We propose to study the structure and mechanism of action of the UNC-45 molecular chaperone, a myosin- affiliated """"""""UCS domain"""""""" protein. Molecular chaperones play key roles in muscle development and function by aiding protein folding and inhibiting protein denaturation and aggregation. We have demonstrated that UNC-45 is critical for skeletal muscle myosin accumulation and myofibril assembly and that it interacts with myosin to protect it from heat-induced aggregation. However, the structure of UNC-45, its mode of interaction with myosin and its roles in skeletal muscle disease are largely unexplored. Our goal is to define the molecular structure of Drosophila UNC-45 and to study its physical interaction with myosin. We will test the hypotheses that 1) ATP binding, which enhances UNC-45 chaperone function, causes structural elements of the protein to undergo conformational change and 2) the UCS domain of UNC-45 interacts with myosin. To this end, we will examine UNC-45 in the apo- and nucleotide-bound states at atomic-level resolution by crystallization, x-ray diffraction and computational analysis. This will be the first high-resolution structure of a UCS domain protein, a class of myosin-associated proteins found in fungi through mammals. We will also image UNC-45 complexed with myosin S-1 by negative staining, electron microscopy and single particle image analysis followed by docking the crystal structures into class averaged projections. This research program will take advantage of our expertise in contractile protein analysis and the capabilities of highly qualified collaborators. Our studies will elucidate the structure of UNC-45, provide an understanding of its interaction with ATP and define ATP- induced conformational changes. Further, our efforts will yield insight into UNC-45's physical interaction with the myosin substrate, which is critical to myofibril assembly and resistance to stress.

Public Health Relevance

. The UNC-45 chaperone is critical for the functional integrity of myosin, the molecular motor of muscle. We will study the structure of the UNC-45 chaperone and its interaction with its myosin target. Our efforts will elucidate the mechanism of action of UNC-45 and provide insight into how it functions in normal muscle as well as an understanding of its role during muscle stress.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR055958-03
Application #
7796735
Study Section
Skeletal Muscle and Exercise Physiology Study Section (SMEP)
Program Officer
Boyce, Amanda T
Project Start
2008-07-01
Project End
2012-03-31
Budget Start
2010-04-01
Budget End
2012-03-31
Support Year
3
Fiscal Year
2010
Total Cost
$260,489
Indirect Cost
Name
San Diego State University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
073371346
City
San Diego
State
CA
Country
United States
Zip Code
92182
Melkani, Girish C; Bhide, Shruti; Han, Andrew et al. (2017) TRiC/CCT chaperonins are essential for maintaining myofibril organization, cardiac physiological rhythm, and lifespan. FEBS Lett 591:3447-3458
Smith, Daniel A; Carland, Carmen R; Guo, Yiming et al. (2014) Getting folded: chaperone proteins in muscle development, maintenance and disease. Anat Rec (Hoboken) 297:1637-49
Lee, Chi F; Melkani, Girish C; Bernstein, Sanford I (2014) The UNC-45 myosin chaperone: from worms to flies to vertebrates. Int Rev Cell Mol Biol 313:103-44
Trujillo, Adriana S; Ramos, Raul; Bodmer, Rolf et al. (2014) Drosophila as a potential model to ameliorate mutant Huntington-mediated cardiac amyloidosis. Rare Dis 2:e968003
Melkani, Girish C; Trujillo, Adriana S; Ramos, Raul et al. (2013) Huntington's disease induced cardiac amyloidosis is reversed by modulating protein folding and oxidative stress pathways in the Drosophila heart. PLoS Genet 9:e1004024
Lee, Chi F; Melkani, Girish C; Yu, Qin et al. (2011) Drosophila UNC-45 accumulates in embryonic blastoderm and in muscles, and is essential for muscle myosin stability. J Cell Sci 124:699-705
Melkani, Girish C; Bodmer, Rolf; Ocorr, Karen et al. (2011) The UNC-45 chaperone is critical for establishing myosin-based myofibrillar organization and cardiac contractility in the Drosophila heart model. PLoS One 6:e22579
Lee, Chi F; Hauenstein, Arthur V; Fleming, Jonathan K et al. (2011) X-ray crystal structure of the UCS domain-containing UNC-45 myosin chaperone from Drosophila melanogaster. Structure 19:397-408
Melkani, Girish C; Lee, Chi F; Cammarato, Anthony et al. (2010) Drosophila UNC-45 prevents heat-induced aggregation of skeletal muscle myosin and facilitates refolding of citrate synthase. Biochem Biophys Res Commun 396:317-22
Sousa, Duncan; Cammarato, Anthony; Jang, Ken et al. (2010) Electron microscopy and persistence length analysis of semi-rigid smooth muscle tropomyosin strands. Biophys J 99:862-8