Sponsor
Portland State University. Department of Chemistry
First Advisor
Steve Reichow
Term of Graduation
Summer 2023
Date of Publication
7-20-2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (Ph.D.) in Chemistry
Department
Chemistry
Language
English
Subjects
alpha-crystallin, amyloids, chaperones, cryo-EM, Small heat shock proteins, structural biology
DOI
10.15760/etd.3650
Physical Description
1 online resource (vii, 224 pages)
Abstract
The protein αB-crystallin represents the archetypical small heat-shock protein (sHSP), and together with αA-crystallin makes up the primary chaperone system in the eye lens, where they play a critical role in maintaining proteostasis. To achieve transparency, the lens undergoes a loss of organelles and both protein turnover halts, requiring that proteins remain functional for long periods of time as they accumulate chemical damage. The α-crystallins are highly abundant in the lens to stabilize proteostasis for multiple decades. Over time though, the small heat-shock proteins become overwhelmed by a high burden of destabilized clients and their own accumulated chemical damage. Mechanistic insights into the function and failure of the α-crystallins remain poorly understood though, largely driven by the high degree of polydispersity exhibited by this family of proteins.
Developments in the understanding of the α-crystallin have relied on insights into model sHSP, which often poorly represent the polydispersity and heterogeneity found in mammalian sHSP. For my dissertation research, I identified a conserved NT-IXI motif of the N-terminal domain and used site-directed mutagenesis to disrupt it and analyzed the NT-IXI motif’s role in stability, oligomeric assembly, and chaperone activity of the α-crystallins. Disruption of this motif in αB-crystallin induced the formation of a dynamic filamentous state that could converted back into the native-like morphology. Notably, the conformational heterogeneity in this filamentous state was reduced as compared to the native-like assembly, rendering it more amenable to CryoEM while maintaining many of the underlying properties of mammalian sHSP that are poorly understood, such as subunit exchange and client binding. I report the first cryo-EM reconstruction and atomic models of αB-crystallin in its full-length state at 3.5 Å, this represents the first structure of the high polydispersity small heat shock proteins. Structural analysis of αB-crystallin reveals insights into the interactions between multiple α-crystallin domains and the disordered N- and C-terminal domains.
During biochemical characterization of the NT-AXA variant of αB-crystallin, I also identified that these constructs underwent amyloid-like aggregation upon heating in near physiological buffer conditions. Upon trying to characterize this process, I observed that the wildtype α-crystallins undergo the same process upon heating for extended periods of time. This directly relates to another outstanding problem in the field of lens biology: the mechanisms of cataract formation and more specifically the contribution of amyloid-like aggregation to the onset of age-related cataract. There have been prior proposals that amyloidogenesis may play a significant role in cataract, though they still suffer from having no identified amyloid peptide or pathways to amyloid formation in the healthy lens. In my second set of research objectives, I characterize the filamentous aggregates formed by the α-crystallins upon heating, test for amyloidogenicity and compare this to amyloid-like species previously identified for the α-crystallins. This research direction supports the broad hypothesis that α-crystallins undergo amyloid (or amyloid-like) aggregation as a part of their contribution to the formation of age-related cataract.
Rights
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Persistent Identifier
https://archives.pdx.edu/ds/psu/40833
Recommended Citation
McFarland, Russell James, "Mechanisms of Fibrillar Aggregation of the Lens Small Heat Shock Proteins, αA- and αB-crystallin" (2023). Dissertations and Theses. Paper 6514.
https://doi.org/10.15760/etd.3650