Department of Chemistry, University of Illinois at Chicago, 845 W. Taylor Street, MC 111, Chicago, IL 60607, USA.
Brain Res. 2013 Oct 2;1533:1-15. doi: 10.1016/j.brainres.2013.08.010. Epub 2013 Aug 12.
Under various physiological and patho-physiological conditions, spectrin breakdown reactions generate several spectrin breakdown products (SBDPs)-in particular SBDPs of 150 kDa (SBDP150) and 120 kDa (SBDP120). Recently, numerous studies have shown that reactions leading to SBDPs are physiologically relevant, well regulated, and complex. Yet molecular studies on the mechanism of the SBDP formation are comparatively scarce. We have designed basic systems to allow us to follow the breakdown of αII-spectrin model proteins by caspase-3 in detail with gel electrophoresis, fluorescence and mass spectrometry methods. Amongst the predicted and reported sites, our results show that caspase-3 cleaves after residues D1185 and D1478, but not after residues D888, D1340 and D1475. We also found that the cleavage at these two sites is independent of each other. It may be possible to inhibit one site without affecting the other site. Cleavage after residue D1185 in intact αII-spectrin leads to SBDP150, and cleavage after D1478 site leads to SBDP120. Our results also show that the cleavage after the D1185 residue is unusually efficient, with a kcat/KM value of 40,000 M(-1) s(-1), and the cleavage after the D1478 site is more similar to most of the other reported caspase-3 substrates, with a kcat/KM value of 3000 M(-1) s(-1). We believe that this study lays out a methodology and foundation to study caspase-3 catalyzed spectrin breakdown to provide quantitative information. Molecular understanding may lead to better understanding of brain injuries and more precise and specific biomarker development.
在各种生理和病理生理条件下，血影蛋白断裂反应会产生几种血影蛋白断裂产物（SBDP）-特别是 150kDa（SBDP150）和 120kDa（SBDP120）的 SBDP。最近，大量研究表明，导致 SBDP 的反应在生理上是相关的、受到良好调节的且复杂的。然而，关于 SBDP 形成机制的分子研究相对较少。我们设计了基本系统，使我们能够通过凝胶电泳、荧光和质谱方法详细研究 caspase-3 导致αII-血影蛋白模型蛋白断裂的机制。在预测和报道的位点中，我们的结果表明 caspase-3 在残基 D1185 和 D1478 后切割，但不在残基 D888、D1340 和 D1475 后切割。我们还发现这两个位点的切割彼此独立。有可能抑制一个位点而不影响另一个位点。在完整的αII-血影蛋白中，残基 D1185 后的切割导致 SBDP150，而残基 D1478 后的切割导致 SBDP120。我们的结果还表明，残基 D1185 后的切割异常有效，kcat/KM 值为 40000M(-1)s(-1)，而残基 D1478 后的切割更类似于大多数其他报道的 caspase-3 底物，kcat/KM 值为 3000M(-1)s(-1)。我们相信，这项研究为研究 caspase-3 催化的血影蛋白断裂提供了一种方法和基础，可以提供定量信息。分子理解可能导致更好地理解脑损伤，并开发更精确和更特异的生物标志物。
