Proteins Interacting in Myosin Assembly

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DESCRIPTION (provided by applicant): The myosins function as protein motors of the actin cytoskeleton in fundamental biological activities including cell division (cytokinesis), plasma membrane dynamics and vesicle trafficking as well as more specialized physiological roles including cardiac and skeletal muscle contraction. A newly identified family of proteins containing UCS-domains (U: unc-45; C: Cro-1; S: She4p) is expressed in eukaryotic organisms from yeast to humans and has been implicated in myosin assembly and function. In animals from Caenorhabditis elegans to humans, two additional protein regions are conserved: an NH2-terminal TPR-domain (Tetratrico Peptide Repeat), which is responsible for binding Hsp90, a molecular chaperone implicated in protein folding. The central region together with the COOH-UCS-domain is sufficient for myosin binding and chaperone activity. The animal proteins are generically termed UNC-45 after the canonical C. elegans molecule. To further understand both the mechanisms by which UNC-45 machines work and the finding in mice and humans of general cell and striated muscle isoforms, we propose the following Specific Aims: 1) What mechanisms operate in UNC-45 activity as a chaperone and as a Hsp90 co-chaperone in myosin S1 folding, full-length myosin assembly, or myosin motor-related activity? 2) What are the structure-function relationships between identified regions of UNC-45 and specific sites within them and their interactions with Hsp90 for myosin S1 folding, full-length myosin assembly, or myosin motor-related activity? 3) What are the functional consequences of the differences in amino acid sequences and patterns of expression between the general cellular and striated muscle isoforms of UNC-45 in mammals? To answer these questions, we will use wild type and mutant recombinant UNC-45 proteins of C. elegans and mice and specific mouse knockout lines. The understanding of these questions is likely to have broad significance not only to multiple problems in cell biology but also with respect to disease. The findings that UNC-45 fungal homologues are necessary for cytokinesis suggests that the UNC-45 general cell isoform may be important in relation to the dysregulation of cell division of cancer. The defective assembly of myosin into filaments by unc-45 mutants in C. elegans suggests that the UNC-45 striated muscle isoform may be important in heart failure and skeletal muscle atrophy in which the contractile machinery is dysfunctional or deficient.