Suppressed Disassembly of Autolyzing p94/CAPN3 by N2A Connectin/Titin in a Genetic Reporter System

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...

Expanding Members and Roles of the Calpain Superfamily and Their Genetically Modified Animals

“…Calpain-3/p94 binds specifically to the gigantic muscle protein connectin/titin through the region close to the IS2 region. The protease activity of calpain-3/p94 is almost suppressed in vivo as it is bound to the N2A region of connectin/titin, where other important subcellular structures of muscle, such as sarcoplasmic reticulum and T-tubules, are aligned [123]. Some alternative splicing products (e.g., Lp82, Up86) of Capn3 are expressed in tissues other than muscles [6,28,77,94,144].…”

Nicastrin Is Dispensable for γ-Secretase Protease Activity in the Presence of Specific Presenilin Mutations

“…The mutagenized PS1 cDNA fragments (ϳ4 g) were cotransformed with 4 g of the KpnI fragment of pBEVY-T into PJ69-4A. The PS1 primers, PS1S and PS1AS, contain 40-bp regions from pBEVY-T (32) at the 5Ј termini, which enable ligation in vivo by homologous recombination (36). About 2 ϫ 10 5 transformants were screened on selection medium plates, SD-LWHUAde (Table 1).…”

Myogenic Stage, Sarcomere Length, and Protease Activity Modulate Localization of Muscle-specific Calpain

“…shown to proceed by "nicking" in NS, IS1, and/or IS2 (21), with the reaction in IS2 regulated by N2A. These proteolytic modifications of p94 itself possibly result in a more "movable" conformation for p94, altering its affinity to connectin and other molecules.…”

“…p94 directly interacts with connectin at both N2A and M regions (19). Interaction between p94 and connectin at N2A stabilizes p94, which otherwise autolyzes very rapidly; thus, connectin may regulate p94 proteolytic functions (20,21). p94 has been also detected in Z (19,22), although a molecular basis for anchoring p94 in Z is unknown.…”