Calpain-2 specifically cleaves Junctophilin-2 at the same site as Calpain-1 but with less efficacy.
Calpain-2 specifically cleaves Junctophilin-2 at the same site as Calpain-1 but with less efficacy.
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DOI：
10.1042/bcj20210629
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发表时间：
2021-10-15
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影响因子：
--
通讯作者：
Song LS
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文献类型：
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作者：
Wang J;Ciampa G;Zheng D;Shi Q;Chen B;Abel ED;Peng T;Hall DD;Song LS
钙蛋白酶蛋白水解有助于心力衰竭的发病机制，但钙蛋白酶同工型及其底物特异性尚未严格定义。结膜亲蛋白-2 （Junctophilin-2, JP2）是维持心脏连接体和兴奋-收缩耦合所必需的底物。我们之前证明了小鼠JP2被calpain-1 （CAPN1）在精氨酸565 （R565）和苏氨酸566 （T566）之间切割。最近，calpain-2 （CAPN2）被报道在甘氨酸482 （G482）和苏氨酸483 （T483）之间的一个新位点上切割JP2。我们的目的是直接比较每个calpain异构体的贡献，它们的Ca2+敏感性和它们对JP2的裂解位点选择。我们发现CAPN1、CAPN2及其必需的CAPNS1调控亚基是由JP2裂解同时发生的压力过载应力诱导的。通过体外calpain裂解实验，我们发现CAPN1和CAPN2将JP2裂解为相似的75kd n端（JP2NT）和25kd c端片段（JP2CT）， CAPNS1共表达促进蛋白水解。缺失突变表明，CAPN1和CAPN2都需要R565/T566，而不需要G482/T483。当异源表达时，与R565/T566裂解相对应的JP2CT肽接近于心脏应激时发现的25 kD物种，而来自G482/T483潜在裂解的c端肽产生35 kD产物。人类JP2也得到了类似的结果。最后，我们发现CAPN1在JP2和其他心脏底物（包括cTnT、cTnI和β2-spectrin）上比CAPN2具有更高的Ca2+敏感性和裂解效率。我们得出结论，CAPN2在与CAPN1相同的功能保守的R565/T566位点切割JP2，但效果较差，并提示可能通过特异性抑制CAPN1来靶向心力衰竭。
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.