Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis Using Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array.
Predictions of Cleavability of Calpain Proteolysis by Quantitative Structure-Activity Relationship Analysis Using Newly Determined Cleavage Sites and Catalytic Efficiencies of an Oligopeptide Array.
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DOI：
10.1074/mcp.m115.053413
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发表时间：
2016-04
期刊：
影响因子：
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通讯作者：
Sorimachi H
中科院分区：
文献类型：
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作者：
Shinkai-Ouchi F;Koyama S;Ono Y;Hata S;Ojima K;Shindo M;duVerle D;Ueno M;Kitamura F;Doi N;Takigawa I;Mamitsuka H;Sorimachi H
钙蛋白酶是细胞内受钙离子调节的半胱氨酸蛋白酶，对多种细胞功能是必不可少的。哺乳动物传统的钙蛋白酶(calain-1和calain-2)通过有限的蛋白分解来调节其底物的结构和功能。因此，确定钙调蛋白在蛋白质中的位置(S)是至关重要的。然而，钙调蛋白的底物特异性仍不清楚，因为其裂解位点周围的氨基酸(AA)序列非常多样化。为了阐明Calpains的底物特异性，用Calpains对84个20聚体寡肽进行了蛋白降解，这些寡肽对应于已报道的裂解位点序列的P10-P10‘，并通过LC/MS测定了催化效率(kcat/Km)。虽然大多数位点都被−-1和−-2以相似的kcat/kM切割，但序列比较显示在P9-P7/P2/P5‘有明显的AA偏好。新位点的氨基酸组成与以前报道的氨基酸组成总体上没有统计学上的不同，这表明Calpain对序列特异性有严格的隐含规则，而完整底物的有限蛋白质分解是由于底物的高阶结构。裂解位置频率表明，与C末端(P‘位点)相比，N端至裂解位(P’位)的较长序列更有利于蛋白质的降解。用偏最小二乘回归和~gt；1,300个氨基酸描述符进行定量构效关系分析，得到了kcat/kM的预测值，相关系数r=0.834，与我们的模型构建无关，二元构效关系模型对132个已报道的calain裂解位点的预测正确率为87.5%。这些结果比以前的calain切割预测指标更好，并揭示了P2、P3‘和P4’位点以及P1-P2协同作用的重要性。此外，使用我们的二元定量构效关系模型，在肌红蛋白中识别了新的切割位点，验证了我们的预测。这项研究增加了我们对钙蛋白酶底物特异性的理解，并为“下一代”，即与活动相关的定量和协作性相关的分析打开了钙蛋白酶的大门。
Calpains are intracellular Ca2+-regulated cysteine proteases that are essential for various cellular functions. Mammalian conventional calpains (calpain-1 and calpain-2) modulate the structure and function of their substrates by limited proteolysis. Thus, it is critically important to determine the site(s) in proteins at which calpains cleave. However, the calpains' substrate specificity remains unclear, because the amino acid (aa) sequences around their cleavage sites are very diverse. To clarify calpains' substrate specificities, 84 20-mer oligopeptides, corresponding to P10-P10′ of reported cleavage site sequences, were proteolyzed by calpains, and the catalytic efficiencies (kcat/Km) were globally determined by LC/MS. This analysis revealed 483 cleavage site sequences, including 360 novel ones. The kcat/Kms for 119 sites ranged from 12.5–1,710 M−1s−1. Although most sites were cleaved by both calpain-1 and −2 with a similar kcat/Km, sequence comparisons revealed distinct aa preferences at P9-P7/P2/P5′. The aa compositions of the novel sites were not statistically different from those of previously reported sites as a whole, suggesting calpains have a strict implicit rule for sequence specificity, and that the limited proteolysis of intact substrates is because of substrates' higher-order structures. Cleavage position frequencies indicated that longer sequences N-terminal to the cleavage site (P-sites) were preferred for proteolysis over C-terminal (P′-sites). Quantitative structure-activity relationship (QSAR) analyses using partial least-squares regression and >1,300 aa descriptors achieved kcat/Km prediction with r = 0.834, and binary-QSAR modeling attained an 87.5% positive prediction value for 132 reported calpain cleavage sites independent of our model construction. These results outperformed previous calpain cleavage predictors, and revealed the importance of the P2, P3′, and P4′ sites, and P1-P2 cooperativity. Furthermore, using our binary-QSAR model, novel cleavage sites in myoglobin were identified, verifying our predictor. This study increases our understanding of calpain substrate specificities, and opens calpains to “next-generation,” i.e. activity-related quantitative and cooperativity-dependent analyses.