Muscle sarcomeres contain a number of giant polypeptides (0.7-4 MDa). Much is currently known about the function of the largest of these polypeptides, vertebrate titin. Much less is known about the newest vertebrate member of this family, obscurin. The pathogenesis of one form of muscular dystrophy may involve obscurin. Obscurin is the homolog of UNC-89 in C. elegans. Essential features of UNC-89 as a signaling / scaffolding protein crucial for A-band/M-line assembly were discovered before obscurin was reported. To understand how UNC-89 is localized, and how it functions, we are taking a systematic approach for identifying and studying the function of its binding partners. We discovered that the Ig1-3 segment of UNC-89 interacts with CPNA-1, a copine domain protein. Although CPNA-1 is not required for initial assembly of UNC-89 at the M-line, it is required for its retention. CPNA-1 is located at both M-lines and dense bodies, whereas UNC-89 is located only at M-lines. Therefore, we hypothesize that there are proteins which direct assembly of UNC-89 solely to the M-line. UNC-89 Ig9-11 interacts with CUL-1 (cullin 1), and UNC-89 Ig2-3 interacts with MEL-26, a substrate recognition protein for CUL-3 (cullin 3). Cullins are scaffolds for assembly of the ubiquitination machinery. One function of the CUL-3/MEL-26 complex is to promote degradation of MEI-1 (katanin). Independently, the coI of this proposal, Stephan Lange, found that in mouse heart, degradation of sAnk1.5 is promoted by a cullin 3 substrate recognition protein, KCTD6, and this is dependent upon obscurin. We hypothesize that UNC-89 (obscurin) negatively regulates the activity of cullin complexes in muscle. We further hypothesize that UNC-89 interacts with other cullins or cullin 3 adaptor proteins, and there are substrates in addition to MEI-1. In humans, muscle atrophy is associated with immobilization, chronic diseases and advanced age. Since in muscle atrophy, degradation of sarcomeric proteins is upregulated, our studies have medical relevance. Also, our collaborator and coI of this grant, Ken Norman, has found that a key function of obscurin for organization of the SR, is conserved for UNC-89. In addition, UNC-89 was found to have a physiological role in EC coupling, and this involves the conserved RacGEF, VAV-1. We hypothesize that UNC-89 and VAV-1 interact, and that VAV-1 localization and function is dependent on UNC-89. Finally, we hypothesize that the binding partners and functional mechanisms discovered for UNC-89 in C. elegans are biologically relevant for mammalian striated muscle.
Specific aims are: (1) determine mechanisms which direct assembly of UNC-89 solely to the M-line; (2) determine if other cullins interact with UNC-89, if there are additional substrates for MEL-26, and determine the biochemical effects of the UNC-89 to MEL-26 interaction;(3) define the biochemistry and functional consequence of interaction between UNC-89 and myosin;(4) investigate the molecular mechanism underlying UNC-89's role in calcium regulation;and (5) determine if functional interactions and molecular mechanisms identified for nematode UNC-89 are conserved for mammalian obscurin.

Public Health Relevance

Understanding the assembly and maintenance of the muscle contractile machinery called the sarcomere is essential for understanding the pathogenesis of muscular dystrophies, myopathies and cardiomyopathies. Using a molecular genetic approach in the model organism C. elegans, we have discovered new information about a key sarcomeric protein, UNC-89, similar to human obscurin. Both UNC-89 and obscurin have been implicated in a novel mechanism controlling protein turnover, and this is relevant to muscle atrophy which occurs in many human diseases (e.g. renal failure, diabetes, cancer). This project will gain new insights into the function of UNC-89 and obscurin.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Skeletal Muscle and Exercise Physiology Study Section (SMEP)
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Boyce, Amanda T
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Emory University
Schools of Medicine
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Qadota, Hiroshi; Matsunaga, Yohei; Bagchi, Pritha et al. (2018) Protein phosphatase 2A is crucial for sarcomere organization in Caenorhabditis elegans striated muscle. Mol Biol Cell 29:2084-2097
Qadota, Hiroshi; Matsunaga, Yohei; Nguyen, Ken C Q et al. (2017) High-resolution imaging of muscle attachment structures in Caenorhabditis elegans. Cytoskeleton (Hoboken) 74:426-442
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Loveless, Timothy; Qadota, Hiroshi; Benian, Guy M et al. (2017) Caenorhabditis elegans SORB-1 localizes to integrin adhesion sites and is required for organization of sarcomeres and mitochondria in myocytes. Mol Biol Cell 28:3621-3633
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Qadota, Hiroshi; Mayans, Olga; Matsunaga, Yohei et al. (2016) The SH3 domain of UNC-89 (obscurin) interacts with paramyosin, a coiled-coil protein, in Caenorhabditis elegans muscle. Mol Biol Cell 27:1606-20
Matsunaga, Yohei; Qadota, Hiroshi; Furukawa, Miho et al. (2015) Twitchin kinase interacts with MAPKAP kinase 2 in Caenorhabditis elegans striated muscle. Mol Biol Cell 26:2096-111
Benian, Guy M; Mayans, Olga (2015) Titin and obscurin: giants holding hands and discovery of a new Ig domain subset. J Mol Biol 427:707-14