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Acidic pH and Detergents Enhance in Vitro Conversion of Human Brain PrPc to a PrPSc-like Form by Wen-Quan Zou and Neil R. Cashman The Journal of Biological Chemistry Vol. 277, No. 46, Issue of November 15, pp. 43953-43947, 2002 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 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. 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. 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. 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) Nature 370, 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 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 selfassociation 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. Acta 1431, 1-13 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 (195213) corresponding to the C-terminal region of PrP. W.Q. Zou, et. al., (2001) Eur. J. Biochem. 268, 4885-4891 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 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. 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. Data/Results The mock-treated samples of PrP were predominantly in the detergent-soluble (S) fraction, and acid/GdnHCltreated 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. 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. 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. 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. Data/Results Immunoprecipitation of treated PrP and PrPSc with anti-PrP antibodies, 6H4 & 3F4. Lanes 1 & 3 are mocktreated, 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). 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. 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. 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 endosomallysosomal system. 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.