Investigating the Structural Pathogenesis of Δ 160E Mutation – Linked Hypertrophic Cardiomyopathy

Persistent Link:
http://hdl.handle.net/10150/605220
Title:
Investigating the Structural Pathogenesis of Δ 160E Mutation – Linked Hypertrophic Cardiomyopathy
Author:
Abdullah, Salwa
Issue Date:
2016
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Embargo:
Release 04-Feb-2017
Abstract:
Hypertrophic cardiomyopathy (HCM) is a primary disease of the myocardium. 4-11% of HCM is caused by mutations in cardiac troponin T (cTnT) and 65% of them are within the tropomyosin (TM)-binding TNT1 domain. Two of the known mutational hotspots within TNT1 are in the N and C-terminal domains. Unlike the N-terminal domain; no high-resolution structure exists for the highly conserved C-terminal domain limiting both our ability to understand the functional role of this extended domain in myofilament activation and molecular mechanism(s) of HCM. The Δ160E mutation is an in-frame deletion of a glutamic acid residue at position 160 of cTnT. This TNT1 C-terminal mutation is associated with an especially poor prognosis. The Δ160E mutation is located in a putative "hinge region" immediately adjacent to the unstructured flexible linker connecting the TM-binding TNT1 domain to the Ca²⁺-sensitive TNT2 domain. Unwinding of this α-helical hinge may provide the flexibility necessary for thin filament function. Previous regulated in vitro motility assay (R-IVM) data showed mutation-induced impairment of weak actomyosin binding. Thus, we hypothesized that the Δ160E mutation repositions the flexible linker which impairs weak electrostatic binding and ultimately leads to severe cardiac remodeling. The goal of our studies is two-fold: 1) to gain high-resolution insight into the position of the cTnT linker with respect to the C-terminus of TM, and 2) to identify Δ160E-induced positional changes using Fluorescence Resonance Energy Transfer (FRET) in a fully reconstituted thin filament. To this end, residues in the middle and distal regions of the cTnT linker were sequentially cysteine-substituted (A168C, A177C, A192C and S198C) and labeled with the energy donor IAEDANS. The energy acceptor, DABMI was attached to cysteine 190 (C190) in the C-terminal region of TM and FRET measurements were obtained in the presence and absence of Ca²⁺ and myosin subfragment 1 (S1). An all-atom thin filament model in the Ca²⁺–on state was employed to predict the pathogenic effects of the Δ160E mutation on the structure and the dynamics of the cTnT linker region. Our data suggest that the linker domain runs alongside the C-terminus of TM and is differentially repositioned by calcium, myosin and the Δ160E mutation. The Δ160E mutation moves the linker closer to the C-terminus of TM. The in silico model supported this finding and demonstrated a mutation-induced decrease in linker flexibility. Moreover, the model predicted a pathogenic change in the orientation of the middle region of the linker and in the position of the Ca²⁺-sensitive TNT2 domain and the TM-binding TNT1 domain in response to Δ160E mutation. Collectively, our findings suggest that the Δ160E mutation-induced changes in the structure, position and dynamics of the linker region cause steric blocking of weak myosin binding sites on actin and subsequent impairment of contraction and disruption of sarcomeric integrity. These studies, for the first time, provided information regarding the role of the extended linker in both myofilament activation and disease.
Type:
text; Electronic Dissertation
Keywords:
Hypertrophic Cardiomyopathy; Sarcomere; Thin Filament; Molecular & Cellular Biology; Cardiac Troponin T
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Molecular & Cellular Biology
Degree Grantor:
University of Arizona
Advisor:
Tardiff, Jil

Full metadata record

DC FieldValue Language
dc.language.isoen_USen
dc.titleInvestigating the Structural Pathogenesis of Δ 160E Mutation – Linked Hypertrophic Cardiomyopathyen_US
dc.creatorAbdullah, Salwaen
dc.contributor.authorAbdullah, Salwaen
dc.date.issued2016en
dc.publisherThe University of Arizona.en
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en
dc.description.releaseRelease 04-Feb-2017en
dc.description.abstractHypertrophic cardiomyopathy (HCM) is a primary disease of the myocardium. 4-11% of HCM is caused by mutations in cardiac troponin T (cTnT) and 65% of them are within the tropomyosin (TM)-binding TNT1 domain. Two of the known mutational hotspots within TNT1 are in the N and C-terminal domains. Unlike the N-terminal domain; no high-resolution structure exists for the highly conserved C-terminal domain limiting both our ability to understand the functional role of this extended domain in myofilament activation and molecular mechanism(s) of HCM. The Δ160E mutation is an in-frame deletion of a glutamic acid residue at position 160 of cTnT. This TNT1 C-terminal mutation is associated with an especially poor prognosis. The Δ160E mutation is located in a putative "hinge region" immediately adjacent to the unstructured flexible linker connecting the TM-binding TNT1 domain to the Ca²⁺-sensitive TNT2 domain. Unwinding of this α-helical hinge may provide the flexibility necessary for thin filament function. Previous regulated in vitro motility assay (R-IVM) data showed mutation-induced impairment of weak actomyosin binding. Thus, we hypothesized that the Δ160E mutation repositions the flexible linker which impairs weak electrostatic binding and ultimately leads to severe cardiac remodeling. The goal of our studies is two-fold: 1) to gain high-resolution insight into the position of the cTnT linker with respect to the C-terminus of TM, and 2) to identify Δ160E-induced positional changes using Fluorescence Resonance Energy Transfer (FRET) in a fully reconstituted thin filament. To this end, residues in the middle and distal regions of the cTnT linker were sequentially cysteine-substituted (A168C, A177C, A192C and S198C) and labeled with the energy donor IAEDANS. The energy acceptor, DABMI was attached to cysteine 190 (C190) in the C-terminal region of TM and FRET measurements were obtained in the presence and absence of Ca²⁺ and myosin subfragment 1 (S1). An all-atom thin filament model in the Ca²⁺–on state was employed to predict the pathogenic effects of the Δ160E mutation on the structure and the dynamics of the cTnT linker region. Our data suggest that the linker domain runs alongside the C-terminus of TM and is differentially repositioned by calcium, myosin and the Δ160E mutation. The Δ160E mutation moves the linker closer to the C-terminus of TM. The in silico model supported this finding and demonstrated a mutation-induced decrease in linker flexibility. Moreover, the model predicted a pathogenic change in the orientation of the middle region of the linker and in the position of the Ca²⁺-sensitive TNT2 domain and the TM-binding TNT1 domain in response to Δ160E mutation. Collectively, our findings suggest that the Δ160E mutation-induced changes in the structure, position and dynamics of the linker region cause steric blocking of weak myosin binding sites on actin and subsequent impairment of contraction and disruption of sarcomeric integrity. These studies, for the first time, provided information regarding the role of the extended linker in both myofilament activation and disease.en
dc.typetexten
dc.typeElectronic Dissertationen
dc.subjectHypertrophic Cardiomyopathyen
dc.subjectSarcomereen
dc.subjectThin Filamenten
dc.subjectMolecular & Cellular Biologyen
dc.subjectCardiac Troponin Ten
thesis.degree.namePh.D.en
thesis.degree.leveldoctoralen
thesis.degree.disciplineGraduate Collegeen
thesis.degree.disciplineMolecular & Cellular Biologyen
thesis.degree.grantorUniversity of Arizonaen
dc.contributor.advisorTardiff, Jilen
dc.contributor.committeememberTardiff, Jilen
dc.contributor.committeememberDoetschman, Thomasen
dc.contributor.committeememberGranzier, Hendrikusen
dc.contributor.committeememberGregorio, Carolen
dc.contributor.committeememberKrieg, Paulen
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