特异性化学干预PDI活性与帕金森病发病机制中的作用研究

Neurodegenerative diseases (ND) affect the most precious qualities of human beings. The hallmark feature of these disorders is protein misfolding and aggregation. Protein disulfide isomerase (PDI) is one of key proteins in keeping cell homeostasis by catalyzing protein folding through reduction and oxidation. However, its function is still unclear and there is still a lack of tools to fully understand the mechanism of PDI in ND. Herein, based on our previous results, by taking advantage of chemical biology, this proposal will aim to develop not only small probes targeting each cysteine in active sites but also hybrid compounds for S-nitrosylation of PDI, to understand the function of PDI in Parkinson’s disease (PD, one of ND). With probes in hand, we will further figure out their patterns on modification of PDI active sites and subsequently explore their working mechanisms in two models of PD. If it works well, this approach impacts on unveiling the relationship between PDI and ND to give insight on new therapy strategies.

神经退行性疾病已对社会造成巨大压力。蛋白质错误折叠及聚集是其共性发病机制。蛋白质二硫键异构酶PDI是一种通过介导氧化还原蛋白质折叠来维持细胞稳态的关键酶。目前，PDI在神经退行性疾病发生发展中的作用尚不清晰，亟需寻找有效方法认知PDI的调控机制。因此，本项目在前期研究的基础上，聚焦帕金森病，拟以PDI为目标物，充分发挥化学生物学研究方法的优势，通过分子理性设计，合成两类PDI靶向性的小分子探针，寻找对PDI活性中心四个半胱氨酸的专一性修饰方法，并结合巯基亚硝基化作用，特异性原位干预PDI活性，探究化学干预前后的PDI对蛋白质聚集、细胞凋亡的调控规律，全面分析PDI与帕金森病发生发展间的内在联系，为疾病的预防、诊断、治疗及药物研发提供更充分的理论依据。

本项目以通过氧化还原介导蛋白质折叠以维持细胞稳态的关键酶--蛋白质二硫键异构酶（Protein disulfide isomerase，PDI）为目标物，利用化学生物学研究方法的优势，通过分子理性设计，结合基于活性的蛋白质组分析技术（Activity-based protein profiling，ABPP）技术、蛋白质水解靶向嵌合体（Proteolysis targeting chimera，PROTAC）、荧光探针等理论，我们设计合成了一氧化氮供体杂合型、荧光开启型、靶向降解型等靶向PDI的小分子，发现乙烯砜、氯乙酰反应基团对PDI活性中心半胱氨酸（C397，C53）靶向具有专一性、选择性，含有结合基团的PDI巯基亚硝基化明显，对SH-SY5Y细胞增殖抑制较强；获得了基于乙烯砜的PDI荧光探针，与PDI结合后发光强度增加了17倍；利用降解型抑制剂，发现PDI的C53位点与突触蛋白的聚集表达相关性更强。整体而言，项目基本按项目书研究计划展开工作，基本完成了课题任务，并根据研究方案进行了拓展，达到了预期目标。