Caspase-independent mechanisms of GzmH-induced target cell death

GzmH 诱导靶细胞死亡的非 Caspase 机制

• 批准号: 76858740
• 负责人: Dr. Edward Fellows
• 金额: --
• 依托单位: Institut für Klinische Neuroimmunologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2008
• 资助国家: 德国
• 起止时间: 2007-12-31 至 2010-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/76858740?language=en
• 关键词: Caspase; independent; mechanisms; GzmH; induced

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.

最近，我们的研究结果表明，重组GzmH作为一种新的细胞毒性效应蛋白酶的重要性。虽然我们无法找到细胞死亡相关的底物，但我们的研究强烈提示靶细胞死亡由对DNA和线粒体造成的半胱天冬酶非依赖性损伤定义。这项初步研究为GzmH作为先天免疫中的重要蛋白酶奠定了基础。该研究还为研究促凋亡底物奠定了基础，该底物控制由这种糜蛋白酶样蛋白酶触发的细胞死亡途径，这是该项目的目标。为了实现我们的目标，我们打算使用最先进的方法。首先，使用蛋白酶底物文库，包括已知的人类胞质蛋白的接头区域的选择，我们将试图更好地破译GzmH的切割特异性，而且其细胞毒性作用，通过搜索被GzmH切割的肽和衍生自促凋亡和抗凋亡蛋白。另外的方法如“蛋白酶切割特异性的蛋白质组范围鉴定”或“CLIP-PICS”采用蛋白质组范围肽文库作为蛋白水解底物筛选，用于表征蛋白酶活性位点特异性。该技术应允许我们选择性地分离新氨基（N）-末端肽，产生后的GzmH处理，从靶细胞裂解物。然后可以通过测序和各种质谱方法鉴定回收的N-末端肽。其次，GzmH相关底物的寻找促使我们尝试GzmH的结构分析。我们的重组、非糖基化和未标记的GzmH是结晶的理想候选物，不仅因为我们能够从大肠杆菌中产生大量基于包涵体的蛋白。大肠杆菌中，但也因为纯的重组GzmH缺乏N-连接的碳水化合物，这有利于结晶。活性GzmH的三维晶体结构将是一个有用的工具，与它获得更好地了解蛋白酶的整体结构和其底物特异性，但也为发展高度特异性抑制剂。事实上，由于所选择的动物模型不具有与人GzmH基因的功能等同物，因此不可能通过敲除方法对GzmH进行功能研究。随着我们对GzmH特异性的了解越来越多，定制GzmH抑制剂的设计应该会大大提高我们对GzmH细胞毒性的理解。