The binding of the molecular chaperone Hsc70 to the prion protein PrP is modulated by pH and copper |
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Authors: | Simon Wilkins Ashraf A. Choglay J. Paul Chapple Jacqueline van der Spuy Alexandre Rhie Christopher R. Birkett Michael E. Cheetham |
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Affiliation: | 1. Department of Neurology and Geriatrics, Gifu University Graduate School of Medicine, Gifu City, Gifu, Japan;2. Life Science Research Center, Center for Molecular Biology and Genetics, Mie University, Tsu City, Mie, Japan;3. Graduate School of Regional Innovation Studies, Mie University, Tsu City, Mie, Japan;1. Department of Cell Systems and Anatomy, The University of Texas Health Science Center, San Antonio, TX, USA;2. Department of Medicine, The University of Texas Health Science Center, San Antonio, TX, USA;3. Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX, USA;4. Barshop Institute for Longevity and Aging Studies, San Antonio, TX, USA;5. Department of Biochemistry, UT Southwestern Medical Center, Dallas, TX, USA |
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Abstract: | Conformational transitions in the prion protein (PrP) are thought to be central to the pathogenesis of the transmissible spongiform encephalopathies (TSE), such as Creutzfeldt-Jacob disease and bovine spongiform encephalopathy. Studies of prion phenomena in yeast have shown that molecular chaperones play an important role in prion related conformational transitions. Here, we investigated the interaction of the molecular chaperone Hsc70 (HSPA8) with recombinant PrP in vitro using an ELISA based assay. Hsc70 bound to PrP in a saturable manner over a range of temperatures and binding was greatest at low pH. Surprisingly, Hsc70 bound more avidly to native recombinant PrP than to denatured PrP or other potential clients, such as denatured luciferase or rhodanese. Hsc70 binding to native PrP was enhanced by incubation with Cu2+ at low pH. The Hsc70 binding sites in PrP were analysed using a synthetic PrP-derived peptide array. The binding of Hsc70 to PrP was reminiscent of the published ovine PrP to bovine PrP binding data and included two potential regions of binding that correspond to the proposed ‘protein X’ binding sites in PrP. Synthetic peptides corresponding to these sites specifically inhibited the Hsc70 interaction with native PrP, further demonstrating that Hsc70 might interact with PrP via this epitope. The data suggest that molecular chaperones could modulate important PrP conformational transitions or protein–protein interactions in TSE pathogenesis. |
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