Calpain proteolysis contributes to the pathogenesis of heart failure but the calpain isoforms responsible and their substrate specificities have not been rigorously defined. One substrate, Junctophilin-2 (JP2), is essential for maintaining junctional cardiac dyads and excitation-contraction coupling. We previously demonstrated that mouse JP2 is cleaved by calpain-1 (CAPN1) between Arginine 565 (R565) and Threonine 566 (T566). Recently, calpain-2 (CAPN2) was reported to cleave JP2 at a novel site between Glycine 482 (G482) and Threonine 483 (T483). We aimed to directly compare the contributions of each calpain isoform, their Ca2+ sensitivity, and their cleavage site selection for JP2. We find CAPN1, CAPN2 and their requisite CAPNS1 regulatory subunit are induced by pressure overload stress that is concurrent with JP2 cleavage. Using in vitro calpain cleavage assays, we demonstrate that CAPN1 and CAPN2 cleave JP2 into similar 75 kD N-terminal (JP2NT) and 25 kD C-terminal fragments (JP2CT) with CAPNS1 co-expression enhancing proteolysis. Deletion mutagenesis shows both CAPN1 and CAPN2 require R565/T566 but not G482/T483. When heterologously expressed, the JP2CT peptide corresponding to R565/T566 cleavage approximates the 25 kD species found during cardiac stress while the C-terminal peptide from potential cleavage at G482/T483 produces a 35 kD product. Similar results were obtained for human JP2. Finally, we show that CAPN1 has higher Ca2+ sensitivity and cleavage efficacy than CAPN2 on JP2 and other cardiac substrates including cTnT, cTnI and β2-spectrin. We conclude that CAPN2 cleaves JP2 at the same functionally conserved R565/T566 site as CAPN1 but with less efficacy and suggest heart failure may be targeted through specific inhibition of CAPN1.

中文翻译：

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Calpain-2 在与 Calpain-1 相同的位点特异性裂解 Junctophilin-2，但功效较低

钙蛋白酶蛋白水解有助于心力衰竭的发病机制，但负责的钙蛋白酶同种型及其底物特异性尚未得到严格定义。一种底物 Junctophilin-2 (JP2) 对维持心脏交界处二分体和兴奋-收缩耦合必不可少。我们之前证明小鼠 JP2 被精氨酸 565 (R565) 和苏氨酸 566 (T566) 之间的钙蛋白酶-1 (CAPN1) 切割。最近，有报道称 calpain-2 (CAPN2) 在甘氨酸 482 (G482) 和苏氨酸 483 (T483) 之间的新位点切割 JP2。我们旨在直接比较每种钙蛋白酶同种型的贡献、它们的 Ca2+ 敏感性以及它们对 JP2 的切割位点选择。我们发现 CAPN1、CAPN2 及其必需的 CAPNS1 调节亚基是由与 JP2 切割同时发生的压力超载应力诱导的。使用体外钙蛋白酶切割测定，我们证明 CAPN1 和 CAPN2 将 JP2 切割成相似的 75 kD N 端 (JP2NT) 和 25 kD C 端片段 (JP2CT)，CAPNS1 共表达增强蛋白水解。缺失突变显示 CAPN1 和 CAPN2 都需要 R565/T566，但不需要 G482/T483。当异源表达时，对应于 R565/T566 切割的 JP2CT 肽接近心脏应激期间发现的 25 kD 种类，而来自 G482/T483 潜在切割的 C 端肽产生 35 kD 产物。人类 JP2 也获得了类似的结果。最后，我们表明 CAPN1 对 JP2 和其他心脏底物（包括 cTnT、cTnI 和 β2-血影蛋白）具有比 CAPN2 更高的 Ca2+ 敏感性和切割功效。