Template-directed protein misfolding in neurodegenerative disease

• Main Author: Guest, William Clay
• Language: English
• Published: University of British Columbia 2012
• Online Access: http://hdl.handle.net/2429/41990

Protein misfolding diseases represent a large burden to human health for which only symptomatic treatment is generally available. These diseases, such as Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, and the systemic amyloidoses, are characterized by conversion of globular, nativelyfolded proteins into pathologic β-sheet rich protein aggregates deposited in affected tissues. Understanding the thermodynamic and kinetic details of protein misfolding on a molecular level depends on accurately appraising the free energies of the folded, partially unfolded intermediate, and misfolded protein conformers. There are multiple energetic and entropic contributions to the total free energy, including nonpolar, electrostatic, solvation, and configurational terms. To accurately assess the electrostatic contribution, a method to calculate the spatially-varying dielectric constant in a protein/water system was developed using a generalization of Kirkwood Frohlich theory along with brief all-atom molecular dynamics simulations. This method was combined with previously validated models for nonpolar solvation and configurational entropy in an algorithm to calculate the free energy change on partial unfolding of contiguous protein subsequences. Results were compared with those from a minimal, topologically-based Gō model and direct calculation of free energies by steered all-atom molecular dynamics simulations. This algorithm was applied to understand the early steps in the misfolding mechanism for β₂-microglobulin, prion protein, and superoxide dismutase 1 (SOD1). It was hypothesized that SOD1 misfolding may follow a template-directed mechanism like that discovered previously for prion protein, so misfolding of SOD1 was induced in cell culture by transfection with mutant SOD1 constructs and observed to stably propagate intracellularly and intercellularly much like an infectious prion. A defined minimal assay with recombinant SOD protein demonstrated the sufficiency of mutant SOD1 alone to trigger wtSOD1 misfolding, reminiscent of the “protein-only” hypothesis of prion spread. Finally, protein misfolding as a feature of disease may extend beyond neurodegeneration and amyloid formation to cancer, in which derangement of protein folding quality control may lead to antibodyrecognizable misfolded protein present selectively on cancer cell surfaces. The evidence for this hypothesis and possible therapeutic targets are discussed as a future direction. === Medicine, Faculty of === Medicine, Department of === Experimental Medicine, Division of === Graduate