SSNMR Lecture - Molecular disease mechanisms in neurodegeneration: solid-state NMR studies of protein aggregation and mitochondrial lipid oxidation
- Dr. Patrick van der Wel (University of Pittsburgh)
- Wednesday 31 May 2017
2333 CC Leiden
Huntington’s Disease (HD) and other neurodegenerative diseases are caused by the misfolding and aggregation of proteins . The proteins’ structural changes induce neuronal death via a poorly understood gain of toxic function. A lack of detailed information on the protein misfolding and aggregation, and the down-stream toxic events, continues to hinder the design of effective drugs. Using solid-state NMR and a range of complementary techniques, we investigate both the protein aggregation and mitochondrial protein-lipid interactions implicated in the toxic mechanism.
Protein aggregates associated with HD are formed by the exon 1 fragment of a mutated form of the huntingtin (HTT) protein, in which a naturally occurring polyglutamine segment is expanded. Though identified in the 1990s, polyglutamine-based amyloids have resisted structural characterization . Through advanced ssNMR experiments, we have determined the architecture of aggregated mutant HTT exon 1 and various polyglutamine-based peptides . We probe the aggregated protein via distance and torsion angle measurements, relaxation and order parameter measurements of local dynamics, solvent exposure measurements, and solution-NMR-like spectroscopy on disordered protein segments outside the aggregate core itself. We find that the aggregates are cytotoxic to neurons, capable of prion-like propagation by inducing the aggregation of polyglutamine protein monomers, and display an unusual type of fibril polymorphism. The structural data also illuminate the mechanism of misfolding, which is invaluable for attempts to devise drugs that modulate the misfolding pathway and for understanding how chaperones prevent aggregation in vivo.
Misfolded HTT appears to cause toxicity in part by inducing mitochondrial dysfunction . Characteristically, this is associated with increases in reactive oxygen species (ROS) that ultimately trigger cell death by ROS-induced lipid peroxidation. Remarkably, mitochondrial cytochrome c (cyt-c) facilitates and catalyzes the peroxidation process, with the resulting oxidized cardiolipin (CL) species acting as pro-apoptotic signals . To elucidate the molecular underpinnings of this process, which appears druggable in animal studies, we use ssNMR and other methods to probe the interactions between cyt-c and mitochondrial membrane-mimicking lipid vesicles. We observe the induction of peroxidase activity, and find it to correlate to the binding of cyt-c to CL lipids. 2D and 3D ssNMR spectra allow for assignment of the protein signals in the membrane-bound state, and reveal local structural and dynamic changes in both protein and lipids. Our findings point to a surprisingly native-like fold in the peripherally bound protein, and led us to propose a novel model for this pivotal pro-apoptotic event. Our model predicts a potential for developing compounds that target the lipid-protein complex and thus could modulate apoptosis and neuronal degeneration.
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