Molecular mechanism of force-sensing in desmoplakin

桥粒不仅在细胞之间建立紧密的连接，而且还将机械应力整合到细胞-细胞界面的生化网络中。桥粒蛋白如何响应机械力，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斑蛋白的机械特性和推定的力感功能。桥粒斑蛋白的血影蛋白重复片段具有SH 3结构域，该结构域具有独特的隐蔽结合位点和目前未知的功能。血影蛋白-SH 3相互作用是涉及皮肤和心脏疾病的突变的热点，强调了其在桥粒斑蛋白功能中的关键作用。我们将把两个假定的作用的SH 3域，从而desmoplakin，测试，即机械稳定功能和机械传感功能，这可能不排除对方。为此，我们将进行平衡和力探针分子动力学模拟，以监测血影蛋白/SH 3片段和更大的结构下的张力桥粒斑蛋白。模拟将使我们能够量化SH 3结构域在受到机械力时可以稳定血影蛋白重复序列对抗展开和/或可以暴露其结合位点以用于参与下游化学信号传导的合作伙伴的程度。我们还将研究桥粒斑蛋白的变体，缺乏SH 3结构域或携带疾病突变体，以进一步阐明桥粒斑蛋白在应激桥粒中的力传递和力敏感作用。为了测试最终的氧化还原调节的力反应的桥粒斑蛋白，我们将受到桥粒斑蛋白血影蛋白/SH 3片段在不同的氧化态的一个新开发的二硫键交换算法和监测氧化后的展开机制的变化。我们的研究结果，验证后，在国王学院伦敦进行的单分子力谱实验，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能够首次揭示桥粒斑蛋白在力传递和机械应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.