细胞铁死亡中新型羰基化修饰的组学发现和修饰率的定量分析

Ferroptosis is a newly defined type of cell death that is morphologically and mechanistically distinct from apoptosis, necrosis and autophagy. It is functionally implicated with cancer and multiple degenerative diseases, however, the molecular mechanism of ferroptosis remains elusive and a defined molecular marker for this new type of cell death is still lacking. Ferroptosis is accompanied by elevated levels of lipid ROS, which in turn attack and degrade polyunsaturated fatty acids in cell membranes, producing a diversified array of lipid-derived electrophiles (LDEs). LDE molecules can covalently modify proteins which result in the so-called “protein carbonylations”. In a previous work, we developed an aniline-based chemical probe that enabled specific capture and site-specific profiling of protein carbonylations in proteomes. Using this probe, we identified several endogenous carbonylation sites in ferroptotic cells that are modified by LDEs with known structures, including 4-hydroxy-2-nonenal (HNE) and 4-oxy-2-nonenal (ONE). Building on the aforementioned work, we proposed here to develop advanced chemoproteomic strategies to discover protein carbonylations with novel molecular structures as well as to quantitatively profile modification stoichiometries of carbonylated proteins in proteomes. Our proposed methods will rely on the aniline-based probes for chemical capture and incorporate new technologies such as mass shift tags, isotope-coded tags and open database search. By applying these novel methods to profile functional carbonylations in ferroptotic proteomes, we aim to uncover potential molecular markers and provide new mechanistic insights for this new type of cell death.

铁死亡是一种有别于凋亡、坏死和自噬的新型细胞死亡方式，与癌症和多种退行性疾病有重要关联，然而目前其分子机制尚不明确。细胞发生铁死亡时胞内产生大量脂源性活性氧自由基，攻击膜内不饱和脂肪酸，降解生成不同类型的脂源性亲电小分子，与蛋白质共价反应产生羰基化修饰。申请人在前期工作中发展了羰基化特异性的苯胺化学捕捉探针，在铁死亡细胞中鉴定到被4-羟基-2-壬烯醛（HNE）和4-氧代-2-壬烯醛（ONE）等已知脂源性亲电分子羰基化修饰的位点。在此基础上，申请人在本项目中拟发展基于苯胺探针的化学蛋白组学方法，结合质量位移标签、同位素质谱特征标签和开放式数据搜索等前沿技术，系统地分析铁死亡细胞中羰基化修饰的修饰率，鉴定有别于HNE和ONE的新型羰基化修饰，发现潜在的基于羰基化修饰的铁死亡分子标志物，助力铁死亡的功能和机制研究。

铁死亡是一种有别于凋亡、坏死和自噬的新型细胞死亡方式，与癌症和多种退行性疾病有重要关联。细胞发生铁死亡时胞内氧化水平升高，生成例如HNE等脂源性亲电小分子，与蛋白质共价反应产生羰基化修饰。在本项目中，我们发展了一种基于质量标签位移的蛋白质翻译后修饰率的定量组学方法，并利用该技术完成了亲电内源代谢物HNE在全细胞蛋白质组中产生羰基化修饰的修饰率的定量分析。我们还将该质量位移策略进一步拓展用于双官能团活性代谢物在蛋白质组内产生蛋白间共价交联的分析，系统地鉴定了甲基乙二醛（MGO）在蛋白质组交联的靶标蛋白。在细胞铁死亡新型修饰的研究中，我们发现蛋白质DJ-1的第106位半胱氨酸上发生了磺酰化修饰，导致DJ-1与20S蛋白酶体解离，激活蛋白酶体活性，促进细胞发生铁死亡。我们的工作不仅为在蛋白质组水平定量分析不同类型蛋白质翻译后提供了有力的工具，还为细胞铁死亡的功能和机制研究提供了丰富的数据和线索。