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Principal Investigator: WILLIAM J LEHMAN
Organization: BOSTON UNIVERSITY MEDICAL CAMPUS
Fiscal Year: 2024
Award: $412,500
Funding agency: National Heart Lung and Blood Institute
Thin filament-linked actin-binding proteins, troponin and tropomyosin, control actomyosin-based muscle
contraction in cardiac and skeletal muscles. To elucidate mechanisms of muscle thin filament function at a
fundamental molecular level, it is crucial to determine the changing structural interactions of these regulatory
proteins that control muscle cooperative activation and relaxation via allosteric communication pathways between
filament components. It follows that disease-related myofibrillar protein mutants can perturb muscle on-off
switching by causing an imbalance in troponin-tropomyosin interactions on actin which, in turn, destabilizes relaxed
or active states and the transitions between them. It is our premise that early stage intervention to correct such
imbalances is paramount in diminishing or reversing resulting inexorable disease progression. In the current work,
we will address these imbalances by taking a multifaceted structural approach to elucidate the mechanism of thin
filament regulation and thus establish root causes of these perturbations. To accomplish this goal: 1. We will use
cryo-electron microscopy, coupled with 3D-image reconstruction, to establish regulatory transitions of troponin and
tropomyosin as well as test the impact of myosin-binding on thin filament actin and tropomyosin. 2. We will refine
this experimental approach with computational tools that we have pioneered to bring cryo-EM structures even
closer to an atomic level using protein-protein docking protocols and molecular dynamics. 3. We will compare
structural interactions that occur in normal thin filaments with those in filaments containing mutant proteins linked
to myopathies in order to assess how mutation-linked aberrant physiology can link to myopathology, while
localizing druggable target pockets at protein-protein interfaces. To achieve our aims, (1) we will focus on
identifying structural domains at the interface between of troponin subunit-T and actin-tropomyosin (Specific Aim
1); (2) we will reveal the structural mechanism used by regulatory domains of troponin subunit-I to trap tropomyosin
in its relaxed-state position on actin (Specific Aim 2); (3) we will determine the impact of myosin structural
interactions on actin-tropomyosin, less recognized but significant effectors of thin filament regulation (Specific Aim
3). The influence of myopathic-linked mutations in troponin, tropomyosin, actin and myosin will not only be predicted
and tested structurally but assayed functionally by measuring in vitro motility and contractility in engineered heart
tissue. Aiming to develop tools to counteract regulatory imbalances, we collaborate with associates at the Boston
University Central for Molecular Discovery to identify small molecules to be trapped at druggable interfaces along
thin filaments in order to potentially manipulate cooperative, regulatory pathways. Thus, our work on the molecular
regulation of cardiac and skeletal muscle thin filaments and muscle contraction lies at the intersection of basic and
translation biomedical research. Here, our overarching goal is to couple understanding of atomic level mutational
“insults” that alter muscle control mechanisms to prospects of reversing early-stage defects in physiological function.
Terms: <3-D Images><3-D image><3D image><3D images><Actin Filaments><Actin-Activated ATPase><Actin-Binding Protein><Actins><Actomyosin><Address><Affect><Assay><Autoregulation><Binding><Bioassay><Biochemical><Biochemistry><Biological Assay><Biological Chemistry><Biomedical Research><Body Tissues><Boston><C-terminal><Cardiac><Cardiac Muscle Contraction><Cardiomyopathies><Clinical><Collaborations><Communication><Complex><Computational toolkit><Computer Models><Computerized Models><Coupled><Cryo-electron Microscopy><Cryoelectron Microscopy><Data><Defect><Disease><Disease Outcome><Disease Progression><Disorder><Docking><Dysfunction><Electron Cryomicroscopy><Face><Filament><Functional disorder><Genes><Genetic><Genetic Alteration><Genetic Change><Genetic defect><Goals><Head><Heart><Homeostasis><In Vitro><Individual><Intervention><Intervention Strategies><Investigators><Lead><Left><Link><Location><Measures><Microfilaments><Modeling><Molecular><Molecular Configuration><Molecular Conformation><Molecular Dynamics Simulation><Molecular Interaction><Molecular Stereochemistry><Motility><Muscle><Muscle Cell Contraction><Muscle Contraction><Muscle Disease><Muscle Disorders><Muscle Proteins><Muscle Relaxation><Muscle Tissue><Muscle relaxation phase><Muscular Contraction><Muscular Diseases><Mutation><Myocardial Diseases><Myocardial Disorder><Myocardiopathies><Myocardium><Myofilaments><Myopathic Conditions><Myopathic Diseases and Syndromes><Myopathic disease or syndrome><Myopathy><Myosin ATPase><Myosin Adenosine Triphosphatase><Myosin Adenosinetriphosphatase><Myosins><Pathologic><Pathway interactions><Pb element><Penetrance><Phenotype><Physiologic><Physiological><Physiological Homeostasis><Physiology><Physiopathology><Play><Position><Positioning Attribute><Process><Proteins><Protocol><Protocols documentation><Regulation><Regulatory Pathway><Regulatory Protein><Relaxation><Research><Research Personnel><Researchers><Restrictive Cardiomyopathy><Role><Severities><Signal Pathway><Single Crystal Diffraction><Site><Skeletal Muscle><Specific qualifier value><Specified><Structure><Symptoms><Tail><Testing><Thin Filament><Three-Dimensional Image><Tissue Model><Tissues><Translations><Tropomyosin><Troponin><Universities><Voluntary Muscle><Work><X Ray Crystallographies><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray/Neutron Crystallography><Xray Crystallography><cardiac function><cardiac muscle><cardiac tissue engineering><cartilage link protein><computational modeling><computational models><computational toolbox><computational tools><computational toolset><computer based models><computerized modeling><computerized tools><conformation><conformational><conformational state><conformationally><conformations><cryo-EM><cryoEM><cryogenic electron microscopy><defined contribution><design><designing><drug development><druggable target><engineered heart tissue><faces><facial><function of the heart><genetic regulatory protein><genome mutation><heart function><heart muscle><heavy metal Pb><heavy metal lead><image construction><image generation><image reconstruction><in vitro Assay><insight><interventional strategy><link protein><molecular dynamics><muscular><muscular disorder><mutant><myocardium disease><myocardium disorder><pathophysiology><pathway><reconstruction><regulatory gene product><response><small molecule><social role><synergism><tool><translation>