Molecular mechanism of force-sensing in desmoplakin

桥粒不仅在细胞之间建立紧密的联系，而且还在细胞-细胞界面将机械应力整合到生化网络中。桥粒蛋白如何对机械力做出反应，从而改变其结构和功能，目前尚不清楚。我们的目标是表征桥粒的主要成分桥粒蛋白的力学性质和可能的力敏感功能。桥粒蛋白的光谱重复片段以SH3结构域为特征，具有一个特殊而隐蔽的结合位点，目前功能未知。光影蛋白-SH3相互作用是皮肤和心脏疾病相关突变的热点，突显了它在桥粒蛋白功能中的关键作用。我们将对SH3结构域以及桥粒蛋白的两个可能的作用进行测试，即机械稳定功能和机械传感功能，这两个功能可能不会相互排斥。为此，我们将进行平衡和力探针分子动力学模拟，以监测SPECTIN/SH3片段和更大结构的桥粒蛋白在张力下的情况。这些模拟将使我们能够量化SH3结构域在多大程度上能够在机械力的作用下稳定血影蛋白重复序列而不被展开，和/或能够为参与下游化学信号传递的伙伴暴露其结合部位。我们还将研究桥粒蛋白变异体，缺失SH3结构域或携带疾病突变体，以进一步阐明桥粒蛋白在应激桥粒中的力携带和力敏感作用。为了测试桥粒蛋白作用力响应的最终氧化还原调节，我们将把不同氧化状态的桥粒蛋白光谱蛋白/SH3片段应用于新开发的二硫键交换算法，并监测氧化时去折叠机制的变化。我们的结果，经过伦敦国王学院将进行的单分子力谱实验的验证，可以帮助解释和指导未来的体外和体内实验。我们希望我们的工作首次揭示桥粒蛋白在力量传递和将机械压力转化为生化信号中的直接作用。

Desmosomes not only establish tight connections between cells but also integrate mechanical stress into biochemical networks at the cell-cell interface. How desmosomal proteins respond to mechanical force such that their structure and function is altered is currently unknown. Our objective is to characterize the mechanical properties and putative force-sensing function of a major desmosomal component, desmoplakin. The spectrin-repeat fragment of desmoplakin features an SH3 domain with a peculiar and cryptic binding site and currently unknown function. The spectrin-SH3 interaction is a hot spot for mutations involved in skin and cardiac diseases, underlining its pivotal role in desmoplakin function. We will put two putative roles of the SH3 domain, and thereby of desmoplakin, to test, namely a mechanically stabilizing function and a mechano-sensing function, which might not exclude each other. To this end, we will perform equilibrium and force-probe Molecular Dynamics simulations to monitor the spectrin/SH3 fragment and larger constructs of desmoplakin under tensile forces. The simulations will allow us to quantify the extent to which the SH3 domain, when being subjected to mechanical force, can stabilize the spectrin repeats against unfolding and/or can expose its binding site for partners involved in downstream chemical signalling. We will also examine desmoplakin variants, lacking the SH3 domain or carrying disease mutants, to shed further light on the force-carrying and force-sensitive role of desmoplakin in stressed desmosomes. To test eventual redox regulation of the force response of desmoplakin, we will subject the desmoplakin spectrin/SH3 fragment at different oxidation states to a newly developed disulfide swapping algorithm and monitor variations in the unfolding mechanism upon oxidation. Our results, after validation by single molecule force spectroscopy experiments to be performed at Kings College London, can help to interpret and guide future in vitro and in vivo experiments. We expect our work to reveal, for the first time, a direct role of desmoplakin in force transmission and conversion of mechanical stress into biochemical signals.