Aggregation Mechanisms of PolyQ-containing Proteins: Structure and Cytotoxicity of the Metastable Intermediate Species
Final Report Abstract
Insoluble fibrillar aggregates are the major histopathological feature of polyglutamlne (polyQ)-repeat diseases, including Huntington's disease, dentatorubropallidoluysian atrophy and several ataxias. The role of the aggregates in the disease pathology is still controversial. The following three hypotheses could explain the reported in the literature poor correlation between toxicity and aggregate formation in vivo: (i) a sequential assembly pathway with multiple metastable species with different toxicity, (ii) multiple competing aggregation pathways with various transient species with different stability and toxicity, and/or (iii) various intra- (e.g., polyQ-flanking sequences) and intermolecular factors (e.g., aging, stress response) modulate the aggregation pathway(s) and toxicity. Using conformation-sensitive labeling, dynamic imaging approaches and orthogonal cross-seeding we studied the cellular dynamics and molecular architecture of aggregates of different polyQ-containing proteins, including Huntingtin exon-1, atrophin-1 and ataxin-3. The analyses were complemented with molecular dynamics simulations and a broad spectrum of in vitro and ex vivo biochemical and biophysical approaches, in ensemble and single-molecule mode. Our results revealed high heterogeneity of the cellular inclusions and can be summarized as follows: 1) The polyQ stretch of the aggregation-initiation species, most likely a monomer, is in ß-sheet or ß-sheetstack conformation. 2) Expansion of the polyQ stretch beyond the pathological threshold destabilizes the adjacent domains and alters the structural integrity of the host protein/adjacent domains. 3) The aggregate size increases incrementally in the aggregation time-course. 4) The architecture of the aggregate core in the cell evolves from non-polyQ based in the early species to polyQ-based in the later fibrillar and detergent-resistant aggregates. 5) Structurally stable elements, either N- or C-terminal (or both) to the polyQ-stretch can initially associate into flexible inclusions with the polyQ sequence excluded from the aggregate core. 6) Nuclear inclusions of poyQ proleins are more dynamic which provides a molecular explanation of the enhanced cellular toxicity of the nuclear aggregates in the polyQ-based neurodegeneration. In response to stress small organic compounds termed osmolytes are ubiquitously accumulated in all cell types lo regulate the intracellular solvent qualily and to counteract the deleterious effect on the stability and function of cellular proteins. Given that polyQ expansion destabilizes the host protein, we therefore dedicated a substantial effort to address the effect of various osmoprotective substances on the polyQ aggregation. We observed a clear difference in the mechanism of action of various osmolytes and their effect of the polyQ aggregation ranged from favoring the fibrillization, through partial solubilization to triggering assembly into amorphous aggregates. Our study reveals new insights into the complex interactions of osmoprotectants with polyQ aggregates. Although none of them completely abrogated the aggregate formation; they redirected the amyloidogenesis into alternative, non-toxic aggregate species.
Publications
- (2008). A fluorescent window into protein folding and aggregation in cells. Meth. Cell. Biol. 89, 59-70
Ignatova, Z. and Gierasch, L.M.
- (2011). Natural osmolytes remodel the aggeragtion pathway of mutant huntingtin exon 1. Biochem. 50, 2048-2060
Ignatova, Z.
