Useful Terms & Abbreviations

1. Acidic pH and Detergents Enhance in Vitro Conversion of Human Brain PrPc to a PrPSc-like Formby Wen-Quan Zou and Neil R. CashmanThe Journal of Biological ChemistryVol. 277, No. 46, Issue of November 15, pp. 43953-43947, 2002

2. Useful Terms & Abbreviations • BSE – Bovine Spongiform Encephalopathy (Mad Cow Disease) • CJD – Creutzfeldt-Jakob Disease • OSE – Ovine Spongiform Encephalopathy (Scrapie) • PrPC – cellular prion protein (normal) • rPrP – recombinant prion protein • PrPSc – pathogenic prion protein • PK – proteinase K • GdnHCl – guanidine hydrochloride • PMCA – protein misfolding cyclic amplification • PBS – phosphate buffered saline • SDS – sodium dodecyl sulfate

3. Introduction • Most diseases can be traced to bacteria, viruses, fungi or parasites. • Prions are pieces of protein, containing no DNA or RNA to direct its activity. • Prions have been linked to BSE, OSE, CJD and Kuru and appear to cross between species.

4. Introduction (continued) • Interspecies transmission is due to feeding diseased carcasses of one animal to another, causing progressive destruction of brain tissue and death. • Pathogenic prions have the same primary structure (amino acid sequence) as normal PrP but differ in the secondary and tertiary structures.

5. Introduction (continued) • Pathogenic prions move neuron to neuron destroying each cell and have the ability to transform normal prions into pathogenic isoforms. • Researchers have not discovered how or why some prions transform into disease-causing vectors or have the power to convert other prions.

6. Review of Literature • Normal soluble, PK sensitive PrPc (rich in α-helices) is converted to infectious, insoluble, proteinase K-resistant PrPSc (rich in β-sheets), by a template-directed process catalyzed by PrPSc. This has been modeled in vitro. • Kocisko, D.A., et. al., (1994) Nature370, 471-474 • Saborio, G.P., et. al., (1999) Biochem. Biophys. Res. Commun. 258, 470-475 • The insoluble, β-sheet form of the prion protein (PrPSc) is the only known component associated with the group of transmissible, fatal neurodegenerative diseases found in humans and other animals. A posttranslational process, changes the conformation of the protein from normal PrPC to PrPSc. • Prusiner, S.B. et. al., (1998) Cell 93, 337-348

7. Review of Literature (continued) • The primary structure of PrPC and PrPSc are identical but secondary and tertiary structures differ, creating different physicochemical properties. PrPC is soluble in detergent and can be degraded by proteinase K (PK). PrPSc is insoluble in detergents and is resistant to PK digestion, forming aggregates. • S.B. Prusiner (1998) Proc. Natl. Acad. Sci. U.S.A.95, 13363-13383 • Recombinant PrP (rPrP) have been used to demonstrate changes of α-helices into β-sheets and concomitant self-association with the use of acidic pH when combined with detergents in vitro. • W. Swietnicki, et. al., (1997) J. Biol. Chem.272, 27517-27520 • S. Hornemann & R. Glockshuber (1998) Proc. Natl. Acad. Sci. U.S.A.95, 6010-6014 • G.S. Jackson, et. al., (1999) Biochim. Biophys. Acta1431, 1-13

8. Review of Literature (continued) • Conversion of PrPC to PrPSc can be promoted by protein misfolding cyclic amplification (PMCA). • G. P. Saborio, et. al., (2001) Nature 411, 810-813 • Acid-induced conformational transition and aggregation may be associated with the protonation of acidic amino acids aspartate (Asp) and glutamate (Glu) using a peptide (195-213) corresponding to the C-terminal region of PrP. • W.Q. Zou, et. al., (2001) Eur. J. Biochem.268, 4885-4891

9. Hypothesis • Acidic pH and guanidine hydrochloride (GdnHCl)-treated brain tissue containing normal PrPC can be converted into abnormal PrPSc in an in vitro environment and is a superior substrate to untreated PrPC. PrPC PrPSc

10. Methodology • Brain tissue samples were obtained from normal human brain, prion-infected brain (CJD patient) and mice (wild-type and PrP-/-). • Samples of brain tissue were treated with GdnHCl at Ph 3.5 and 7.4 to create Acid/GdnHCl-treated PrP and mock-treated samples. • Assay of Detergent Insolubility and Proteinase K resistance was performed using immunoblotting.

11. Methodology (continued) • Immunoprecipitation of PrP samples was performed using anti-PrP monoclonal antibodies 6H4 & 3F4 and magnetic Dynabeads. • In vitro conversion of Acid/GdnHCl-treated PrP to a form similar to PrPSc was performed using the CJD brain tissue as a template.

12. Data/Results • The mock-treated samples of PrP were predominantly in the detergent-soluble (S) fraction, and acid/GdnHCl-treated PrP in the insoluble pellet fraction (P). • Acidic pH and GdnHCl induces conformational transition of recombinant PrP into a detergent-insoluble PrPSc-like species, even when the sample is returned to physiological pH for 7 days.

13. Data/Results • At pH < 3.5 PrP is insoluble, while at pH > 4.5 it is soluble. This corresponds to the pKa of the side chains of Aspartate & Glutamate, suggesting the solubility of PrP may be associated with protonation of acidic residues.

14. Data/Results • Most brain proteins are soluble in acidic environments. • GdnHCl makes PrP insoluble at low pH (3.5); only becomes soluble as the concentration increases. • Acidic pH-treated PrP may possess a unique structure at 1.5M GdnHCl.

15. Data/Results • Partial proteinase K (PK) resistance is a hallmark of PrPSc. • Both mock-treated and Acid/GdnHCl- treated brain PrP do not demonstrate this same resistance at concentrations > 1μg/ml PK.

16. Data/Results • Immunoprecipitation of treated PrP and PrPSc with anti-PrP antibodies, 6H4 & 3F4. Lanes 1 & 3 are mock-treated, 2 & 4 acid/GdnHCl treated, 5 & 7 normal brain, 6 & 8 CJD brain. There was no significant difference in binding of 6H4 & 3F4 in mock or acid-treated samples. There was a significant decrease of precipitate in CJD samples. (Molecular masses are shown in kDa).

17. Data/Results • Lanes 1 & 2: trace PrPSc with mock or acid pH/GdnHCl treated samples in SDS (a denaturing anionic detergent) • Lanes 3 & 4: no PrPSc is added to samples, SDS is added • Figure a suggests SDS may induce conformational change in treated brain PrP. • Figure b shows converted PrP display a protease-induced gel mobility shift similar to that displayed in CJD brain. • Figure c shows no amplification of PrPSc was observed when human template was incubated with brain homogenates from treated wild-type or PrP-/- mice.

18. Discussion • This study supports the hypothesis that acidic pH & GdnHCl induces a physical transition of cellular PrP in normal brain homogenates. • Treated PrP becomes detergent-insoluble (similar to PrPSc) but still remains PK-sensitive and epitope accessible. Only a small portion of the acidic pH treated PrP acquires PK resistance if treated with low concentrations of SDS, which is enhanced if in the presence of trace amounts of native PrPSc.

19. Discussion (continued) • The pH range of 3.5-4.5 in which these transitions occur is consistent with protonation of acidic amino acids. • Obscuration in PrPSc of the 3F4 epitope (residues 109-112) is consistent with the theory that PrPSc formation is dependent upon structural changes in codons ~90-120. • PrP is synthesized in the rough endoplasmic reticulum and transits through the Golgi apparatus to the cell surface. It is recycled in the endosomal-lysosomal pathway. PrPSc forms on the cell surface and accumulates in the endosomal-lysosomal system.

20. Conclusion/Further Research • Conversion of PrPC to PrPSc may pass through two discrete stages: 1. Low pH & denaturants induce the first stage (making it more “recruitable” than native PrPC.) 2. SDS assists in the second stage of rearrangement in the presence of a PrPSc template. • Further research should focus on the possibility that acidic pH PrP may be useful to determine the conformational events of underlying prion protein conversion in disease, the molecular co-factors and posttranslational modifications critical in conversion and pharmaceutical agents that might prevent PrPSc formation in vitro and in vivo.