Division of Allergy, Pulmonary, and Critical Care Medicine, Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232, USA.
J Biol Chem. 2010 Dec 17;285(51):39655-62. doi: 10.1074/jbc.M110.170290. Epub 2010 Oct 11.
Peptidylarginine deiminases (PADs) are enzymes that convert arginine to citrulline in proteins. In this study, we examined PAD-mediated citrullination and its effect on pro-inflammatory activity in the macrophage cell line RAW 264.7. Citrullination of 45-65-kDa proteins was induced when cells were treated with lipopolysaccharide (LPS; 1 μg/ml). Protein citrullination was suppressed by the intracellular calcium chelator BAPTA/AM (30 μM). LPS treatment up-regulated COX-2 levels in cells. Interestingly, overexpressing PAD2 reduced LPS-mediated COX-2 up-regulation by 50%. PAD2 overexpression also reduced NF-κB activity, determined by NF-κB-driven luciferase activity. The effect of PAD2 on NF-κB activity was further examined by using HEK 293 cells transfected with NF-κB luciferase, IκB β/γ kinase (IKKβ/γ) subunits, and PAD2. IKKβ increased NF-κB activity, but this increase was markedly suppressed when PAD2 was present in cells. IKKβ-mediated NF-κB activation was further enhanced by IKKγ in the presence of calcium ionophore A23187. However, this stimulatory effect of IKKβ/γ was abolished by PAD2. Coimmunoprecipitation of cell lysates showed that IKKγ and PAD2 can coimmunoprecipitate in the presence of the Ca(2+) ionophore. IKKγ coimmunoprecipitated truncation mutants, PAD2(1-385) and PAD2(355-672). The substitution of Gln-358 (a putative ligand for Ca(2+) binding) with an Ala abolished coimmunoprecipitation. Conversely, PAD2 coimmunoprecipitated truncation mutants IKKγ(1-196) and IKKγ(197-419). In other experiments, treating RAW 264.7 cells with LPS induced citrullination in the immunoprecipitates of IKKγ. In vitro citrullination assay showed that incubation of purified PAD2 and IKKγ proteins in the presence of Ca(2+) citrullinated IKKγ. These results demonstrate that PAD2 interacts with IKKγ and suppresses NF-κB activity.
肽基精氨酸脱亚氨酶（PADs）是一种将蛋白质中的精氨酸转化为瓜氨酸的酶。在这项研究中，我们研究了 PAD 介导的瓜氨酸化及其对 RAW 264.7 巨噬细胞系促炎活性的影响。当细胞用脂多糖（LPS；1μg/ml）处理时，诱导了 45-65kDa 蛋白的瓜氨酸化。细胞内钙螯合剂 BAPTA/AM（30μM）抑制 LPS 处理诱导的蛋白瓜氨酸化。LPS 处理上调了细胞中环氧化酶-2（COX-2）水平。有趣的是，过表达 PAD2 可使 LPS 介导的 COX-2 上调减少 50%。PAD2 过表达还降低了 NF-κB 活性，这是通过 NF-κB 驱动的荧光素酶活性来确定的。通过用 NF-κB 荧光素酶、IκBβ/γ 激酶（IKKβ/γ）亚基和 PAD2 转染 HEK 293 细胞进一步研究了 PAD2 对 NF-κB 活性的影响。IKKβ 增加了 NF-κB 活性，但当细胞中存在 PAD2 时，这种增加明显受到抑制。在钙离子载体 A23187 的存在下，IKKβ 介导的 NF-κB 激活进一步增强了 IKKγ。然而，当存在 PAD2 时，这种 IKKβ/γ 的刺激作用被消除。细胞裂解物的共免疫沉淀表明，在钙离子载体存在下，IKKγ 和 PAD2 可以共免疫沉淀。IKKγ 共免疫沉淀截短突变体 PAD2(1-385)和 PAD2(355-672)。用丙氨酸取代 Gln-358（一种假定的 Ca2+结合配体），则共免疫沉淀被消除。相反，PAD2 共免疫沉淀截短突变体 IKKγ(1-196)和 IKKγ(197-419)。在其他实验中，用 LPS 处理 RAW 264.7 细胞诱导了 IKKγ 的免疫沉淀中的瓜氨酸化。体外瓜氨酸化实验表明，在 Ca2+存在下孵育纯化的 PAD2 和 IKKγ 蛋白使 IKKγ 瓜氨酸化。这些结果表明 PAD2 与 IKKγ 相互作用并抑制 NF-κB 活性。
