UNC-45 is a molecular chaperone that is required for myosin accumulation and myofibril assembly in striated muscle. Its C-terminal UCS domain interacts directly with myosin, while its N-terminal TPR domain binds the chaperone Hsp90. Although its mechanism of action is unknown, UNC-45 appears to be critical both for myosin folding in vivo and for protecting myosin from stress-induced denaturation. Further, changes in UNC-45 levels correlate with skeletal muscle inclusion body myopathy and cardiac failure, implicating UNC-45 in human disease. To begin to understand structure-function relationships in this enigmatic protein, we solved the first crystal structure of UNC-45. This proposal builds upon this Drosophila melanogaster structure to identify the molecular mechanisms and consequences of UNC-45 dimerization, UNC-45 interaction with myosin and UNC-45's relationship with yet to be identified partners.
Aim 1 will map the structural and functional basis of our recent discovery that UNC-45 dimerizes. We will employ high-resolution electron microscopy, molecular modeling, cross-linking studies and functional analyses to test the hypothesis that dimerization of UNC-45 is a critical step in its mechanism of action.
Aim 2 a will be the first structure-functio based mutagenesis of UNC-45 and will test the role of a highly-conserved surface groove that we defined in the UCS domain. Mutant versions of the protein will be analyzed in vitro through myosin-binding and aggregation assays, and in vivo by muscle structure and function analysis in transgenic Drosophila. This will test the hypothesis that the conserved cleft in the UCS domain of UNC-45 binds myosin, aids in myosin accumulation in muscle and/or protects myosin from stress-induced denaturation.
Aim 2 b will explore our observed differential localization of UNC-45 within sarcomeres of different muscle types along with our electron microscopy results showing that UNC-45 can bind to the neck region of myosin. We will use transgenic fly lines expressing alternative versions of the neck converter region along with confocal microscopy to test the hypothesis that UNC-45 binds specifically to the converter domain of the myosin neck and preferentially binds to specific versions of this myosin domain.
Aim 3 will employ both genetic and biochemical approaches to define new partners for UNC-45 and test their importance in muscle structure and function. We will use flies with a depleted UNC-45 background in conjunction with the powerful genetic techniques of deficiency mapping and microRNA-enabled knockdown to define these partners. Further, we will use mass spectrometry to identify proteins isolated from developing and stressed muscles by UNC-45-based protein pull-down. We will examine the roles of these proteins during muscle development and stress by RNAi-based transient knockdown in vivo.
This aim will test the hypothesis that UNC-45 has different binding partners and functions during myosin folding, during its occupancy of the muscle sarcomere and during muscle stress. Overall, our integrative analysis will provide important insights into the mechanism of action of UNC-45 and its role in muscle development, stasis and stress.

Public Health Relevance

UNC-45 is protein that is critical for accumulation of the molecular motor myosin in skeletal and cardiac muscles and it protects this key contractile protein during muscle stress~ its abnormal expression is affiliated with skeletal muscle inclusion body myopathy as well as heart failure. Our project is designed to test hypotheses about the function of UNC-45 protein domains and interactions and should provide fundamental knowledge regarding UNC-45's mechanism of action. This will help elucidate UNC-45's role in normal muscle development and function as well as provide insight into how it might be used therapeutically to alleviate symptoms of muscle disease and stress.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR055958-04
Application #
8489071
Study Section
Special Emphasis Panel (ZRG1-MOSS-C (02))
Program Officer
Boyce, Amanda T
Project Start
2008-04-01
Project End
2018-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
4
Fiscal Year
2013
Total Cost
$317,688
Indirect Cost
$105,188
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
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
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