【摘要】 hhLIM是LIM蛋白家族成员之一,该蛋白质含有两个LIM结构域,在基因表达调节、细胞骨架组构及细胞肥大过程中发挥重要作用.构建hhLIM不同LIM结构域的突变体,探讨其两个LIM结构域在与actin相互结合中的作用及其可能机制.GST-pull down和hhLIM及其突变体与actin细胞定位关系的免疫荧光分析结果表明,C端的LIM结构域2是hhLIM与actin结合所必需的,该结构域中的两个Cys置换为Ser后可使hhLIM结合actin的功能完全丧失,N端的LIM结构域1突变使hhLIM结合actin的能力下降.F-actin交联实验结果显示,hhLIM通过LIM结构域2与actin直接结合并起到交联F-actin的作用.结果表明,LIM结构域2在hhLIM与actin相互作用及调节actin细胞骨架组构中起决定性作用.更多还原

【Abstract】 hhLIM, a member of LIM protein family, has two LIM domains and plays an important role in gene regulation, cytoskeleton organization and cell hypertrophy. To understand the functional importance of hhLIM in cytoskeleton organization and muscle hypertrophy, hhLIM and its mutants of two LIM domains were constructed and function of each LIM domain and its interaction with actin were studied. GST pull down assay and immunofluorescence assays showed that LIM domain 2 at the C-terminus of hhLIM is critical for its interaction with actin. The mutant of the LIM domain 2 in which two important Cys are replaced by Ser lost the capacity of hhLIM to interact with actin. Mutation of the LIM domain 1 at the N-terminus of hhLIM impaired the capacity of hhLIM to interact with actin. F-actin cross-linking assay identified that hhLIM could make F-actin to cross-link into bundles by interaction between LIM domain 2 and actin. In conclusion, LIM domain 2 at the C-terminus of hhLIM plays a central role in F-actin polymerization and cytoskeleton stabilization, whereas the first LIM domain is essential for the nuclear localization of hhLIM.更多还原