Dissertation
ROLE OF PROTEIN-PROTEIN INTERACTIONS IN PROPER FORMATION AND FUNCTION OF CARDIAC THIN FILAMENTS
Doctor of Philosophy (PhD), Washington State University
01/2018
Handle:
https://hdl.handle.net/2376/118130
Abstract
In striated muscle, actin-based thin filaments and myosin-based thick filaments are organized into basic contractile units called sarcomeres. Tropomyosin (Tpm), a two-stranded coiled-coil protein, assembles in a head-to-tail manner to extend along the thin filament. Tpm stabilizes the thin filament and, along with the troponin complex (Tn), regulates Ca2+-dependent actomyosin interactions which are molecular prerequisites for muscle contraction. Thin filaments are polar with the pointed ends directed toward the M band and the barbed ends anchored to the Z disks. At the pointed end, the N-terminus of Tpm interacts with tropomodulin (Tmod) and leiomodin (Lmod). Tmod is an efficient pointed-end capping protein, whereas Lmod, its larger homologue, does not completely prevent elongation of the thin filament from the pointed end.
The dissertation determines the arrangement and functional significance of Tpm- and actin-binding sites within the N-terminal region of Lmod2, a major Lmod isoform in cardiac muscle. We found that Lmod2 contains only one Tpm-binding site and binds to only one Tpm molecule. The N-terminal actin-binding site of Lmod2 is located within residues 43-90, with additional actin-binding interactions within residues 124-201. We found that the N-terminal Tpm- and actin-binding sites are important for Lmod2’s assembly to the pointed end in cardiomyocytes where it acts as a leaky cap to regulate actin dynamics.
The dissertation investigates effects of cardiomyopathy-linked mutations K15N and R21H in Tpm1.1, a striated muscle Tpm isoform, on the Ca2+-dependent regulation and pointed-end dynamics of cardiac thin filaments. We found that the K15N mutation, but not the R21H mutation, decreases the Ca2+ sensitivity of thin filaments and the rate of structural changes within cTn triggered by Ca2+ dissociation from cTn. The R21H mutation significantly destabilizes the structural integrity of Tpm1.1 near its N-terminus and reduces Tpm1.1’s affinity for F-actin and Lmod2. The R21H mutation also affects the in vitro pointed-end capping ability of Lmod2 and changes how Lmod2 binds to the sides of F-actin. Our data suggest that the K15N mutation causes DCM mostly by altering the Ca2+-dependent thin filament regulation, and one possible HCM-causing mechanism of the R21H mutation is through alteration of Lmod2’s function.
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Details
- Title
- ROLE OF PROTEIN-PROTEIN INTERACTIONS IN PROPER FORMATION AND FUNCTION OF CARDIAC THIN FILAMENTS
- Creators
- Thu Ngoc Anh Ly
- Contributors
- Alla S Kostyukova (Advisor)Wen-ji Dong (Committee Member)Nehal I Abu-Lail (Committee Member)Anita Vasavada (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Voiland College of Engineering and Architecture
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Number of pages
- 230
- Identifiers
- 99900581507301842
- Language
- English
- Resource Type
- Dissertation