蛋白质SUMO化调节PAX6功能的分子机制

Pax6 is a master regulator of eye development.It is expressed in the region of anterior surface ectoderm corresponding to the future adenohypophyseal,olfactory and lens placodes,optic vesicle and other parts of the future brain.Pax6 null mice do not form olfactory and lens placodes and show disrupted pituitary gland development.Pax6 mutations and other factors causing altered expression of Pax6 lead to a series of ocular diseases such as absence of iris, cataract, glaucoma,retinoblastoma,and WAGR,etc.Thus,it is extremely important to have a thorough understanding of the functional mechanisms of Pax6 and it's regulation in order to elucidate the mechanisms mediating various ocular diseases.Pax6 is localized at 11p13 of human chromosome 13,and the genomic gene with 25 kb codes for 14 exons.Pax6 protein exists in 4 diffferent isoforms:p48,p46,p43 and p32.While the functions of p46 Pax6 has been extensively studied and investigation of P48 Pax6 function has also been reported,little is known about the functional mechanisms of p32 and p43 Pax6.We have recently shown that activation of p32 Pax6 requires SUMO1-mediated sumoylation.Whether sumoylation regulates expression and functions of other Pax6 isoforms remains to be determined.. Sumoylation,a post-translational modification is a process in which the small ubiquitin-like modifers (SUMOs) are conjugated to target proteins by three ligases.Three SUMO isoforms have been identified.While SUMO2 and SUMO3 share 97% sequence homology,they have significantly difference with SUMO1 (45% sequence difference between SUMO1 and SUMO2/3).. Our recent study reveals that SUMO1 and SUMO2/3 have distinct functions in regulating lens differentiation.While SUOM1 promotes lens differentiation,SUMO2/3 inhibits this process. The underlying mechanism mediating this contrast functions of different SUMOs is derived from their differential regulation of the transcription factor SP-1 and likely other factors.Whether SUMO1 and SUMO2/3 can also differentially control the functions of Pax-6 remains to be studied.. In the present application,we propose to study the sumoylation regulation of Pax6 expression and functions.Using molecular and cellular biology technology,we want to determine:1) what are the sumoylation patterns of different isoforms of Pax6 in the normal ocular tissues and those from patients with various ocular diseases?2) does sumoylation regulate their expression levels?3)what is the function of P43 Pax6?4) does sumoylation regulate the functions of various isoforms of Pax6 in vitro and in vivo?The proposed study will determine how sumoylation controls Pax6 expression and functions.Moreover, this study will help to understand the molecular mechanisms mediating various Pax-6 related ocular diseases,and shed light to their clinic diagnosis and therapeutic treatment.

人类配对盒基因（Paired Box Gene，简称Pax6）是控制眼睛和大脑发育的关键基因。Pax6缺失导致胚胎发育异常，大脑发育残缺，眼睛和鼻子缺失。由Pax6突变或其它因素造成的表达水平改变导致一系列眼睛疾病: 虹膜缺失、白内障、青光眼及黄斑病变等。因此，对Pax6功能及其调控机制的研究在临床医学上有十分重要的意义。我们最近的研究表明P32 Pax6的激活需要经过类泛素化修饰（Sumoylation, 简称SUMO化）。SUMO化修饰是否能够调节其它Pax6异构体(P48，P46和P43 Pax6)则有待进一步研究。本项目拟用分子细胞生物学的方法来研究SUMO化对上述三种Pax6异构体的表达水平及其功能的调节机制，并探讨其与上述各种眼睛疾病之间的关系，其结果将阐明SUMO化对Pax6表达和功能的调控机理，并进一步帮助了解与Pax6相关疾病的病理机制，为它们的诊断和治疗提供新的切入点。

人类配对盒基因（Paired Box Gene 6，简称Pax6）是控制眼睛和大脑发育的关键基因。Pax6缺失导致胚胎发育异常，大脑发育残缺，眼睛和鼻子缺失。由Pax6突变或其它因素造成的表达水平改变导致一系列眼睛疾病: 虹膜缺失、白内障、青光眼、黄斑病变、视网膜母细胞瘤及WAGR综合症。因此，对Pax6功能及其调控机制的研究在临床医学上有十分重要的意义。 本项目在国家基金委的资助下，首次全面分析了SUMO化对Pax6表达和功能的调节作用。以小鼠模式动物、白内障病人超声乳化手术分离的晶状上皮细胞及HLE、FHL124、aTN4-1、N/N1003A、RLEC、和ARPE-19细胞系为材料，并运用多种分子细胞生物学和生物化学的方法，进行了一系列的实验研究，对SUMO化调节Pax6的表达和功能，阐明了如下重要信息。首先，在小鼠体内和各种细胞系中, SUMO1介导的SUMO化能修饰P46和P32 Pax6，增加其稳定性；其次，无论在体内还是细胞系中，用CRISPR/CAS9技术构建的Pax6K91R突变体，岀现十分显著的转录组改变，导致Pax6 K91R小鼠在角膜、晶体状和视网膜组织发生严重的病理表型，由此证明SUMO化对Pax6的正常功能有十分重要的调节作用；再次，在不同年龄白内障和视网膜母细胞瘤中，P46 Pax6的SUMO化发生显著改变，表明Pax6的 SUMO化与重要眼疾明显相关。这些结果表明Pax6 SUMO化可以作为某些眼病治疗的切入点。同时，特征性SUMO化状态可以作为白内瘴和视网膜瘤的分子标记,为其临床转化医学研究提供了论据。本课题资助发表了16篇SCI研究论文 (其中10篇论文为第一批注论文），8篇SCI综述论文（其中6篇为第一批注论文）。 此外，正在发表的研究论文4篇。本课题帮助培养了3名青年教师，4名博士生和4名硕士生。