Structure-function analysis of mammalian formins FMNL1 and FMNL2

肌动蛋白细胞骨架的调控是真核细胞稳定性和运动性的关键过程。近年来，一类不同的Forin蛋白家族和包含WH2结构域的蛋白被认为是肌动蛋白的核因子和肌动蛋白细丝的有效聚合因子。福尔明是由福尔明同源2(FH2)结构域的存在定义的，然而，在人类中发现的八个福尔明家族的模块化结构域结构似乎有很大的不同。本研究项目致力于哺乳动物福尔马林调节域的生化和结构分析。白细胞中的Forin相关基因2(FMNL2)是肉豆蔻酰化的，这是一种N末端的脂类修饰，靶向于细胞膜上的蛋白质。在前期工作中，我们建立了一种体外肉豆蔻化试验，允许FMNL2以其天然形式进行表征。我们计划分析在脂质体存在的情况下，三磷酸镉42的激活如何影响肉豆蔻酸盐的构象。将进行脂化蛋白质的结构分析，并分析与C末端自动调节结构域的相互作用。此外，我们还能够纯化FMNL2的N-末端和C-末端结构域的复合体，它构成了58 kDa和54 kDa的双同源二聚体。最后，功能实验将解决原肌球蛋白对细丝在伸长过程中稳定性的影响。

Regulation of the actin cytoskeleton is a key process for the stability and motility of eukaryotic cells. A diverse family of formin proteins and WH2 domain containing proteins have recently been recognized as actin nucleators and potent polymerization factors of actin filaments. Formins are defined by the presence of a formin homology 2 (FH2) domain, yet, the modular domain architecture appears significantly different for the eight formin families found in man. This research project focuses on the biochemical and structural analysis of the regulatory domains of mammalian formins. The formin-related gene in leukocytes 2 (FMNL2) is myristoylated, an N-terminal lipid modification which targets a protein to cellular membranes. In preliminary work, we have established an in vitro myristoylation assay that allows characterization of FMNL2 in its native form. We plan to analyse how activation by triphosphate Cdc42 influences the conformation of the myristate in the presence of liposomes. Structural analysis of the lipidated protein will be performed and the interaction with the C-terminal autoregulation domain analysed. Also, we are able to purify a complex of the N- and C-terminal domains of FMNL2 which constitute a double homodimer of 58 kDa and 54 kDa. Finally, functional experiments will address the impact of tropomyosin on filament stabilization during elongation.