MODULATION OF CALPASTATIN-CALPAIN INTERACTIONS BY ETHANOL

PC12细胞暴露在乙醇中会导致钙刺激的蛋白酶活性发生很大变化。由于这些酶是神经递质释放以及细胞毒性和死亡的关键调节器，我们正在探索一种假设，即乙醇介导的神经毒性可能是由于钙激活的蛋白酶失调所致。钙调蛋白是一种钙蛋白酶的抑制剂，是一种酸性的疏水蛋白，它与u-和m-钙蛋白酶的疏水活性部位(S)相互作用。已经描述了一系列钙调蛋白的翻译后修饰，这些修饰改变了与钙蛋白酶的结合亲和力，其中包括PKC介导的磷酸化。利用PC12模型，我们正在研究酒精暴露和戒断对蛋白质-蛋白质相互作用的影响。由于calastatin-calain相互作用的疏水性，我们研究了乙醇可能改变蛋白酶-calastatin(抑制剂)复合体稳定性的可能性。我们发现，PC12细胞暴露在乙醇中会导致钙蛋白酶和含有钙蛋白酶的蛋白质复合体的分子量增加，这与蛋白酶活性的变化有关。我们通过使用免疫细胞化学技术扩展了这些观察。我们开发了一种方法来量化感兴趣的免疫反应蛋白的空间组织变化，并成功地将其应用于荧光免疫组织化学图像。这种方法不仅使我们能够在原位定量信号的大小，而且还可以确定暴露于酒精后空间信号是否发生了变化。我们发现，与Mu-calain和calastatin相关的信号大小会因酒精暴露而改变，但在Mu-calain的情况下，信号的质地也会改变。这一发现表明，在体内，PC12细胞暴露在酒精中可能会通过蛋白质-蛋白质相互作用或翻译后修饰改变Calain表位识别。我们现在正在扩展这些发现，并开发在酒精暴露后立即拍摄感兴趣的蛋白质在活细胞中的时间进程和移位的能力。这将增加我们对酒精调节钙蛋白酶活性的机制的理解。

Exposure of PC12 cells to ethanol results in large changes in calcium-stimulated protease activities. Since these proteases are critical modulators of both neurotransmitter release as well as cellular toxicity and death, we are exploring the hypothesis that ethanol-mediated neural toxicity may result from calcium- activated protease dysregulation. Calpastatin, an inhibitor of calpain, is an acidic, hydrophobic protein which interacts with the hydrophobic active site(s) of mu- and m-calpains. A series of post-translational modifications of calpastatin have been described which alter the binding affinity to calpains, among them, PKC-mediated phosphorylations. Using a PC12 model, we are examining the effects of ethanol exposure and withdrawal on protein-protein interactions. Because of the hydrophobic nature of calpastatin-calpain interactions, we examined the possibility that ethanol might modify protease-calpastatin (inhibitor) complex stability. We found that exposure of PC12 cells to ethanol results in an increase in the molecular weight of calpain and calpastatin-containing protein complexes, and that this is associated with a change in protease activity. We have extended these observations by use of immunocytochemical techniques. We developed a method to quantitate alterations in spatial organization of the immunoreactive proteins of interest and applied it successfully to fluorescent immunohistochemical images. This method allows us to not only quantitate signal magnitude in situ, but also to determine if the spatial signal is altered after exposure to alcohol. We found that signal magnitudes associated with mu-calpain and calpastatin are altered by alcohol exposure, but that in the case of mu-calpain, the texture of the signal is also altered. This finding suggests that in vivo, exposure of PC12 cells to alcohol may alter calpain epitope recognition by either a protein-protein interaction, or by a post-translational modification. We are now in the process of extending these findings, and developing the capability of filming the time course and translocation of the proteins of interest in live cells immediately after exposure to alcohol. This will increase our understanding of the mechanisms involved in the regulation of calpain activity by alcohol.