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效应蛋白酶的重要性。尽管我们无法找到与细胞死亡相关的底物，但我们的研究强烈暗示，靶细胞死亡是由caspase对DNA和线粒体造成的非依赖性损伤所定义的。这项初步研究为GzmH作为一种重要的先天免疫蛋白水解酶奠定了基础。这项研究还为寻找促凋亡底物奠定了基础，该底物控制着由这种类糜蛋白酶引发的细胞死亡途径，这也是该项目的目标。为了实现我们的目标，我们打算使用最先进的方法。首先，通过使用由已知的人类胞浆蛋白的连接区组成的蛋白酶底物文库，我们将试图更好地破译GzmH的切割特异性，以及它的细胞毒作用，通过寻找被GzmH切割的多肽，这些多肽来自促凋亡和抗凋亡蛋白。另外一些方法，如“蛋白组学鉴定蛋白酶裂解特异性”或“CLIP-PICS”，使用蛋白质组广泛的多肽库作为蛋白分解底物筛选，用于表征蛋白酶活性部位的特异性。这项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(N)末端多肽。回收的N端肽可以通过测序和各种质谱学方法进行鉴定。其次，对GzmH相关底物的寻找促使我们尝试对GzmH进行结构分析。我们的重组、非糖基化和未标记的GzmH是结晶的完美候选者，这不仅是因为我们能够从大肠杆菌中生产大量基于包涵体的蛋白质，还因为纯重组GzmH缺乏促进结晶的N-连接碳水化合物。活性GzmH的三维晶体结构将有助于更好地了解该酶的整体结构及其底物专一性，也有助于开发高专一性的抑制剂。事实上，由于所选择的动物模型不具备与人类GzmH基因相同的功能，因此用基因敲除方法对GzmH进行功能研究是不可能的。随着我们更多地了解GzmH的特异性，量身定制的GzmH抑制剂的设计将极大地提高我们对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.