The UNC-45 Chaperone as a Modulator of Myosin Biogenesis and Function

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 DESCRIPTION (provided by applicant): In this proposal we aim to elucidate the precise molecular mechanisms by which the chaperones UNC-45 and Hsp90 assist in the folding of the myosin head which is critical for sarcomere assembly during development and repair of stress-induced damage to sarcomeres in mature muscle. Developing an understanding of these mechanisms is a problem at the core of muscle development and function. It is also highly relevant to the folding and repair of the many classes of myosin molecules that are expressed in non-muscle tissues. Our recent discovery that myosin attains a novel conformation, which hydrolyzes ATP at the normal rate but with a greatly reduced ability to translocate actin filaments, in the presence of UNC-45 and is reversed by Hsp90, raises important questions as to the exact roles of these chaperones in myosin folding and repair and myofilament assembly. Guided by our recent published and preliminary studies, we hypothesize that during myosin assembly or repair, UNC-45 assists the myosin head to attain its native conformation and subsequently locks it in a state that prevents premature powerstrokes; this state is relieved by Hsp90 upon successful assembly into the sarcomere or repair of myosin heads. This hypothesis will be tested by using a combination of in vitro and in vivo analyses through a unique collaboration between two expert research labs. In Aim 1 we will use mutagenesis, biochemical and biophysical assays, including myosin-dependent actin filament gliding assays, to establish the UNC-45 domains and key amino acid regions that mediate the different unique functions of UNC-45. In Aims 2 and 3 we will use the power of C. elegans as a model system to examine the functional relationships between mutant UNC-45 proteins and sarcomere assembly (Aim 2), and the dynamics of association of UNC-45 and Hsp90 with themselves and with myosin during repair of damaged myosin heads (Aim 3). Our hypothesis, if confirmed, will represent a new paradigm in the biology of myosins, with potential novel therapeutic approaches not only for striated muscle disorders stemming from mutations in sarcomeric proteins (skeletal myopathies and cardiomyopathies), but also for the problem of tumor invasiveness.