钙蛋白酶是负责肌肉中许多生物学作用的半胱氨酸蛋白酶,包括蛋白质降解,肌肉生长,和成肌细胞融合.钙蛋白酶被钙蛋白酶抑制,内源性抑制剂。其他因素,例如pH值的变化,离子强度,和氧化影响钙蛋白酶活性。这项研究旨在确定氧化对钙蛋白酶抑制的影响程度-1。使用一系列添加测定的顺序来确定暴露于氧化剂(N-乙基马来酰亚胺[NEM]或过氧化氢[H2O2]后的钙蛋白酶-1钙活化和自溶。在第一个系列中,在165mMNaCl的氧化暴露之前或之后,将纯化的calpastatin添加到测定中,pH6.5。在第二个系列中,评估孵育缓冲液离子强度(165mM或295mMNaCl)。纯化的猪calpastatin的抑制活性,纯化的人calpastatin结构域I,或亚结构域B抑制剂肽在第三系列中进行了评估。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在每种条件下评估游离巯基的可及性和钙蛋白酶-1的标记。这项研究的结果表明,钙蛋白酶-1的自溶,当用作激活的指标时,当钙蛋白酶-1/calpastatin复合物暴露于氧化剂或半胱氨酸修饰剂如NEM时发生。然而,当calpain-1在calpastatin之前暴露于半胱氨酸修饰剂时,钙蛋白酶-1的自溶没有发生或明显下降(P<0.05)。NEM对半胱氨酸残基的不可逆修饰可防止在不存在钙蛋白酶的情况下激活钙蛋白酶-1,但是如果半胱氨酸修饰是潜在可逆的(H2O2),calpain-1活性可以恢复。这项研究的结果表明,当钙蛋白酶蛋白与钙蛋白酶-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也会激活。Calpain-1在有calpastatin的情况下没有用马来酰亚胺-聚乙二醇(MAL-PEG)标记,表明calpastatin阻断或覆盖钙蛋白酶-1上的游离半胱氨酸。此外,暴露于具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,对钙蛋白酶蛋白的调节作用不是抑制性的,而是对钙蛋白酶-1的保护作用。
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.
Protein degradation in skeletal muscle is a key component of protein turnover and maintenance of muscle function. Protein degradation in postmortem muscle is commonly observed and is associated with the accumulation of degradation products and improved meat tenderness. Because there is significant evidence that calpain-1 is involved with proteolysis of muscle proteins in both situations, defining the factors that regulate calpain activity will position scientists to improve calpain-1 activity in both contexts. Calpain-1 is a neutral calcium-dependent proteinase that is inhibited by calpastatin, oxidation, and slightly acidic pH environments. Because oxidation of the calpain/calpastatin complex with hydrogen peroxide appeared to activate calpain-1, we hypothesize that calpastatin binding to calpain may protect the active site cysteine. In the current study, we tested this hypothesis and investigated how n-ethyl maleimide (NEM), an alkylating agent, affects the regulation of calpain in the presence and absence of calpastatin molecules. The results suggest that calpastatin can protect calpain-1 from reacting with maleimide-polyethylene glycol but that exposure of calpain-1/calpastatin complex to NEM or hydrogen peroxide resulted in autolysis and activation of calpain. Under some circumstances, calpastatin appears to protect calpain-1 from inhibition by modification of active site cysteine. These novel observations show a different role for calpastatin and give reason to interpret calpastatin abundance and activity data in a different light.