白血病靶点蛋白PML-RARa等的结构和功能研究

The goal of targeted therapy is to interfere with specific molecules such as the oncogenic PML-RARa fusion protein in acute promyelocytic leukemia (APL). Over the last three decades Shanghai Institute of Hematology together with other laboratories/hospitals have successfully developed an all-trans retinoic acid (ATRA)/arsenic trioxide (ATO) synergistic therapy with a CR rate of 95% in APL patients. However, due to the lack of structural information, very little is known about the oligomerization, sumoylation and assembly of PML and PML-RARa. Thus far, we have determined the crystal structure of PML Ring domain by X-ray crystallography (MAD phasing). The 2.0 Å crystal structure reveals a novel oligomerisation mechanism by PML protein: four Ring monomers fold into a doughnut-shape tetramer mediated by a N-terminal molecular Velcro loop and a disulphide bridge. Structure-based site-directed mutagenesis together with in vitro and in vivo assays show that the oligomerisation of PML Ring domain is critical to the formation of PML nuclear bodies, and the efficacy of ATO upon PML-RARa protein. In this proposal, we will determine several structures of PML-RARa and other oncogenic proteins such as AML1-ETO and NUP98-IQCG etc. Based on these novel structures, we will use in vitro biochemical/cellular assays and animal experiment/model to extend our understanding towards the structure, function, assembly, and leukemogenesis of these oncogenic proteins. More importantly, this work should provide important structural basis/information for novel targeted cancer therapy.

伴随着对白血病发病机理的深入了解，结合蛋白结构设计小分子药物的靶向治疗的思路也越来越被科研和临床工作者所接受。以早幼粒细胞白血病（APL）为例， PML-RARα在APL发病和临床治疗中都起了至关重要的作用。然而，因为一直缺少相关的结构生物学信息，对于其分子水平的发病机制和靶点治疗中的重要环节还不是很清楚。至今，我们已通过X-射线晶体学手段确定PML氮端结构域的结构。高分辨率的晶体结构揭示了一种通过新的PML蛋白的组装机制。基于结构的定点突变、超速离心法和荧光显微镜等实验表明，PML的寡聚是PML核体的形成至关重要环节。初期动物实验表明，该癌蛋白的功能、结构与砷的治疗有密切关系。在此基础上，本项目计划通过以结构生物学，分子生物学、细胞生物学和动物模型等技术平台，对PML-RARa等白血病重要靶点蛋白的结构和功能进行系统的分析，设计和改善现有的小分子药物，寻找新的治疗药物和治疗诊断方法。

PML-RARa在APL发病中起着关键的作用，影响并改变野生型PML的结构和功能。而砷剂可以靶向PML/PML-RARa, 促使其多聚化进而促进其泛素化及蛋白的降解。但由于缺乏结构信息，PML-RARa的发病机理和靶向治疗机理尚不清楚。在本项目中，我们通过同晶置换的方法，解析出PML-RARα四聚体晶体结构，是关于PML多聚体的首次报道，从原子水平上揭示了PML本身如何通过N端结构域展开分子间聚合，进而形成PML核体的组装机制。同时，进一步的分子生物学、生物物理学、细胞生物学、动物模型实验均提示，该聚合方式在APL白血病发病和治疗（ATO）也有重要影响。这是上海血液学研究所在APL领域的又一重大发现和延伸, 为白血病治疗和预后提供新的理论(Wang et al, Nature Communications, 2017)。

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