酿酒酵母中Asr1p与肌动蛋白结合蛋白Crn1p新的相互作用

Actin cytoskeleton, a network structure in cells composed of monomeric actin, has to undergo rapid remodeling for controlling most cellular movements such as cell locomotion, endocytosis, vesicle and organelle transport, cytokinesis, and polarized cell growth. Coronin, a very conserved component of the actin cytoskeleton found in all eukaryotes from yeast to mammals, functions as the well-known regulator in the processes of either actin polymerization or depolymerization. Furthermore, it has been suggested involved in the diseases such as immunodefeciency, neurodegeneration, and Down syndrom. In our previous study, it has been demonstrated that Asr1p, which is a newly identified E3 ligase with two Ring finger/PHD domains, could interact with Crn1p in vivo and in vitro in Saccharomyces cerevisiae by yeast two-hybrid assay, pull-down, Co-Ip as well as subcellular localization of Asr1p. Thus, we here try to confirm the domains involved in the interactions between Asr1p and Crn1p. It will be also demonstrated if their binding results to ubiquitinoylation of Crn1p or influences the interactions of Crn1p with other proteins, which consequently change its stability or activity. Furthermore, our current research will reveal if their interaction can significantly influence the dynamic assembly of actin cytoskeleton as well as actin-based cellular movements. This findings would suggest a novel regulatory mechanism to control the activity of Crn1p, and firstly confirm the biological function of Asr1p in actin dynamics.

微丝骨架是动态存在于细胞中由肌动蛋白构成的网络状结构, 它参与了维持细胞形态运动、信号传导、增殖分化以及基因表达调控等几乎所有的细胞生理过程。Coronin是一种真核生物中保守、重要的肌动蛋白结合蛋白，人类中与免疫缺陷、神经退行病变、唐氏综合症等密切相关。我们前期通过酵母双杂交、pull-down、Co-IP及亚细胞定位实验已发现，模式生物酿酒酵母中的Crn1p在体内外均可以与泛素化E3酶Asr1p蛋白结合。因此本研究拟进一步确认二者的相互作用分子基础，揭示二者的相互作用是否会导致Crn1p产生泛素化修饰或者影响Crn1p与其它蛋白的结合，从而调控Crn1p的稳定性或活性；在此基础上，探索二者相互作用在肌动蛋白细胞骨架形成和分布、以及细胞形态、运动和有丝分裂等相关生物学行为中的功能。该研究可阐明重要的肌动蛋白结合蛋白Crn1新的调控方式，并首次证实Asr1p在细胞微丝骨架动态平衡中的作用。

微丝骨架是动态存在于细胞中由肌动蛋白构成的网络状结构, 它参与了维持细胞形态运动、信号传导、增殖分化以及基因表达调控等几乎所有的细胞生理过程。Coronin是一种真核生物中保守、重要的肌动蛋白结合蛋白，人类中与免疫缺陷、神经退行病变、唐氏综合症等密切相关，然而Coronin在体内可以通过哪些方式进行调控还一无所知。我们的研究以模式生物酿酒酵母为对象，首次发现Asr1p (Alcohol Sensitive Ring/PHD finger 1 protein)可以通过２个完整的Ring finger/PHD结构域与酿酒酵母体内唯一的Coronin家族成员Crn1p相互结合，它们的结合可以导致Crn1p的572位赖氨酸残基发生单泛素化修饰，影响细胞骨架相关保守蛋白Crn1p的稳定性和活性，从而影响微丝骨架的动态平衡及其相关细胞生物学功能。总之，我们的研究数据揭示了Asr1p一个新的生物学功能——参与调控肌动蛋白骨架的动态平衡和分布。在此基础上，发现Asr1p与Crn1p之间的相互作用是Asr1p调节肌动蛋白骨架的动态平衡的分子基础。最终，我们提出了Asr1p参与体内肌动蛋白细胞骨架动态平衡调节的作用模型：即Asr1p的表达单泛素化修饰Crn1p，提高Crn1p的体内稳定性，增加后者的浓度，高浓度Crn1p负调控Arp2/3复合物的活性或正调控Cofilin活性、从而抑制F-肌动蛋白合成、同时促进F-肌动蛋白的解聚。反之亦然。