∆159-175 4% Sarcosyl Sc-Infected Non-infected Sup Pellet Sup Pellet 3F4 (Transfected ∆PrPs) ∆159-175 0 1 2 4 8 These findings suggests that ∆PrPs might be different from wildtype PrP but there was no difference among ∆PrPs. Results Co-transfected (3F4)MoPrP with the Internal-deletion mutants and observed the levels of (3F4)PrPSc ∆159-175 ∆159-171 ∆159-164 ∆159-163 ∆159-162 ∆159 ∆159-175 Film Vector *There was a leak in “Δ-164” ∆159-171 ∆159-164 ∆159-163 ∆159-162 ∆159 Vector PK-resistant core (3F4)MoPrP+ ∆159 and ∆159-162 showed most efficient inhibition and the inhibitory effects were reduced as the deletions were extended Discussion • Reproducible differences between ∆PrPs in efficiencies of dominant-negative inhibition were observed • The differences suggests that the loop region between the second beta strand and the second alpha helix might be important for efficient inhibition • This supports the point of view that the loop region is important for propagation of prion/PrPSc (3F4)MoPrP MoPrP Δ159 Δ159-163 Δ159-167 Δ159-171 Δ159-175 -YRYPNQVYYRPVDQYSNQNNFVHD-YRYPN VYYRPVDQYSNQNNFVHD-YRYPN PVDQYSNQNNFVHD-YRYPN YSNQNNFVHD-YRYPN NNFVHD-YRYPN HD-- --- --- ------------------------------ --------------------------------------------------------------- -------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------ To be continued…. • Isolation of the critical amino acids that are absolutely needed for binding (proteins following the B-sheet) • Finding efficient incPrPs that can bind to PrPSc effectively may be used therapeutically by the inhibition of PrPC to PrPSc conversion. Acknowledgments • • • • • • • • • • Prof. Hermann Schatzl Dr. Yuzuru Taguchi Dr. Sabine Gilch Missy Stuart Fawn Pickard Ted John Leah Kyle Xiaotang Du Shaihla Khan Lauren Millet • Honors Program • Rick Matlock
