Spungin Dina, Berman-Frank Ilana
Department of Marine Biology, Leon H. Charney School of Marine Sciences, University of Haifa, Haifa, Israel.
Bio Protoc. 2019 Aug 20;9(16):e3341. doi: 10.21769/BioProtoc.3341.
Programmed cell death (PCD) is an irreversible, genetically-controlled form of cell suicide in which an endogenous biochemical pathway leads to morphological changes and ultimately, cellular demise. PCD is accompanied by protein synthesis of a family of proteases-"caspases" that are often used as a diagnostic marker of PCD. Although phytoplankton do not contain true caspases, caspase-like activity (hypothetical proteins with analogous activity) has been traditionally used as a diagnostic marker of PCD in marine phytoplankton. Increased caspase-like proteolytic activity was demonstrated when synthetic fluorogenic activity substrates specific for caspases (with an Asp at the P1 position) were applied upon PCD induction. Metacaspases, cysteine proteases, share structural properties with those of caspases, yet they are highly specific for Arg and Lys cleavage site at the P1 position implying that caspase specific substrates are not indicative of metacaspase catalytic activity. This method specifically tests direct metacaspase activity in phytoplankton by the cleavage of the fluorogenic metacaspase substrate Ac-VRPR-AMC. Metacaspase activity was tested by the addition of a metacaspase specific peptide that is conjugated to the fluorescent reporter molecule. The cleavage of the peptide by the metacaspase releases the fluorochrome that, when excited by light, emits fluorescence. The level of metacaspase enzymatic activity in the cell lysate is directly proportional to the fluorescence signal detected. The use of specific standards in this test enables the quantification of the fluorescence results. This assay directly allows monitoring the metacaspase cleavage products and thereby tracing evidence for programmed cell death.
程序性细胞死亡（PCD）是一种不可逆的、由基因控制的细胞自杀形式，其中内源性生化途径会导致形态变化并最终导致细胞死亡。PCD伴随着一类蛋白酶——“半胱天冬酶”的蛋白质合成，这些蛋白酶常被用作PCD的诊断标志物。尽管浮游植物不含真正的半胱天冬酶，但半胱天冬酶样活性（具有类似活性的假设蛋白质）传统上一直被用作海洋浮游植物中PCD的诊断标志物。当在PCD诱导时应用对半胱天冬酶具有特异性的合成荧光活性底物（P1位置为天冬氨酸）时，可证明半胱天冬酶样蛋白水解活性增加。金属半胱天冬酶，即半胱氨酸蛋白酶，与半胱天冬酶具有共同的结构特性，但它们对P1位置的精氨酸和赖氨酸切割位点具有高度特异性，这意味着半胱天冬酶特异性底物并不能指示金属半胱天冬酶的催化活性。该方法通过荧光金属半胱天冬酶底物Ac-VRPR-AMC的切割来特异性检测浮游植物中的直接金属半胱天冬酶活性。通过添加与荧光报告分子偶联的金属半胱天冬酶特异性肽来测试金属半胱天冬酶活性。金属半胱天冬酶对肽的切割会释放荧光染料，当被光激发时会发出荧光。细胞裂解物中金属半胱天冬酶的酶活性水平与检测到的荧光信号成正比。在该测试中使用特定标准能够对荧光结果进行定量。该测定法直接允许监测金属半胱天冬酶的切割产物，从而追踪程序性细胞死亡的证据。
