Calpain-3 - Human Molecular Genetics, 2008, Vol. 17, No. 21

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Novel role of calpain-3 in the triad-associated protein complex regulating calcium release in skeletal muscle Irina Kramerova 1 , Elena Kudryashova 1 , Benjamin Wu 1 , Coen Ottenheijm 2 , Henk Granzier 2 and Melissa J. Spencer 1, ± 1 Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA and 2 Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85724, USA Received June 17, 2008; Revised and Accepted July 30, 2008 Calpain-3 (CAPN3) is a non-lysosomal cysteine protease that is necessary for normal muscle function, as mutations in CAPN3 result in an autosomal recessive form of limb girdle muscular dystrophy type 2A. To elucidate the biological roles of CAPN3 in skeletal muscle, we performed a search for potential substrates and interacting partners. By yeast-two-hybrid analysis we identified the glycolytic enzyme aldolase A (AldoA) as a binding partner of CAPN3. In co-expression studies CAPN3 degraded AldoA; however, no accumulation of AldoA was observed in total extracts from CAPN3-deficient muscles suggesting that AldoA is not an in vivo substrate of CAPN3. Instead, we found CAPN3 to be necessary for recruitment of AldoA to one specific location, namely the triads, which are structural components of muscle responsible for calcium transport and excitation–contraction coupling. Both aldolase and CAPN3 are present in the triad-enriched fraction and are able to interact with ryanodine receptors (RyR) that form major calcium release channels. Levels of triad-associated AldoA and RyR were decreased in CAPN3-deficient muscles compared with wild-type. Consistent with these observations we found calcium release to be significantly reduced in fibers from CAPN3-deficient muscles. Together, these data suggest that CAPN3 is necessary for the structural integrity of the triad-associated protein complex and that impairment of calcium transport is a phenotypic feature of CAPN3-deficient muscle. INTRODUCTION Limb girdle muscular dystrophy type 2A (LGMD2A) caused by mutations in the non-lysosomal cysteine protease calpain-3 (CAPN3), is one of the most frequently occurring forms of LGMD,adiseasecharacterizedbyhighgeneticandclinicalvaria- bility (1). It is often assumed that enzymatic activity of CAPN3 is essential for its physiological function. However, pathogenic mutations are not concentrated only in the catalytic domain of CAPN3 but are spread along the entire length of the protein (1). In a recent study, it was estimated that about one-third of the LGMD2A biopsies had normal levels of CAPN3 proteolytic activity suggesting that CAPN3 serves other physiological roles besides that of a protease (2). It has been demonstrated that at least some mutations which do not affect proteolytic activity, decreasethe abilityofCAPN3 tointeract withtitin,anestablished CAPN3-binding partner (3). The anchorage to titin occurs at two places, at the N2-line and the M-line regions of titin (4,5). Inter- estingly, mutations at both of these regions of titin cause muscle disease in humans and mice (6–8).
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Calpain-3 - Human Molecular Genetics, 2008, Vol. 17, No. 21

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