Molecular mechanisms of bacterial toxin translocation across membranes

细菌毒素跨膜易位的分子机制

• 批准号: RGPIN-2017-06817
• 负责人: Melnyk, Roman
• 金额: $ 2.04万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=682590
• 关键词: Molecular; mechanisms; bacterial; toxin; translocation

Protein translocases are ubiquitous structures in nature that enable transport of proteins across biological membranes. These membrane-embedded structures are critical for a number of important biological processes in eukaryotic cells including protein secretion, membrane protein integration into membranes, and organelle biosynthesis. Certain microbial pathogens secrete protein toxins, which naturally enter target cells where they cause damage to the host. Toxins such as diphtheria, botulinum, tetanus and the large Clostridial toxins possess a protein translocase domain, which inserts itself into the membranes of host cells to create membrane-spanning pores. These pores are thought to facilitate translocation of an associated toxic enzyme into the cytosol of the host cell, resulting in host cell death. In my laboratory, we are interested in understanding the process of protein translocation across membranes using bacterial toxins as model systems. Bacterial toxins are ideal systems to study this complex process as they: (1) possess all of the necessary elements for translocation within a single protein, (2) are highly efficient translocases; and (3) can be produced in large quantities to facilitate biophysical characterization. *** The objectives of the proposed research program are to elucidate the structural architecture and the mechanism of translocation for Clostridium difficile toxin B (TcdB), a prototypic translocating toxin. Under on-going NSERC Discovery Grant funding (2012-2017) we identified both the pore-forming region, and, unexpectedly, and a receptor-binding region within the translocation domain of TcdB. These findings allowed us to propose a model for the toxin translocase (Zhang et al. PNAS; Chumbler et al. Nature Micro, Hamza et al. Pathog Dis), and the binding site for a newly discovered receptor (Zhang et al. in preparation). Together, these findings have advanced our understanding of the structure and function of a toxin translocase, and at the same time have served to generate new hypotheses, which form the foundation of the current proposal. In the current proposed program of research, we seek to build on these discoveries to understand how the translocase “pore”, is assembled and how it then ushers proteins across membranes. We will employ a series of biochemical and biophysical techniques to address these questions. The proposed work is expected to extend our understanding of TcdB translocation into cells and further help define the principles that underlie protein transport - a ubiquitous process in all living systems that remains poorly characterized.

蛋白质移位酶是自然界中普遍存在的结构，其使得蛋白质能够跨生物膜运输。这些膜包埋结构对于真核细胞中的许多重要生物过程是至关重要的，包括蛋白质分泌、膜蛋白整合到膜中以及细胞器生物合成。某些微生物病原体分泌蛋白质毒素，这些蛋白质毒素自然进入靶细胞，在那里它们对宿主造成损害。白喉、肉毒杆菌、破伤风和大型梭菌毒素等毒素具有蛋白质移位酶结构域，该结构域将自身插入宿主细胞的膜中以产生跨膜孔。这些孔被认为促进相关的毒性酶易位到宿主细胞的胞质溶胶中，导致宿主细胞死亡。在我的实验室，我们有兴趣了解蛋白质跨膜转运的过程中使用细菌毒素作为模型系统。细菌毒素是研究这一复杂过程的理想系统，因为它们：（1）在单个蛋白质内具有易位的所有必要元素，（2）是高效的易位酶;（3）可以大量产生以促进生物物理表征。* 拟议研究计划的目标是阐明艰难梭菌毒素B（TcdB）（一种原型易位毒素）的结构架构和易位机制。在正在进行的NSERC发现资助（2012-2017）下，我们发现了TcdB易位结构域中的孔形成区域和出乎意料的受体结合区域。这些发现使我们能够提出毒素移位酶的模型（Zhang等，PNAS; Chumbler等，Nature Micro，Hamza等，Pathog Dis），以及新发现的受体的结合位点（Zhang等，准备中）。总之，这些发现促进了我们对毒素移位酶的结构和功能的理解，同时也产生了新的假设，这些假设构成了当前提案的基础。在目前提出的研究计划中，我们试图建立在这些发现的基础上，以了解易位酶“孔”是如何组装的，以及它如何引导蛋白质穿过膜。我们将采用一系列的生物化学和生物物理技术来解决这些问题。这项拟议的工作有望扩展我们对TcdB易位到细胞中的理解，并进一步帮助定义蛋白质转运的基本原理-这是所有生命系统中普遍存在的过程，但其特征仍然很差。