Molecular mechanism of force-sensing in desmoplakin

桥粒不仅在细胞之间建立紧密的连接，而且在细胞-细胞界面将机械应力整合到生化网络中。桥粒体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。我们的目的是表征一种主要桥粒成分桥粒蛋白的机械特性和假定的力感应功能。desmoplakin的spectrin-repeat片段具有一个SH3结构域，该结构域具有一个特殊而神秘的结合位点，目前功能未知。spectrin-SH3相互作用是涉及皮肤和心脏疾病的突变热点，强调了其在桥蛋白功能中的关键作用。我们将对SH3结构域以及desmoplakin的两个假定作用进行测试，即机械稳定功能和机械传感功能，它们可能不会相互排斥。为此，我们将进行平衡和力探针分子动力学模拟，以监测张力作用下的光谱蛋白/SH3片段和更大的desmoplakin结构。模拟将使我们能够量化SH3结构域在多大程度上受到机械力的影响，可以稳定谱蛋白重复序列，防止其展开和/或可以将其结合位点暴露给下游化学信号传导的伙伴。我们还将研究缺乏SH3结构域或携带疾病突变体的桥粒蛋白变异体，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desmoplakin力响应的最终氧化还原调节，我们将在不同氧化状态下将desmoplakin光谱蛋白/SH3片段置于新开发的二硫交换算法中，并监测氧化时展开机制的变化。我们的结果将在伦敦国王学院进行的单分子力谱实验验证后，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desmoplakin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.