Kanazawa Y, Makino M, Morishima Y, Yamada K, Nabeshima T, Shirasaki Y
Biological Research Laboratories 1, Daiichi Sankyo Co., Ltd., Tokyo, Japan.
Neuroscience. 2008 Jun 23;154(2):473-81. doi: 10.1016/j.neuroscience.2008.03.044. Epub 2008 Mar 26.
Excessive elevation of intracellular Ca2+ levels and, subsequently, hyperactivation of Ca2+/calmodulin-dependent processes might play an important role in the pathologic events following cerebral ischemia. PEP-19 is a neuronally expressed polypeptide that acts as an endogenous negative regulator of calmodulin by inhibiting the association of calmodulin with enzymes and other proteins. The aims of the present study were to investigate the effect of PEP-19 overexpression on cell death triggered by Ca2+ overload and how the polypeptide levels are affected by glutamate-induced excitotoxicity and cerebral ischemia. Expression of PEP-19 in HEK293T cells suppressed calmodulin-dependent signaling and protected against cell death elicited by Ca2+ ionophore. Likewise, primary cortical neurons overexpressing PEP-19 became resistant to glutamate-induced cell death. In immunoprecipitation assay, wild type PEP-19 associated with calmodulin, whereas mutated PEP-19, which contains mutations within the calmodulin binding site of PEP-19, failed to associate with calmodulin. We found that the mutation abrogates both the ability to suppress calmodulin-dependent signaling and to protect cells from death. Additionally, the endogenous PEP-19 levels in neurons were significantly reduced following glutamate exposure, this reduction precedes neuronal cell death and can be blocked by treatment with calpain inhibitors. These data suggest that PEP-19 is a substrate for calpain, and that the decreased PEP-19 levels result from its degradation by calpain. A similar reduction of PEP-19 also occurred in the hippocampus of gerbils subjected to transient global ischemia. In contrast to the reduction in PEP-19, no changes in calmodulin occurred following excitotoxicity, suggesting the loss of negative regulation of calmodulin by PEP-19. Taken together, these results provide evidence that PEP-19 overexpression enhances resistance to Ca2+-mediated cytotoxicity, which might be mediated through calmodulin inhibition, and also raises the possibility that PEP-19 degradation by calpain might produce an aberrant activation of calmodulin functions, which in turn causes neuronal cell death.
细胞内钙离子水平的过度升高以及随后钙调蛋白依赖性过程的过度激活，可能在脑缺血后的病理事件中起重要作用。PEP-19是一种在神经元中表达的多肽，通过抑制钙调蛋白与酶及其他蛋白质的结合，作为钙调蛋白的内源性负调节剂。本研究的目的是探讨PEP-19过表达对钙超载引发的细胞死亡的影响，以及该多肽水平如何受到谷氨酸诱导的兴奋性毒性和脑缺血的影响。PEP-19在HEK293T细胞中的表达抑制了钙调蛋白依赖性信号传导，并保护细胞免受钙离子载体引发的细胞死亡。同样，过表达PEP-19的原代皮质神经元对谷氨酸诱导的细胞死亡具有抗性。在免疫沉淀试验中，野生型PEP-19与钙调蛋白结合，而在PEP-19的钙调蛋白结合位点内含有突变的突变型PEP-19未能与钙调蛋白结合。我们发现该突变消除了抑制钙调蛋白依赖性信号传导和保护细胞免于死亡的能力。此外，谷氨酸暴露后神经元内源性PEP-19水平显著降低，这种降低先于神经元细胞死亡，并且可以被钙蛋白酶抑制剂治疗所阻断。这些数据表明PEP-19是钙蛋白酶的底物，并且PEP-19水平的降低是由于其被钙蛋白酶降解所致。在经历短暂全脑缺血的沙鼠海马中也发生了类似的PEP-19降低。与PEP-19降低相反，兴奋性毒性后钙调蛋白没有变化，这表明PEP-19对钙调蛋白的负调节作用丧失。综上所述，这些结果提供了证据，即PEP-19过表达增强了对钙离子介导的细胞毒性的抗性，这可能是通过抑制钙调蛋白介导的，并且还增加了钙蛋白酶降解PEP-19可能导致钙调蛋白功能异常激活的可能性，进而导致神经元细胞死亡。
