氧化修饰诱导APIP蛋白积聚的分子机制及其生物学功能研究

Protein aggregation is a biological phenomenon in which a unusual subset of proteins aggregate and form oligomers, fibers, or even highly ordered structures like amyloid or prion within cells. Mounting evidence suggests that protein aggregation is often part of the cellular response to an imbalanced protein homeostasis and is normally prevented by complex cellular quality control mechanisms. However, under certain circumstances, protein aggregation into abnormal cellular puncta may cause the loss of critical protein functions and change cellular protein-protein interaction networks. Therefore, it has emerged as the hallmarks of many diseases, including neurodegenerative diseases, inflammation, and cancer. We have recently discovered that APIP (apoptotic protease activating factor APAF1 interacting protein), a key negative regulator in apoptotic cell death, is able to aggregate in cells under oxidative stress. To date, our preliminary studies suggest that oxidants may modify reactive cysteines on APIP protein to enable assembly of APIP aggregates. The aim of the proposal will further elucidate the molecular mechanism and the biological consequences of oxidation-dependent APIP aggregation. To this end, we will first perform in-depth mass-spectrometry-based proteomics analyses to map and quantify oxidation of specific amino acid residues on APIP protein in vitro and in situ. We will also utilize chemical cross-linking mass spectrometry to study how oxidized APIP proteins form aggregates. Then, we will define how APIP aggregation relates to oxidant-induced apoptosis or cellular dysfunction. Finally, we will explore the role of oxidation-dependent APIP aggregation in cancer development or intervention. The discovery of the role of biological regulation of APIP aggregation is expected to increase our understanding of the upstream regulatory mechanism of apoptosis. More importantly, this project may be ultimately associated with translational potential for clinical medicine by discovering a novel biomarker in the key areas of cancer diagnosis and therapeutics.

蛋白积聚是蛋白通过自身相互作用形成聚合体甚至难溶性纤维状积聚体的过程。这种积聚作用通常会破坏蛋白的正常生物学功能并且改变蛋白-蛋白相互作用网络，故其与许多疾病的发生密切相关，如神经退行性疾病、炎症或癌症。本课题组在前期研究中首次发现细胞内一种重要功能蛋白APIP（细胞凋亡蛋白酶激活因子APAF1作用蛋白）在氧化应激下能够快速发生积聚。为了进一步阐明APIP蛋白氧化依赖性积聚发生的分子机制乃至生物学功能，本项目将以蛋白组学技术为核心并综合运用多学科技术手段研究（1）APIP蛋白氧化修饰及其对积聚发生的影响；（2）APIP蛋白氧化积聚对氧化应激诱导细胞凋亡亦或它分子通路的调控作用；（3）APIP蛋白氧化积聚与肿瘤发展、干预的关联性。这些研究有望丰富人们对细胞凋亡关键分子APAF1蛋白上游调控机制的认识，并且可能发现基于全新机理（蛋白氧化积聚）的肿瘤生物标识物乃至新型抗肿瘤策略。

蛋白积聚是蛋白通过自身相互作用形成聚合体甚至难溶性纤维状积聚体的过程。这种积聚作用通常会破坏蛋白的正常生物学功能并且改变蛋白-蛋白相互作用网络，故其与许多疾病的发生密切相关，如神经退行性疾病、炎症或癌症。在本项目的研究中， 我们开发了一种新型定量化学蛋白质组技术（巯基反应性定量技术QTRP），并且运用该技术发现了一种名为APIP的蛋白（全称：细胞凋亡蛋白酶激活因子APAF1作用蛋白，Apoptotic Protease activating factor Interacting Protein）在体外和细胞内均具有较强的氧化敏感性。进一步研究发现APIP蛋白能发生氧化依赖性积聚，形成一系列寡聚体乃至难溶性多聚体。通过质谱分析，我们发现该蛋白的C97位半胱氨酸残基上形成了亚磺酸。体外生化研究表明该位点突变会影响APIP蛋白的酶活性，并且含有该突变的APIP蛋白在氧化剂处理下的积聚程度显著下降，提示C97是APIP发生氧化依赖型积聚的关键位点。为了进一步证实这一点，我们还进行了化学交联-质谱分析，结果发现APIP蛋白C97所在的beta-helix结构能够导致该蛋白发生同源二聚化，并且最终导致蛋白形成难溶性堆叠。我们还在HEK293和A549细胞中分别表达了野生型和C97A/S突变的APIP蛋白，结果表明C97突变体在细胞中的积聚程度相较于野生型也存在显著下降。我们通过免疫共沉淀实验发现APIP C97位的氧化能抑制其与APAF1蛋白的结合，进而影响后者形成凋亡小体。我们还通过亲和纯化-质谱法发现多个APIP相互作用蛋白与前者的结合水平以及复合物拓扑结构在过氧化氢刺激下出现显著性变化并且采用生物信息学构建了APIP氧化依赖型聚集调控的蛋白互作网络。综上所述，我们通过本项目的研究发现介导APIP氧化依赖型积聚的关键位点及其功能调控作用，这些研究丰富了人们对细胞凋亡关键分子APAF1蛋白上游调控机制的认识。