Molecular mechanisms of bacterial toxin translocation across membranes

蛋白质转座是自然界中普遍存在的结构，它使蛋白质能够跨生物膜运输。这些膜嵌入结构对真核细胞中许多重要的生物过程至关重要，包括蛋白质分泌、膜蛋白整合到膜和细胞器生物合成。某些微生物病原体会分泌蛋白质毒素，这些毒素会自然进入目标细胞，对宿主造成损害。白喉、肉毒杆菌、破伤风和大型梭状菌毒素等毒素具有一种蛋白质转位酶结构域，该结构域将自身插入宿主细胞的膜中，形成跨膜孔。这些孔被认为是促进相关的有毒酶易位到宿主细胞的细胞质中，导致宿主细胞死亡。在我的实验室里，我们感兴趣的是利用细菌毒素作为模型系统来理解蛋白质跨膜转运的过程。细菌毒素是研究这一复杂过程的理想系统，因为它们：(1)拥有单个蛋白质内易位的所有必要元素；(2)是高效的易位酶；(3)可大量生产，便于生物物理表征。***本研究旨在阐明艰难梭菌毒素B （Clostridium difficile toxin B, TcdB）的结构结构及其易位机制。在正在进行的NSERC发现基金资助下（2012-2017），我们在TcdB的易位域中发现了孔隙形成区域，出乎意料的是，还有一个受体结合区域。这些发现使我们能够提出毒素转位酶的模型（Zhang等人）。《美国国家科学院学报》上发表;Chumbler等人。Nature Micro， Hamza等。病原菌)，以及新发现受体的结合位点（Zhang et al. in preparation）。总之，这些发现提高了我们对毒素转位点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建议的基础。在目前提出的研究计划中，我们寻求建立在这些发现的基础上，以了解转位酶“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化和生物物理技术来解决这些问题。这项工作有望扩展我们对TcdB转运到细胞中的理解，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，但仍然缺乏特征。

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.