Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University;
Faculty of Medicine, Lomonosov Moscow State University.
J Vis Exp. 2022 Oct 13(188). doi: 10.3791/64206.
Apoptosis is a type of programmed cell death that eliminates damaged cells and controls the development and tissue homeostasis of multicellular organisms. Caspases, a family of cysteine proteases, play a key role in apoptosis initiation and execution. The maturation of caspases and their activity is fine-tuned by post-translational modifications in a highly dynamic fashion. To assess the effect of post-translational changes, potential sites are routinely mutated with residues persistent to any modifications. For example, the serine residue is replaced with alanine or aspartic acid. However, such substitutions could alter the caspase active site's conformation, leading to disturbances in catalytic activity and cellular functions. Moreover, mutations of other amino acid residues located in critical positions could also break the structure and functions of caspases and lead to apoptosis perturbation. To avoid the difficulties of employing mutated residues, molecular modeling approaches can be readily applied to estimate the potential effect of amino acid substitutions on caspase structure. The present protocol allows the modeling of both the wild-type caspase and its mutant forms with the biomolecular simulation package (Amber) and supercomputer facilities to test the effect of mutations on the protein structure and function.
细胞凋亡是一种程序性细胞死亡，它可以清除受损细胞，并控制多细胞生物的发育和组织稳态。半胱氨酸蛋白酶家族中的胱天蛋白酶在凋亡的起始和执行中起着关键作用。胱天蛋白酶的成熟及其活性通过高度动态的翻译后修饰进行微调。为了评估翻译后变化的影响，通常将潜在的位点突变为对任何修饰都具有抗性的残基。例如，丝氨酸残基被丙氨酸或天冬氨酸取代。然而，这种取代可能会改变胱天蛋白酶活性位点的构象，导致催化活性和细胞功能紊乱。此外，位于关键位置的其他氨基酸残基的突变也可能破坏胱天蛋白酶的结构和功能，并导致细胞凋亡失调。为了避免使用突变残基的困难，可以应用分子建模方法来估计氨基酸取代对胱天蛋白酶结构的潜在影响。本方案允许使用生物分子模拟包（Amber）和超级计算机设施对野生型胱天蛋白酶及其突变形式进行建模，以测试突变对蛋白质结构和功能的影响。
