Del Carmen Lafita-Navarro Maria, Conacci-Sorrell Maralice
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Methods Mol Biol. 2019;1915:149-160. doi: 10.1007/978-1-4939-8988-1_12.
As opposed to proteasome-mediated proteolysis that leads to protein degradation, calpain proteases carry out limited proteolytic cleavages of their substrates. The cleavage of some substrates can produce active fragments that perform functions that are different from those performed by the full-length proteins. Therefore, cleavage by calpains can operate as a posttranslational modification and increase the functional diversity of target proteins. Nevertheless, activation of protein function by calpain cleavage is still an understudied area in molecular biology. Identifying and functionally characterizing by products generated by calpain cleavage could lead to the discovery of biomarkers and the identification of novel drug targets for the treatment of human diseases. This chapter contains a workflow designed to experimentally characterize novel calpain substrates, including identification of potential calpain targets via Western blotting, characterization of calpain cleavage sites, and the study of cellular functions played by such cleaved products. We will employ MYC as an example for these experiments.
与导致蛋白质降解的蛋白酶体介导的蛋白水解相反，钙蛋白酶对其底物进行有限的蛋白水解切割。一些底物的切割可以产生活性片段，这些片段执行的功能不同于全长蛋白质所执行的功能。因此，钙蛋白酶的切割可以作为一种翻译后修饰，并增加靶蛋白的功能多样性。然而，钙蛋白酶切割对蛋白质功能的激活在分子生物学中仍然是一个研究不足的领域。鉴定钙蛋白酶切割产生的产物并对其进行功能表征，可能会导致生物标志物的发现以及用于治疗人类疾病的新型药物靶点的鉴定。本章包含一个旨在通过实验表征新型钙蛋白酶底物的工作流程，包括通过蛋白质印迹法鉴定潜在的钙蛋白酶靶点、表征钙蛋白酶切割位点，以及研究此类切割产物所发挥的细胞功能。我们将以MYC为例进行这些实验。
