Variation in the calpain 10 gene has recently been shown to be associated with type 2 diabetes by positional cloning. Since then, studies on calpain 10 have been started in correlation with diabetes and insulinmediated signaling. In this review, the activation mechanism of calpain by calcium ions, which is essential to understand its physiological functions, is discussed on the basis of recent X-ray structural analyses. Further, special features of the structure of calpain 10 that differ from those of typical -or m-calpain used in most studies are summarized together with discussion of the physiological function of calpain with respect to type 2 diabetes. Diabetes 53 (Suppl. 1)

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a genetic disease that is caused by mutations in the calpain 3 gene (CAPN3), which encodes the skeletal muscle-specific calpain, calpain 3 (also known as p94). However, the precise mechanism by which p94 functions in the pathogenesis of this disease remains unclear. Here, using p94 knockin mice (termed herein p94KI mice) in which endogenous p94 was replaced with a proteolytically inactive but structurally intact p94:C129S mutant protein, we have demonstrated that stretch-dependent p94 distribution in sarcomeres plays a crucial role in the pathogenesis of LGMD2A. The p94KI mice developed a progressive muscular dystrophy, which was exacerbated by exercise. The exercise-induced muscle degeneration in p94KI mice was associated with an inefficient redistribution of p94:C129S in stretched sarcomeres. Furthermore, the p94KI mice showed impaired adaptation to physical stress, which was accompanied by compromised upregulation of muscle ankyrin-repeat protein-2 and hsp upon exercise. These findings indicate that the stretchinduced dynamic redistribution of p94 is dependent on its protease activity and essential to protect muscle from degeneration, particularly under conditions of physical stress. Furthermore, our data provide direct evidence that loss of p94 protease activity can result in LGMD2A and molecular insight into how this could occur.

No abstract

p94/calpain 3 is a skeletal muscle-specific member of the Ca 2؉ -regulated cytosolic cysteine protease family, the calpains. Defective p94 protease activity originating from gene mutations causes a muscular dystrophy called calpainopathy, indicating the indispensability of p94 for muscle survival. Because of the existence of the p94-specific regions IS1 and IS2, p94 undergoes very rapid and exhaustive autolysis. To elucidate the physiological relevance of this unique activity, the autolytic profiles of p94 and the effect of the p94 binding protein, connectin/titin, on this process were investigated. In vitro analysis of p94 autolysis showed that autolysis in IS1 proceeds without immediate disassembly into fragments and that the newly identified cryptic autolytic site in IS2 is critical for disassembling autolyzed fragments. As a genetic system to assay p94 autolysis semiquantitatively, p94 was expressed in yeast as a hybrid protein between the DNA binding and activation domains of the yeast transcriptional activator Gal4. Transcriptional activation by the Gal4-p94:WT hybrid protein is precluded by p94 autolysis. Complete or partial loss of autolytic activity by C129S active site mutation, limb girdle muscular dystrophy type 2A pathogenic missense mutations, or PCR-based random mutagenesis could be detected by semiquantitative restoration of Gal4-dependent ␤-galactosidase gene expression. Using this system, the N2A connectin fragment that binds to p94 was shown to suppress p94 autolytic disassembly. The proximity of the IS2 autolytic and connectin-binding sites in p94 suggested that N2A connectin suppresses IS2 autolysis. These data indicate the importance of p94-connectin interaction in the control of p94 functions by regulating autolytic decay of p94.Calpains (named after the first discovered and best characterized member, Ca 2ϩ -dependent and papain-like protease, EC 3.4.22.18, clan CA, family C2) correspond to a diverse gene family whose members all share the characteristic "calpain protease domain" and comprise a unique branch of the cysteine proteases (1, 2). To date, calpains have been identified in many different mammalian tissues and in almost all types of living organism (3).Studies on calpain protease activity, from enzymatic characterization to clarification of its roles in cellular phenomena, have mainly advanced knowledge of the conventional calpains, the -and m-calpains. These ubiquitously expressed calpains can be conveniently assayed in vitro using established and reproducible methods, such as casein hydrolysis assay, facilitating development of more specific inhibitors as well as more sensitive substrates.In contrast, the skeletal muscle-specific calpain, p94/calpain 3, has remained poorly characterized with regard to its protease activity. The expression of p94 predominates over other calpain species in skeletal muscle, and a defect in p94 proteolytic activity originating from gene mutations causes muscular dystrophy (4 -6). In this context, p94 exemplifies the critical importance of tissue-spe...