Myopodin is an F-actin bundling protein with multiple independent actin-binding regions
Myopodin is an F-actin bundling protein with multiple independent actin-binding regions
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DOI：
10.1007/s10974-012-9334-5
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发表时间：
2013-02-01
影响因子：
2.7
通讯作者：
Fuerst, Dieter O.
中科院分区：
文献类型：
--
作者：
Linnemann, Anja;Vakeel, Padmanabhan;Fuerst, Dieter O.
横纹肌肌原纤维的组装是一个涉及多种蛋白质的多步骤过程。肌原纤维形成的第一步也是最重要的一步是将细小的肌丝排列成有序的I-Z-I刷子，这需要许多肌动蛋白结合蛋白的协调活动。肌动蛋白的早期表达先于肌节α-肌动蛋白，以及它与肌动蛋白、α-肌动蛋白和丝氨酸的结合，表明该蛋白在肌动蛋白细胞骨架重构中起着重要作用，但肌桥蛋白在这一过程中的确切功能仍有待研究。虽然Myopodin之前被描述为一种能够在瞬时转染时将肌动蛋白细丝交联成粗束的蛋白质，但目前尚不清楚myopodin是否能够单独结合肌动蛋白，或者是否涉及其他蛋白质。因此，我们研究了Myopodin的体外肌动蛋白结合特性。与骨骼肌肌动蛋白的高速共沉积分析证实了肌动蛋白与F-肌动蛋白的直接结合，并表明这种相互作用是由至少两个独立的肌动蛋白结合位点介导的，这两个结合位点在迄今发现的所有肌动蛋白亚型中都存在。此外，低速共沉淀分析表明，不仅全长的myopodin，而且只包含第二结合位点的片段，在没有辅助蛋白的情况下，会捆绑微丝。超微结构分析表明，这种结合活性类似于α-肌动蛋白。生化实验表明，捆绑不是通过myopodin的二聚化能力实现的，这表明在第二结合片段中存在两个单独的F-肌动蛋白结合位点。因此，全长myopodin至少包含三个F-肌动蛋白结合位点。这些数据有助于进一步了解肌钙蛋白在肌原纤维组装过程中对肌动蛋白重组的作用机制。
The assembly of striated muscle myofibrils is a multistep process in which a variety of proteins is involved. One of the first and most important steps in myofibrillogenesis is the arrangement of thin myofilaments into ordered I-Z-I brushes, requiring the coordinated activity of numerous actin binding proteins. The early expression of myopodin prior to sarcomeric alpha-actinin, as well as its binding to actin, alpha-actinin and filamin indicate an important role for this protein in actin cytoskeleton remodelling with the precise function of myopodin in this process yet remaining to be resolved. While myopodin was previously described as a protein capable of cross-linking actin filaments into thick bundles upon transient transfections, it has remained unclear whether myopodin alone is capable of bundling actin, or if additional proteins are involved. We have therefore investigated the in vitro actin binding properties of myopodin. High speed cosedimentation assays with skeletal muscle actin confirmed direct binding of myopodin to F-actin and showed that this interaction is mediated by at least two independent actin binding sites, found in all myopodin isoforms identified to date. Furthermore, low-speed cosedimentation assays revealed that not only full length myopodin, but also the fragment containing only the second binding site, bundles microfilaments in the absence of accessory proteins. Ultrastructural analysis demonstrated that this bundling activity resembled that of alpha-actinin. Biochemical experiments revealed that bundling was not achieved by myopodin's ability to dimerize, indicating the presence of two individual F-actin binding sites within the second binding segment. Thus full length myopodin contains at least three F-actin binding sites. These data provide further understanding of the mechanisms by which myopodin contributes to actin reorganization during myofibril assembly.