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Calpains: Proteolytic switches thrown by calcium and short-circuited by an auto-inactivation mechanism.

机译：钙蛋白酶：钙引起的蛋白水解开关和自动灭活机制引起的短路。

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摘要

Calpains are Ca2+-dependent cysteine proteases found in organisms ranging from bacteria to humans. With their endogenous inhibitors they drive and fine-tune Ca2+ signaling pathways through limited, specific proteolysis that modifies the activity of their targets. Calpains regulate processes as common as cell migration and as exclusive as sex determination in nematodes. But how do they respond to Ca2+ signaling when the known Ca2+-binding domains (C2-like and EF-hand) are not even present in all calpain isoforms? The breakthrough in this thesis was the observation that the protease core of calpain (domains I--II) also binds Ca2+. Bacterial expression and biochemical analysis of the cores from both mu- and m-calpain, showed that in isolation they are Ca2+-dependent proteases (minicalpains) with isoform-specific properties reminiscent of their full-length isoform. The protease core is the common element that defines the superfamily. The 2.07A-resolution structure of Ca2+-bound muI--II revealed the conserved Ca2+ switch that aligns the active site. This switch involves cooperative binding of Ca2+ at two non-EF-hand sites, one in each domain, to reposition several loop structures and induce papain-like active site assembly. A mechanism was proposed and tested using mutagenesis and domain swaps. It supported the order and cooperativity of Ca2+ binding, and suggested a hierarchical functional importance for the sequential steps in the mechanism. Accordingly, domain II site assembly is rate-determining, followed in importance by the salt-bridge between sites that provides the basis for cooperativity. Surprisingly, domain I site is more peripheral to activation than originally thought. The 1.95A-resolution structure of mI--II revealed an intrinsic mechanism of silencing of this and similar mini-calpains. A strategically positioned glycine collapses a key alpha-helix leading to rearrangement of the hydrophobic core whereby the conserved tryptophan next to the active site cysteine swings out into the active site cleft and prevents substrate binding. The resistance of non-glycine-containing muI--II to calpastatin inhibition reinforces the patho-physiological consequences of these structure-based mechanisms on calpain signaling. This suggests that active cores produced by autolysis could induce tissue damage during ischemia and neurodegeneration. Minicalpains are, therefore, useful targets for drug screening and design, as shown by the crystal structure of the muI--II-E64 inhibitor complex.
机译：钙蛋白酶是在从细菌到人类的生物体中发现的依赖Ca 2+的半胱氨酸蛋白酶。借助其内源性抑制剂，它们通过有限的特异性蛋白水解来驱动和微调Ca2 +信号传导途径，从而改变其靶标的活性。钙蛋白酶调节与线虫一样普遍的细胞迁移过程和对性别的决定过程。但是，当已知的Ca2 +结合域（C2样和EF-hand）甚至不存在于所有钙蛋白酶异构体中时，它们如何响应Ca2 +信号传导？本论文的突破是观察到钙蛋白酶的蛋白酶核心（域I-II）也与Ca2 +结合。对来自mu-calpain和m-calpain的核心的细菌表达和生化分析表明，它们分别为Ca2 +依赖性蛋白酶（minicalpains），具有异构体特异性特性，使人联想到其全长异构体。蛋白酶核心是定义超家族的常见元素。 Ca2 +结合的muI--II的2.07A分辨率结构揭示了与活性位点对齐的保守Ca2 +开关。此开关涉及在两个非EF手部位（每个结构域一个）中Ca2 +的协同结合，以重新定位多个环结构并诱导木瓜蛋白酶样活性部位装配。提出了一种机制，并使用诱变和域交换进行了测试。它支持Ca2 +结合的顺序和协同作用，并建议该机制中的后续步骤具有分级的功能重要性。因此，域II位点的组装是决定速率的，重要的是位点之间的盐桥为协作提供了基础。出乎意料的是，域I站点比最初想象的更受激活的影响。 mI-II的1.95A分辨率结构揭示了这种和类似迷你钙蛋白酶沉默的内在机制。有策略地定位的甘氨酸会使关键的α-螺旋塌陷，从而导致疏水性核的重排，从而使靠近活性位点半胱氨酸的保守色氨酸向外摆动进入活性位点裂隙并阻止底物结合。非甘氨酸的muI-II对钙蛋白酶抑制的抗性增强了这些基于钙蛋白酶信号的基于结构的机制的病理生理后果。这表明自溶产生的活性核心可能在缺血和神经变性期间诱导组织损伤。因此，如muI-II-E64抑制剂复合物的晶体结构所示，米痛是药物筛选和设计的有用靶标。

著录项

  • 作者

    Moldoveanu, Tudor.;

  • 作者单位

    Queen's University (Canada).;

  • 授予单位 Queen's University (Canada).;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2003
  • 页码 223 p.
  • 总页数 223
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
  • 关键词

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