细胞骨架调控蛋白 MICAL1 和 CLASP2 的靶蛋白识别机制研究

细胞迁移过程中，细胞需要与基质形成黏着斑以介导细胞迁移。黏着斑参与细胞机械传导、细胞周期、增殖、分化等过程的调控。黏着斑是细胞内动态适应性的结构，细胞需要不断调整黏着斑的生长和分解，以适应细胞内的各种活动。黏着斑的形成受到黏着斑相关蛋白、微丝和微管的调控。微管和微丝调控黏着斑相关蛋白的转运和信号通路影响黏着斑的形成。微丝引导微管的生长，使之定位到黏着斑周围，并通过黏着斑周围的皮质微管稳定复合物锚定微管，促进黏着斑的生成和降解。而黏着斑的形成过程中除了受到细胞骨架自身的动力学调控外，还受到许多细胞骨架调控因子的调节。比如MICAL蛋白可以氧化微丝上特定的甲硫氨酸残基来促进肌动蛋白的解聚，导致微丝分解并阻止微丝再聚合，以调控黏着斑附近微丝的解聚，进而促进微管的招募。CLASP蛋白作为黏着斑周围的皮质微管稳定复合物的核心蛋白可以通过招募其他蛋白帮助微管锚定在黏着斑附近，促进黏着斑形成。本论文对黏着斑周围如何招募微丝调控蛋白MICAL和微管调控蛋白CLASP如何介导皮质微管稳定复合物的形成进行了深入的探究。

本论文的第一部分，以微丝解聚蛋白MICAL家族成员MICAL1为研究对象，利用生化和结构生物学的方法研究了MICAL1如何通过特异性蛋白质-蛋白质相互作用识别黏着斑上的膜结合因子ASAP1，参与微丝调控和囊泡运输。MICAL1包含4个结构域。其中，MO结构域起到氧化微丝的作用；而其他结构域结合包括肌动蛋白在内的多种蛋白质，并参与MICAL1的自抑制作用来调控MICAL1的活性。为了更好地理解MICAL1如何结合不同蛋白质来发挥功能，我们首先利用Co-IP实验鉴定出了MICAL1的一个新型结合蛋白ASAP1。ASAP1是一个通过与Src和磷脂结合来协调膜运输、微丝重塑的膜结合因子。ASAP1通过与黏着斑上的FAK结合，调控黏着斑的形成。生化实验显示，MICAL1通过其loop区上一段富含脯氨酸的基序（Proline Rich motif，PRM）与ASAP1上SH3结构域以高亲和力相结合的。通过解析MICAL1-PRM/ASAP1-SH3复合物的晶体结构并结合生化实验验证，我们阐明了MICAL1与ASAP1之间发生高亲和力结合的分子机制，即ASAP1-SH3的结合口袋上包含两个带负电的区域，以特异性识别MICAL1的PRM序列，从而加强相互作用。我们发现这种独特的PRM结合口袋也存在于GRAF和SKAP1的SH3结构域中。因此我们将这些SH3结构域命名为 SH3^AGS。此外，我们利用PRM的序列特征，通过搜索Swiss-Prot蛋白数据库，发现了约130个可能具有SH3^AGS结合能力的蛋白质。生物信息学分析表明，这些蛋白质与含有SH3^AGS的蛋白质之间的相互作用可能在肌动蛋白细胞骨架调节和囊泡运输中发挥作用。这一发现有力地推动了我们理解黏着斑附近的膜结合因子是如何招募细胞骨架调控因子来调节微丝的解聚。

本论文的第二部分，以微管调控蛋白CLASP家族成员CLASP2α为研究对象，利用生化和结构生物学的方法研究了CLASP2α如何通过蛋白质-蛋白质之间的相互作用调控黏着斑周围的皮质微管稳定复合物的组装。CLASP2α是一类微管正末端追踪蛋白，含有4个TOG结构域，通过抑制微管正末端的解聚来稳定微管。CLASP2α也是皮质微管稳定复合物的核心成分，通过与LL5β、ELKS、CLIP等蛋白结合帮助微管停靠在黏着斑周围。为了更好地了解CLASP2α如何介导皮质微管稳定蛋白复合物的组装，我们解析了CLASP2α第4个TOG结构域（TOG4）与LL5β和CLIP1复合物的晶体结构。结构分析与生化实验显示，LL5β和CLIP1使用类似的序列与CLASP2α-TOG4上的同一结合口袋结合。这帮助我们更好地理解黏着斑周围的皮质微管稳定复合物组装以招募微管的机制。

通过以上两部分的研究，我们阐释了细胞骨架调控蛋白MICAL1和CLASP2α如何通过不同的蛋白质-蛋白质相互作用被特异性地招募到黏着斑附近，来参与肌动蛋白细胞骨架的调节和囊泡的运输等的分子机制；同时，我们利用结构与序列分析，扩展了对MICAL1和CLASP2α的潜在结合蛋白的认识，推动我们对细胞骨架调控因子在黏着斑形成中的作用的认识。

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