Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON K7L 3N6, Canada.
Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON K7L 3N6, Canada.
Biochim Biophys Acta Mol Cell Res. 2018 Feb;1865(2):221-230. doi: 10.1016/j.bbamcr.2017.10.013. Epub 2017 Nov 14.
Calpain-1 and -2 are Ca-activated intracellular cysteine proteases that regulate a wide range of cellular functions through the cleavage of their protein substrates. Unlike degradative proteases, calpains make limited, transformative cleavages, typically in accessible sequences linking discrete subdomains, to irreversibly alter substrate functions. The biological roles of calpain and their interplay with calcium signaling are of significant biomedical interest as biomarkers and potential therapeutic targets in a growing number of diseases including Alzheimer's, cancer and fibrosis. Unfortunately, many of the colorimetric and fluorimetric assays that have been developed to study calpain activity suffer from low sensitivity and/or poor calpain specificity. To address the need for a highly sensitive and calpain-specific substrate suitable for in vitro and in vivo calpain activity analysis, we have developed a protein FRET probe. We inserted the optimized calpain cleavage sequence PLFAAR between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) and modulated its flanking sequences for optimal calpain cleavage. We demonstrate greater sensitivity and calpain-specificity of an optimal 16-residue PLFAAR-based FRET substrate compared to a standard α-spectrin-based probe. The 16-residue PLFAAR protein FRET substrate is not significantly cleaved by trypsin, chymotrypsin, cathepsin-L or caspase-3, and is highly sensitive to both calpain-1 and -2. After transfection of the substrate gene into breast cancer cells the PLFAAR protein FRET product was cut in lysed wild-type cells but not in those with a calpain knock-out phenotype. Blockage of substrate cleavage in the lysates by endogenous and exogenous calpastatin was observed, and was overcome by adding extra calpain.
钙蛋白酶-1 和 -2 是 Ca2+ 激活的细胞内半胱氨酸蛋白酶，通过切割其蛋白质底物来调节广泛的细胞功能。与降解性蛋白酶不同，钙蛋白酶进行有限的、转化性的切割，通常在连接离散亚结构域的可及序列中进行，以不可逆地改变底物功能。钙蛋白酶的生物学作用及其与钙信号转导的相互作用作为生物标志物和越来越多疾病（包括阿尔茨海默病、癌症和纤维化）的潜在治疗靶点具有重要的生物医学意义。不幸的是，已经开发出许多用于研究钙蛋白酶活性的比色法和荧光法检测存在灵敏度低和/或钙蛋白酶特异性差的问题。为了解决对高度敏感和钙蛋白酶特异性底物的需求，该底物适用于体外和体内钙蛋白酶活性分析，我们开发了一种蛋白质 FRET 探针。我们在青色荧光蛋白（CFP）和黄色荧光蛋白（YFP）之间插入了优化的钙蛋白酶切割序列 PLFAAR，并调节其侧翼序列以实现最佳钙蛋白酶切割。与标准的α- spectrin 探针相比，我们证明了优化的基于 16 个残基的 PLFAAR 的 FRET 底物具有更高的灵敏度和钙蛋白酶特异性。基于 16 个残基的 PLFAAR 蛋白质 FRET 底物不会被胰蛋白酶、糜蛋白酶、组织蛋白酶-L 或半胱天冬酶-3 显著切割，对钙蛋白酶-1 和 -2 均高度敏感。将该底物基因转染到乳腺癌细胞中后，PLFAAR 蛋白质 FRET 产物在裂解的野生型细胞中被切割，但在钙蛋白酶敲除表型的细胞中没有被切割。观察到内源性和外源性钙蛋白酶抑制素对裂解物中底物切割的阻断作用，并通过添加额外的钙蛋白酶克服了这种阻断作用。
