Table S1. Compounds mixtures (ten-in-one) information. from Small molecule activates citrullination through targeting PAD2

Citrullination is a post-translational modification catalysed by peptidyl arginine deiminase (PAD) enzymes, dysregulation of protein citrullination is involved in various pathological disorders. During the last decade, a panel of citrullination inhibitors have been developed, while small molecules activating citrullination are rarely reported so far. In the study, we screened citrullination activator using an antibody against citrullinated histone H3 (cit-H3), and a natural compound demethoxycurcumin (DMC) significantly activated citrullination. The requirement of PAD2 for DMC-activated citrullination was confirmed by loss of functional assay. Notably, DMC directly engaged with PAD2, and showed binding selectivity among PAD family enzymes. Point mutation assay indicated that residue E352 is essential for DMC targeting PAD2. Consistently, DMC induced typical phenotypes of cells with dysregulation of PAD2 activity, including citrullination-associated cell apoptosis and DNA damage. Overall, our study not only presents a strategy for rationally screening citrullination activator, but also provides a chemical approach for activating protein citrullination.This article is part of the Theo Murphy meeting issue ‘The virtues and vices of protein citrullination’.瓜氨酸化（citrullination）是由肽基精氨酸脱亚胺酶（peptidyl arginine deiminase, PAD）家族酶催化的一类翻译后修饰，蛋白质瓜氨酸化的失调参与多种病理疾病的发生发展。近十年来，学界已开发出多款瓜氨酸化抑制剂，但截至目前，可激活瓜氨酸化的小分子化合物仍鲜有报道。本研究通过靶向瓜氨酸化组蛋白H3（citrullinated histone H3, cit-H3）的抗体开展瓜氨酸化激活剂筛选，发现天然化合物去甲氧基姜黄素（demethoxycurcumin, DMC）可显著激活瓜氨酸化过程。功能缺失实验证实，PAD2是DMC介导瓜氨酸化激活的必需因子。值得注意的是，DMC可直接与PAD2结合，且在PAD家族酶中展现出结合选择性。定点突变实验结果显示，氨基酸残基E352是DMC靶向结合PAD2的关键必需位点。一致性实验表明，DMC可诱导PAD2活性失调细胞出现典型表型，包括瓜氨酸化相关的细胞凋亡与DNA损伤。总体而言，本研究不仅提出了一套理性筛选瓜氨酸化激活剂的研究策略，同时也为激活蛋白质瓜氨酸化提供了一种化学研究手段。本文属于《西奥·墨菲会议专题：蛋白质瓜氨酸化的利与弊》（The virtues and vices of protein citrullination）的特刊文章。