Fischer Audrey, Montal Mauricio
Section of Neurobiology, Division of Biological Sciences, University of California, San Diego, La Jolla, California 92093-0366, USA.
J Biol Chem. 2007 Oct 5;282(40):29604-11. doi: 10.1074/jbc.M703619200. Epub 2007 Jul 31.
Clostridial botulinum neurotoxins (BoNTs) exert their neuroparalytic action by arresting synaptic exocytosis. Intoxication requires the disulfide-linked, di-chain protein to undergo conformational changes in response to pH and redox gradients across the endosomal membrane with consequent formation of a protein-conducting channel by the heavy chain (HC) that translocates the light chain (LC) protease into the cytosol. Here, we investigate the role of the disulfide bridge in the dynamics of protein translocation. We utilize a single channel/single molecule assay to characterize in real time the BoNT channel and chaperone activities in Neuro 2A cells under conditions that emulate those prevalent across endosomes. We show that the disulfide bridge must remain intact throughout LC translocation; premature reduction of the disulfide bridge after channel formation arrests translocation. The disulfide bridge must be on the trans compartment to achieve productive translocation of LC; disulfide disruption on the cis compartment or within the bilayer during translocation aborts it. We demonstrate that a peptide linkage between LC and HC in place of a disulfide bridge is insufficient for productive LC translocation. The disulfide linkage, therefore, dictates the outcome of translocation: productive passage of cargo or abortive channel occlusion by cargo. Based on these and previous findings we suggest a sequence of events for BoNT LC translocation to be HC insertion, coupled LC unfolding, and protein conduction through the HC channel in an N to C terminus orientation and ultimate release of the LC from the HC by reduction of the disulfide bridge concomitant with LC refolding in the cytosol.
肉毒梭菌神经毒素（BoNTs）通过阻止突触囊泡外排发挥其神经麻痹作用。中毒需要二硫键连接的双链蛋白响应跨内体膜的pH和氧化还原梯度发生构象变化，随后重链（HC）形成一个蛋白质传导通道，将轻链（LC）蛋白酶转运到细胞质中。在此，我们研究二硫键在蛋白质转运动力学中的作用。我们利用单通道/单分子检测方法，在模拟内体普遍存在的条件下，实时表征Neuro 2A细胞中的BoNT通道和伴侣活性。我们发现，在整个LC转运过程中，二硫键必须保持完整；通道形成后二硫键过早还原会阻止转运。二硫键必须位于反式区室才能实现LC的有效转运；转运过程中顺式区室或双层内的二硫键破坏会导致转运中止。我们证明，用肽键连接LC和HC代替二硫键不足以实现LC的有效转运。因此，二硫键决定了转运的结果：货物的有效通过或货物导致的通道闭塞失败。基于这些以及之前的发现，我们提出了BoNT LC转运的一系列事件顺序，即HC插入、LC伴随展开、蛋白质以N端到C端的方向通过HC通道传导，以及通过二硫键还原伴随LC在细胞质中重新折叠，最终使LC从HC中释放。
