Melloni Edon, Averna Monica, Stifanese Roberto, De Tullio Roberta, Defranchi Enrico, Salamino Franca, Pontremoli Sandro
Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV, 1, 16132 Genova, Italy.
J Biol Chem. 2006 Aug 25;281(34):24945-54. doi: 10.1074/jbc.M601449200. Epub 2006 Jun 27.
It is generally accepted that the Ca(2+)-dependent interaction of calpain with calpastatin is the most relevant mechanism involved in the regulation of Ca(2+)-induced proteolysis. We now report that a calpain-calpastatin association can occur also in the absence of Ca(2+) or at very low Ca(2+) concentrations, reflecting the physiological conditions under which calpain retains its inactive conformational state. The calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4-7. This calpastatin region recognizes a calpain sequence located near the end of the DII-domain. Interaction of calpain with calpastatins lacking these sequences becomes strictly Ca(2+)-dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca(2+)-free calpain and various recombinant calpastatin forms has been demonstrated by the following experimental results. Addition of calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when calpastatin was immunoprecipitated. The calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-calpastatin interaction was perturbed by protein kinase C phosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-calpastatin interaction at a physiological concentration of Ca(2+) represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca(2+) influx.
一般认为，钙蛋白酶与钙蛋白酶抑制蛋白之间依赖钙离子的相互作用是参与钙离子诱导的蛋白水解调节的最相关机制。我们现在报告，在没有钙离子或极低钙离子浓度的情况下，钙蛋白酶与钙蛋白酶抑制蛋白也会发生结合，这反映了钙蛋白酶保持其无活性构象状态的生理条件。钙蛋白酶抑制蛋白的结合区域位于非抑制性L结构域，该结构域包含由外显子4 - 7编码的氨基酸序列。该钙蛋白酶抑制蛋白区域识别位于DII结构域末端附近的钙蛋白酶序列。钙蛋白酶与缺乏这些序列的钙蛋白酶抑制蛋白的相互作用变得严格依赖钙离子，因为在这些条件下，蛋白酶转变为活性状态是一个必要条件。以下实验结果证明了无钙离子的钙蛋白酶与各种重组钙蛋白酶抑制蛋白形式之间分子结合的发生。添加钙蛋白酶抑制蛋白可保护钙蛋白酶免受胰蛋白酶消化。当免疫沉淀钙蛋白酶抑制蛋白时，钙蛋白酶会共沉淀。暴露于钙蛋白酶后，钙蛋白酶抑制蛋白的分子大小增加。在酶谱分析中，这两种蛋白质一起迁移。此外，位于参与结合的外显子处的蛋白激酶C磷酸化会干扰钙蛋白酶 - 钙蛋白酶抑制蛋白的相互作用。在功能水平上，在生理浓度的钙离子条件下，钙蛋白酶 - 钙蛋白酶抑制蛋白的相互作用代表了一种新的机制，用于控制可能因细胞内钙离子内流而产生的蛋白酶活性形式的量。
