多梳基团(PGs)是转录抑制因子,由几种蛋白质的复合物组成,参与多细胞发育和癌症表观遗传学。这些蛋白质之一是E3泛素蛋白连接酶RING1(或RING1B),与转录抑制的调节相关,并负责组蛋白H2A的单倍体化。另一方面,PADI4是与精氨酸转化为瓜氨酸有关的酶家族的人类同种型之一。它也参与了胶质母细胞瘤的发展,在其他类型的癌症中。在这项工作中,我们通过免疫荧光和邻近连接分析显示了几种癌细胞系中细胞核和细胞质中PADI4和RING1B的关联。此外,我们证明了在酶促PADI4抑制剂GSK484的存在下结合受到阻碍,这表明RING1B可以与PADI4的活性位点结合,正如蛋白质-蛋白质对接模拟所证实的那样。体外和计算机发现表明,与RING1B的N末端(残基1-221)和C末端(残基228-336)区域相对应的分离片段发生了与PADI4的结合。GSK484也阻碍了与PADI4的结合,如等温滴定量热法(ITC)实验所示,C末端的NMR和ITC。PADI4和任何两个分离的RING1B片段之间的解离常数在低微摩尔范围内(〜2-10μM),通过荧光和ITC测量。RING1B和PADI4之间的相互作用可能意味着前者的瓜氨酸化,导致了一些生物学后果,以及通过产生新抗原改善癌症治疗的潜在治疗相关性。
Polycomb groups (PcGs) are transcriptional repressors, formed by a complex of several proteins, involved in multicellular development and cancer epigenetics. One of these proteins is the E3 ubiquitin-protein ligase RING1 (or RING1B), associated with the regulation of transcriptional repression and responsible for monoubiquitylation of the histone H2A. On the other hand, PADI4 is one of the human isoforms of a family of enzymes implicated in the conversion of arginine to citrulline, and it is also involved in the development of glioblastoma, among other types of cancers. In this work, we showed the association of PADI4 and RING1B in the nucleus and cytosol in several cancer cell lines by using immunofluorescence and proximity ligation assays. Furthermore, we demonstrated that binding was hampered in the presence of GSK484, an enzymatic PADI4 inhibitor, suggesting that RING1B could bind to the active site of PADI4, as confirmed by protein-protein docking simulations. In vitro and in silico findings showed that binding to PADI4 occurred for the isolated fragments corresponding to both the N-terminal (residues 1-221) and C-terminal (residues 228-336) regions of RING1B. Binding to PADI4 was also hampered by GSK484, as shown by isothermal titration calorimetry (ITC) experiments for the sole N-terminal region, and by both NMR and ITC for the C-terminal one. The dissociation constants between PADI4 and any of the two isolated RING1B fragments were in the low micromolar range (~2-10 μM), as measured by fluorescence and ITC. The interaction between RING1B and PADI4 might imply citrullination of the former, leading to several biological consequences, as well as being of potential therapeutic relevance for improving cancer treatment with the generation of new antigens.