Calpains are cysteine proteinases responsible for many biological roles in muscle, including protein degradation, muscle growth, and myoblast fusion. Calpains are inhibited by calpastatin, an endogenous inhibitor. Other factors, such as variations in pH, ionic strength, and oxidation influence calpain activity. This study aimed to determine the extent to which oxidation influences calpastatin inhibition of calpain-1. A series of order of addition assays were used to determine calpain-1 calcium activation and autolysis after exposure to an oxidizing agent (N-ethylmaleimide [NEM] or hydrogen peroxide [H2O2]. In the first series, purified calpastatin was added to the assay before or after oxidizing exposure at 165 mM NaCl, pH 6.5. In the second series, incubation buffer ionic strength (165 mM or 295 mM NaCl) was evaluated. The inhibitory activities of purified porcine calpastatin, purified human calpastatin domain I, or a subdomain B inhibitor peptide were evaluated in the third series. In the fourth series, a maleimide-polyethylene glycol molecule (MAL-PEG;MW=5000 Dalton) was used to evaluate the accessibility of free sulfhydryl groups and tagging of calpain-1 under each condition through a molecular weight shift assay. Results from this study indicate that autolysis of calpain-1, when used as an indicator of activation, occurred when the calpain-1/calpastatin complex was exposed to an oxidant or cysteine modifier such as NEM. However, when calpain-1 was exposed to the cysteine modifier before calpastatin, autolysis of calpain-1 did not occur or was significantly decrased (P < 0.05). Irreversible modification of cysteine residues by NEM prevented activation of calpain-1 in the absence of calpastatin, but if the cysteine modification is potentially reversible (H2O2), calpain-1 activity can be recovered. Results from this study indicate that when calpastatin is bound to calpain-1, calpain-1 activation can occur even after being exposed to a cysteine modifier (NEM) or hydrogen peroxide (H2O2). Calpain-1 is not tagged with maleimide-polyethylene glycol (MAL-PEG) in the presence of calpastatin, indicating that calpastatin blocks or covers free cysteines on calpain-1 from modification. Moreover, exposure to calpain-1/calpastatin complex with a cysteine modifier allows activation of calpain-1, indicating that the inhibitory action of calpastatin is compromised. These results indicate a regulatory role for calpastatin that is not inhibitory but protective for calpain-1.

中文翻译：

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Calpain-1/Calpastatin 复合物的形成促进 Calpain-1 在氧化条件下的激活


钙蛋白酶是半胱氨酸蛋白酶，在肌肉中发挥许多生物学作用，包括蛋白质降解、肌肉生长和成肌细胞融合。钙蛋白酶被内源性抑制剂钙蛋白酶抑制素抑制。其他因素，例如 pH 值、离子强度和氧化的变化也会影响钙蛋白酶的活性。本研究旨在确定氧化对 calpastatin 对 calpain-1 抑制的影响程度。使用一系列添加测定顺序来测定暴露于氧化剂（N-乙基马来酰亚胺 [NEM] 或过氧化氢 [H2O2] 后的 calpain-1 钙激活和自溶。在第一个系列中，将纯化的钙蛋白酶抑制剂添加到测定中在第二个系列中，在氧化暴露于 165 mM NaCl、pH 6.5 之前或之后，评估了纯化的猪钙蛋白酶抑制剂、纯化的人钙蛋白酶抑制剂结构域 I 或子结构域的抑制活性。在第三个系列中评估了 B 抑制剂肽，在第四个系列中，使用马来酰亚胺-聚乙二醇分子（MAL-PEG；MW=5000 道尔顿）来评估每种条件下游离巯基的可及性和 calpain-1 的标记。这项研究的结果表明，当将 calpain-1/calpastatin 复合物暴露于氧化剂或半胱氨酸修饰剂（如 NEM）时，会发生 calpain-1 的自溶作用。然而，当 calpain-1 在 calpastatin 之前暴露于半胱氨酸修饰剂时，calpain-1 不会发生自溶或显着减少 (P < 0.05)。 NEM 对半胱氨酸残基的不可逆修饰阻止了在没有 calpastatin 的情况下 calpain-1 的激活，但如果半胱氨酸修饰是潜在可逆的 (H2O2)，则 calpain-1 活性可以恢复。这项研究的结果表明，当 calpastatin 与 calpain-1 结合时，即使暴露于半胱氨酸修饰剂 (NEM) 或过氧化氢 (H2O2) 后，也会发生 calpain-1 激活。在 Calpain-1 存在的情况下，Calpain-1 不会被马来酰亚胺聚乙二醇 (MAL-PEG) 标记，这表明 Calpain-1 会阻断或覆盖 calpain-1 上的游离半胱氨酸，使其免遭修饰。此外，暴露于带有半胱氨酸修饰剂的 calpain-1/calpastatin 复合物可以激活 calpain-1，表明 calpastatin 的抑制作用受到损害。这些结果表明 calpastatin 的调节作用不是抑制而是保护 calpain-1。