The Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 29208, USA.
ACS Chem Biol. 2011 May 20;6(5):466-76. doi: 10.1021/cb1003515. Epub 2011 Feb 7.
The protein arginine deiminases (PADs), which catalyze the hydrolysis of peptidyl-arginine to form peptidyl-citrulline, are potential targets for the development of a rheumatoid arthritis (RA) therapeutic, as well as other human diseases including colitis and cancer. Additionally, these enzymes, and in particular PAD4, appear to play important roles in a variety of cell signaling pathways including apoptosis, differentiation, and transcriptional regulation. To better understand the factors that regulate in vivo PAD4 activity, we set out to design and synthesize a series of activity-based protein profiling (ABPP) reagents that target this enzyme. Herein we describe the design, synthesis, and evaluation of six ABPPs including (i) FITC-conjugated F-amidine (FFA1 and 2) and Cl-amidine (FCA1 and 2), and (ii) biotin-conjugated F-amidine (BFA) and Cl-amidine (BCA). We further demonstrate the utility of these probes for labeling PAD4 in cells, as well as for isolating PAD4 and PAD4 binding proteins. These probes will undoubtedly prove to be powerful tools that can be used to dissect the factors controlling the dynamics of PAD4 expression, activity, and function.
精氨酸脱亚氨酶（PADs）可催化肽基精氨酸水解形成肽基瓜氨酸，它们是开发类风湿关节炎（RA）治疗药物以及包括结肠炎和癌症在内的其他人类疾病的潜在靶点。此外，这些酶，尤其是 PAD4，似乎在多种细胞信号通路中发挥重要作用，包括细胞凋亡、分化和转录调控。为了更好地了解调节体内 PAD4 活性的因素，我们着手设计并合成了一系列针对该酶的基于活性的蛋白质分析（ABPP）试剂。在此，我们描述了六种 ABPP 的设计、合成和评估，包括（i）FITC 缀合的 F-脒基（FFA1 和 2）和 Cl-脒基（FCA1 和 2），以及（ii）生物素缀合的 F-脒基（BFA）和 Cl-脒基（BCA）。我们进一步证明了这些探针在细胞中标记 PAD4、分离 PAD4 和 PAD4 结合蛋白方面的用途。这些探针无疑将被证明是强大的工具，可用于剖析控制 PAD4 表达、活性和功能动态的因素。
