Laboratory of Applied Biochemistry, Division of Biotechnology Review and Research III, Office of Biotechnology Products, Center for Drug Evaluation and Research, U.S. Food and Drug Administration, Silver Spring, Maryland 20993, United States.
Oncology Center of Excellence and Center for Drug Evaluation and Research, Office of Oncologic Diseases, U.S. Food and Drug Administration, Silver Spring, Maryland 20993, United States.
Biochemistry. 2023 Feb 21;62(4):893-898. doi: 10.1021/acs.biochem.2c00551. Epub 2023 Feb 9.
Post-translational modification of arginine to citrulline is catalyzed by members of the peptidylarginine deiminase (PAD) family. Dysregulation of this catalysis is a significant driver of the pathogenesis of numerous inflammatory diseases, including cancer. However, dysregulation of PAD activity has not been examined in breast cancer with respect to hormone receptor status. In this study, we measured PAD enzyme levels using Western blotting and investigated protein citrullination using a mass spectrometry-based proteomics approach in primary estrogen receptor negative (ER-) or positive (ER+) breast tumor and matched adjacent normal tissue. Our findings reveal 72 and 41 citrullinated proteins in ER- tumor and adjacent healthy tissue, respectively, where 20 of these proteins are common between the two groups. We detected 64 and 49 citrullinated proteins in ER+ tumor and adjacent healthy tissue, respectively, where 32 proteins are common. Interestingly, upon comparison of ER- and ER+ tumor tissue, only 32 citrullinated proteins are shared between the two and the rest are unique to the tumor's receptor status. Using the STRING database for protein-protein interaction network analysis, these proteins are involved in protein-folding events (i.e., heat shock proteins) in ER- samples and blood-clotting events (i.e., fibulin) in ER+ samples. Constituents of the extracellular matrix structure (i.e., collagen and fibrinogen) were found in both. Herein, we establish evidence that supports the role of this unique post-translational modification in breast cancer biology. Finally, to aid drug discovery against citrullination, we developed a liquid chromatography-ultraviolet method to measure PAD enzymatic activity and optimized glucagon-like peptide II to quantitatively measure the ability of PADs to citrullinate its substrate.
精氨酸的翻译后修饰为瓜氨酸是由肽基精氨酸脱亚氨酶（PAD）家族成员催化的。这种催化的失调是许多炎症性疾病（包括癌症）发病机制的重要驱动因素。然而，在乳腺癌中，尚未针对激素受体状态检查 PAD 活性的失调。在这项研究中，我们使用 Western blot 测量了 PAD 酶水平，并使用基于质谱的蛋白质组学方法研究了原发性雌激素受体阴性（ER-）或阳性（ER+）乳腺癌肿瘤和匹配的相邻正常组织中的蛋白质瓜氨酸化。我们的发现揭示了 ER-肿瘤和相邻健康组织中分别有 72 和 41 种瓜氨酸化蛋白，其中 20 种蛋白在两组之间是共同的。我们在 ER+肿瘤和相邻健康组织中分别检测到 64 和 49 种瓜氨酸化蛋白，其中 32 种蛋白是共同的。有趣的是，在 ER-和 ER+肿瘤组织之间进行比较时，只有 32 种瓜氨酸化蛋白在两组之间共享，其余蛋白则是肿瘤受体状态所特有的。使用 STRING 数据库进行蛋白质-蛋白质相互作用网络分析，这些蛋白质参与 ER-样本中的蛋白质折叠事件（即热休克蛋白）和 ER+样本中的血凝事件（即纤维蛋白）。细胞外基质结构的组成部分（即胶原蛋白和纤维蛋白原）在两者中都有发现。在此，我们提供了支持这种独特的翻译后修饰在乳腺癌生物学中作用的证据。最后，为了辅助针对瓜氨酸化的药物发现，我们开发了一种液相色谱-紫外法来测量 PAD 酶活性，并优化了胰高血糖素样肽 II 以定量测量 PAD 使自身底物瓜氨酸化的能力。
