Structure-function analysis of mammalian formins FMNL1 and FMNL2

肌动蛋白骨架的调控是真核细胞稳定和运动的关键过程。一个不同的双胍蛋白家族和含有WH2结构域的蛋白最近被认为是肌动蛋白成核因子和肌动蛋白丝的有效聚合因子。formmin由formin同源2 （FH2）结构域的存在来定义，然而，在人类中发现的8个formin家族的模块化结构域结构明显不同。本研究项目的重点是哺乳动物形态调控域的生化和结构分析。白细胞2中的形成相关基因（FMNL2）是肉豆酰化的，这是一种n端脂质修饰，其目标是细胞膜上的蛋白质。在初步工作中，我们建立了一种体外肉豆蔻酰化实验，可以表征其天然形式的FMNL2。我们计划分析三磷酸Cdc42活化如何影响脂质体存在下肉豆肉酸酯的构象。将对脂化蛋白进行结构分析，并分析其与c端自动调节结构域的相互作用。此外，我们能够纯化FMNL2的N端和c端结构域的复合体，构成58 kDa和54 kDa的双同型二聚体。最后，功能实验将探讨原肌球蛋白对长丝稳定性的影响。

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.