Department of Animal Sciences, North Dakota State University, Fargo, ND, 58108, USA.
Department of Animal Science, Iowa State University, Ames, IA, 50010, USA.
J Anim Sci. 2024 Jan 3;102. doi: 10.1093/jas/skae135.
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 = 5,000 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 decreased (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.
钙蛋白酶是半胱氨酸蛋白酶，负责肌肉中的许多生物学作用，包括蛋白质降解、肌肉生长和成肌细胞融合。钙蛋白酶受钙蛋白酶抑制蛋白（一种内源性抑制剂）抑制。其他因素，如 pH 值、离子强度和氧化的变化，会影响钙蛋白酶的活性。本研究旨在确定氧化对钙蛋白酶抑制钙蛋白酶-1的影响程度。一系列添加顺序测定用于确定暴露于氧化剂（N-乙基马来酰亚胺[NEM]或过氧化氢[H2O2]）后钙蛋白酶-1的钙激活和自解。在第一系列中，在 165 mM NaCl、pH 6.5 下进行氧化暴露之前或之后将纯化的钙蛋白酶抑制蛋白添加到测定中。在第二个系列中，评估了孵育缓冲液的离子强度（165 mM 或 295 mM NaCl）。在第三个系列中，评估了纯化的猪钙蛋白酶抑制蛋白、纯化的人钙蛋白酶抑制蛋白 I 域或亚基 B 抑制剂肽的抑制活性。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）通过分子量转移测定评估在每种条件下游离巯基的可及性和钙蛋白酶-1的标记。本研究的结果表明，当钙蛋白酶-1自解用作激活的指示剂时，当钙蛋白酶-1/钙蛋白酶抑制蛋白复合物暴露于氧化剂或半胱氨酸修饰剂（如 NEM）时，钙蛋白酶-1 会发生自解。然而，当钙蛋白酶-1在钙蛋白酶抑制蛋白之前暴露于半胱氨酸修饰剂时，钙蛋白酶-1 的自解不会发生或显着降低（P<0.05）。NEM 不可逆修饰半胱氨酸残基可防止钙蛋白酶-1在没有钙蛋白酶抑制蛋白的情况下激活，但如果半胱氨酸修饰是潜在可逆的（H2O2），则可以恢复钙蛋白酶-1的活性。本研究的结果表明，当钙蛋白酶抑制蛋白与钙蛋白酶-1结合时，即使暴露于半胱氨酸修饰剂（NEM）或过氧化氢（H2O2），钙蛋白酶-1 的激活也可能发生。钙蛋白酶-1在存在钙蛋白酶抑制蛋白时不与马来酰亚胺-聚乙二醇（MAL-PEG）结合，表明钙蛋白酶抑制蛋白阻止或覆盖钙蛋白酶-1上的游离半胱氨酸免受修饰。此外，暴露于钙蛋白酶-1/钙蛋白酶抑制蛋白复合物与半胱氨酸修饰剂允许钙蛋白酶-1激活，表明钙蛋白酶抑制蛋白的抑制作用受损。这些结果表明钙蛋白酶抑制蛋白具有调节作用，而不是抑制作用，而是对钙蛋白酶-1具有保护作用。
