Impact of MFM disease mutations on the assembly mechanism and network formation of muscle-specific intermediate filament proteins

在肌肉中，中间丝(IF)蛋白结蛋白是肌瘤外细胞骨架的主要成分之一。在胚胎发育和发育的后期阶段，这一系统被额外的IF-蛋白，即波形蛋白、同步素和合成酶蛋白所加强和区域功能，因为这些蛋白都与整个细丝或在具有战略意义的重要位置，如衣壳。虽然波形蛋白似乎与结蛋白形成了真正的共聚物，但与Synminin和Syncolin的情况尚不清楚。对于SYNMIN，我们有证据表明它不形成IFProteins的主要分子复合体，即卷曲的螺旋，既不是单独形成的，也不是与波形蛋白或结蛋白形成的复合体。相反，它与形成细丝的高亲和力结合。根据Syncolin的结构域组织，它与标准IF-蛋白的结构有相当大的偏离，我们预计该蛋白也会有类似的分子场景。因此，我们希望通过分析超速离心法和化学交联法在分子水平上研究结蛋白与异构体和异构体的相互作用。为此，我们将从这两个蛋白质中产生特定的重组亚域，并确定它们与结蛋白的结合特性。从这项研究开始，我们将确定仍然与结蛋白结合的最小多肽，并确定它们在结蛋白分子上的结合位置。此外，我们还将研究是否会同时与合成素和合成素以及plectin以及这两个分子中的任何一个分子与细丝发生双重结合。然后，我们将使用这些蛋白质的结合模块来研究它们对结蛋白细丝的组装动力学和稳定性的影响，无论是在体外还是在肌母细胞到肌管转化的细胞系统中。

In muscle, the intermediate filament (IF) protein desmin is one of the major components of the extra-sarcomeric cytoskeleton. During embryogenesis and later stages of development, this system is enforced and regionally functionalized by additional IF-proteins, namely vimentin, synemin and syncoilin, as these proteins all colocalize either with the entire filament or at strategically important sites such as costamers. Whereas vimentin appears to form authentic copolymers with desmin, the situation with synemin and syncoilin is not clear. For synemin we have evidence that it does not form the principal molecular complex of IFproteins, i.e. the coiled coil, neither on its own nor in complex with vimentin or desmin.Instead, it binds with high affinity to forming filaments. According to the domain organization of syncoilin, which deviates considerably from that of standard IF-proteins, we expect a similar molecular scenario for this protein too. Therefore, we want to investigate the interaction of desmin with both synemin and syncoilin at the molecular level employing analytical ultracentrifugation and chemical cross-linking techniques. For this purpose, we will generate from these two proteins specific recombinant subdomains and determine their binding properties to desmin. Starting from this investigation, we will determine the minimal peptides that still bind to desmin and determine where on the desmin molecule they bind. In addition, we will investigate if dual binding of both synemin and syncoilin as well as plectin and either of these molecules to filaments is taking place. We will then employ the binding modules of these proteins to investigate their impact on the assembly kinetics as well as the stability of desmin filaments, both in vitro and in a cellular system of myoblast-to-myotube conversion.