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Title page for ETD etd-07142018-144149


Type of Document Dissertation
Author Mishra, Sanjay
URN etd-07142018-144149
Title Molecular model of αB-crystallin function in the transparency of the eye lens and the stress resistance of the heart
Degree PhD
Department Chemical and Physical Biology
Advisory Committee
Advisor Name Title
Wenbiao Chen, Ph.D. Committee Chair
D. Borden Lacy, Ph.D. Committee Member
Kevin L. Schey, Ph.D. Committee Member
Patrick S. Page-McCaw, Ph.D. Committee Member
Keywords
  • cardiomyopathy
  • sHSP
  • chaperones
  • crystallins
  • small heat shock proteins
  • zebrafish
  • cataract
Date of Defense 2018-05-22
Availability restricted
Abstract
αB-crystallin is a member of the small heat shock protein (sHSP) superfamily. Mutations in αB-crystallin cause autosomal cataracts and cardiomyopathy, but the exact role of αB-crystallin in the lens and heart remains unclear. Zebrafish (ZF) is well suited for studying the role of αB-crystallin because it expresses two paralogs of αB-crystallin: αBa and αBb, with conserved sequence homology with human αB-crystallin. In vitro, αBa displayed higher chaperone activity and formed larger oligomers than mammalian homologs. sHSPs have a highly conserved α-crystallin domain (ACD) flanked by sequentially divergent C- and N-terminal regions. We hypothesized that the elevated activity of αBa is linked to an expanded oligomeric state, modulated by the N-terminal domain (NTD). To map the sequence elements underlying the expansion of the αBa oligomer, we carried out homolog scanning mutagenesis by swapping parts of NTD and C-terminal domain (CTD) from human αB- to zebrafish αBa-crystallin. Swapping of the NTD shifted the equilibrium towards smaller oligomers and reduced the chaperone activity. We show that this approach can identify the role of sequence elements in the evolutionary tuning of the chaperone function of αBa. To understand the role of αB crystallin in vivo, we established ZF knockout (KO) lines using CRISPR/Cas9 system which frame-shifted the coding sequences of the αB-crystallin genes, cryaba and cryabb, generating premature stop codons. Cataractous lens defects were observed in the KO embryos. A fraction of mutant embryos developed pericardial edema and showed heart phenotypes resembling dilated cardiomyopathy when challenged by crowding stress or exposed to elevated cortisol stress, both of which activate glucocorticoid receptor signaling. Our results suggest that αB-crystallin plays an important role in embryonic lens development in zebrafish and is potentially involved in the cellular responses that protect cardiac muscles under stress conditions.
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