胚胎干细胞特异性Hsp90/Epichaperome复合体的功能与作用机制研究

It remains elusive about the molecular mechanisms that govern embryonic stem cell (ESCs) identity, thus hindering safe use of their differentiated derivatives in regenerative medicine. How Hsp90-centered protein quality control regulates ESCs identity has not been reported. Our preliminary results revealed that Hsp90 forms a stable Epichaperome complex in mouse ESCs, but not in the terminally differentiated mouse cells. Inhibition of Hsp90/ Epichaperome complex leads to Nanog degradation, reduction in self-renewal and induction of ESCs differentiation. Affinity purification coupled with mass spectrometry identified Chip E3 ligase as an interacting factor of pluripotency factor, Nanog. Based on these findings, we propose that formation of Hsp90/Epichaperome complex is essential for ESCs maintenance and the complex achieves its function through proper folding and preventing Chip-dependent degradation of Nanog in mouse ESCs. We will integrate biochemical, molecular and cellular approaches to further delineate the role and molecular mechanisms of Hsp90/Epichaperome in quality control of Nanog, ESCs maintenance as well as during somatic reprogramming. Our results will hopefully open a new research orientation and from a new perspective to understand mechanisms in ESCs regulation. Additionally, results from the proposed studies will provide theoretic basis and lay a solid foundation for safe application of ESCs and their derivatives in disease treatment in regenerative medicine.

胚胎干细胞自我更新和多向分化潜能维持的分子机制仍不清楚，妨碍了再生医学中安全有效地利用其分化的细胞。以Hsp90为核心的蛋白质量控制系统如何调控胚胎干细胞功能尚未见报道。 我们前期研究发现，与终末分化细胞不同，Hsp90在小鼠的胚胎干细胞中以独特的Hsp90/Epichaperome复合体形式存在。抑制该复合体功能导致Nanog蛋白质降解、细胞增殖减慢以及分化。亲和纯化结合质谱技术发现泛素连接酶Chip能与Nanog结合。基于这些发现，我们推测Hsp90／Epichaperome复合体能够帮助Nanog蛋白质形成正确构象从而阻止Chip介导其降解来维持胚胎干细胞特性。本项目将进一步探究该复合体在胚胎干细胞特性维持、体细胞重编程以及Nanog蛋白质量稳态控制中的功能与机制，以期从崭新的视角认识胚胎干细胞特性建立、维持的分子机制，为再生医学中安全高效应用胚胎干细胞治疗疾病提供理论基础。

胚胎干细胞自我更新和多向分化潜能维持的分子机制仍不清楚，妨碍了再生医学中安全有效地利用其分化的细胞。以Hsp90为核心的蛋白质量控制系统如何调控胚胎干细胞功能尚未见报道。在该项目的资助下，我们发现在细胞抗应激反应过程中发挥重要的分子伴侣Hsp90， Hsc70, Cdc37, Hop, Aha1， Hsp110等蛋白在多能性的胚胎干细胞中表达上调，并且相互结合形成一个稳定的Hsp90/Epichaperome复合体。后续对其生物学功能研究发现Hsp90/Epichaperome复合体在维持胚胎干细胞的自我更新过程中发挥重要功能，抑制其功能破坏了小鼠胚胎干细胞的自我更新。后续深入的分子机制研究发现，Hsp90/Epichaperome复合体通过帮助 Nanog 进行正确折叠， 提高其 Ser/Thr-Pro(SP/TP)基序磷酸化修饰水平。 (SP/TP)基序磷酸化修饰一方面增强了 Nanog 与 Pin1 之间的相互作用， 帮助 Nanog 形成有功能的三维空间结构。同时，（SP/TP)基序磷酸化修饰减少了 Nanog 与 E3 泛素连接酶 Chip 之间的结合， 阻止 Nanog 通过泛素蛋白酶体途径降解， 从而增强了胚胎干细胞的自我更新的能力。该项目从一个崭新的角度认识胚胎干细胞特性建立和维持的分子机制，并为再生医学中体外更好的维持胚胎干细胞特性以及提高体细胞重编程效率提供新的靶点与思路。

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