Caspase-independent mechanisms of GzmH-induced target cell death

最近，我们的发现证明了重组GzmH作为一种新的细胞毒性效应蛋白酶的重要性。虽然我们无法找到与细胞死亡相关的底物，但我们的研究强烈提示靶细胞死亡是由DNA和线粒体受到半胱天冬酶非依赖性损伤所定义的。这项初步研究为GzmH作为先天免疫中重要的蛋白酶奠定了基础。该研究还为寻找控制这种凝乳胰蛋白酶样蛋白酶引发的细胞死亡途径的促凋亡底物奠定了基础，这是该项目的目标。为了达到我们的目的，我们打算采用最先进的方法。首先，利用蛋白酶底物文库，包括已知的人类细胞质蛋白的连接区域，我们将试图更好地破译GzmH的切割特异性；通过寻找由促凋亡和抗凋亡蛋白衍生的GzmH切割的肽，研究其细胞毒性作用。其他方法，如“蛋白质组范围内的蛋白酶裂解特异性鉴定”或“CLIP-PICS”，使用蛋白质组范围内的肽库作为蛋白酶活性位点特异性表征的蛋白水解底物筛选。该技术应该允许我们有选择地从靶细胞裂解物中分离GzmH处理后产生的新氨基末端肽。回收的n端肽可以通过测序和各种质谱方法进行鉴定。其次，寻找GzmH相关底物促使我们尝试对GzmH进行结构分析。我们的重组，非糖基化和未标记的GzmH是结晶的完美候选物，不仅因为我们能够从大肠杆菌中生产大量基于包体体的蛋白质，而且因为纯重组GzmH缺乏有利于结晶的n链碳水化合物。活性GzmH的三维晶体结构将是一个有用的工具，它可以更好地了解蛋白酶的整体结构和底物特异性，也可以开发高度特异性的抑制剂。事实上，由于选择的动物模型不具有与人类GzmH基因等效的功能，因此通过敲除方法对GzmH进行功能研究是不可能的。随着我们对GzmH特异性的了解越来越多，量身定制的GzmH抑制剂的设计将大大提高我们对GzmH细胞毒性的理解。

Recently, our findings demonstrated the importance of recombinant GzmH as a novel cytotoxic effector protease. Although we were unable to find a cell death related substrate, our study was strongly suggestive of target cell death defined by caspase independent damage inflicted upon DNA and mitochondria. This initial study sets the scene for GzmH as an important protease in innate immunity. The study also lays down the foundations for the search into the pro-apoptotic substrates that govern the cell death pathway triggered by this chymotrypsin-like protease, the goal of this project. To achieve our aim, we intent to use state of the art methods. Firstly, with the use of protease substrate libraries, comprised of a selection of linker regions of known human cytosolic proteins, we will attempt to better decipher the cleavage specificity of GzmH; but also its cytotoxic action, by searching for peptides that are cleaved by GzmH and which are derived from pro- and anti-apoptotic proteins. Additional methods such as “proteome-wide identification of protease cleavage specificity” or “CLIP-PICS” employ proteome-wide peptide libraries as proteolytic substrate screens for the characterization of protease active site specificity. The technique should allow us to selectively isolate neo amino (N)-terminal peptides, generated following treatment with GzmH, from target cell lysates. The recovered N-terminal peptides can then be identified by sequencing and various mass spectrometric methods. Secondly, the search for GzmH related substrates has prompted us to attempt the structural analysis of GzmH. Our recombinant, non glycosylated and untagged GzmH is a perfect candidate for crystallization, not only because we are able to produce high quantities of inclusion body based protein from E. coli, but also because pure recombinant GzmH lacks N-linked carbohydrates which facilitates crystallization. The three-dimensional crystal structure of active GzmH would be a helpful tool with which to gain a better understanding of the protease’s overall structure and its substrate specificity but also for the development highly specific inhibitors. Indeed, since the animal models of choice do not possess a functional equivalent to the human GzmH gene, functional studies on GzmH by knock-out methodology are not possible. As we learn more about the specificity of GzmH, the design of tailored GzmH inhibitors should dramatically improve our understanding of GzmH cytotoxicity.