Peptidylarginine Deiminase 2 Suppresses Inhibitory κB Kinase Activity in Lipopolysaccharide-stimulated RAW 264.7 Macrophages
Peptidylarginine Deiminase 2 Suppresses Inhibitory κB Kinase Activity in Lipopolysaccharide-stimulated RAW 264.7 Macrophages
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DOI：
10.1074/jbc.m110.170290
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发表时间：
2010-12-17
影响因子：
4.8
通讯作者：
Christman, Brian W.
中科院分区：
文献类型：
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作者：
Lee, Hye Jeong;Joo, Myungsoo;Christman, Brian W.
肽基精氨酸脱亚胺酶（PAD）是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介导的瓜氨酸及其对巨噬细胞系RAW 264.7中促炎活性的影响。当用脂多糖（LPS; 1 μ g/ml）处理细胞时，45-65-kDa蛋白质的瓜氨酸被诱导。瓜氨酸蛋白被细胞内钙螯合剂BAPTA/AM（30 μ M）抑制。LPS处理上调细胞中的考克斯-2水平。有趣的是，过表达PAD 2使LPS介导的考克斯-2上调减少50%。PAD 2过表达也降低NF-κ B B活性，这通过NF-κ B驱动的荧光素酶活性来确定。通过使用用NF-κ B荧光素酶、I κ B β/γ激酶（IKK β/γ）亚基和PAD 2转染的HEK 293细胞进一步检查PAD 2对NF-κ B活性的影响。IKK β增加NF-κ B活性，但当细胞中存在PAD 2时，这种增加被显著抑制。在钙离子载体A23187存在下，IKK β可进一步增强IKK κ介导的NF-κ B活化。然而，这种IKK β/γ的刺激作用被PAD 2消除。细胞裂解物的免疫共沉淀表明，IKK γ和PAD 2可以在Ca 2+离子载体的存在下免疫共沉淀。IKK γ共免疫沉淀截短突变体，PAD 2（1-385）和PAD 2（355-672）。用Ala取代Gln-358（Ca 2+结合的假定配体）消除了免疫共沉淀。相反，PAD 2共免疫沉淀截短突变体IKK γ（1-196）和IKK γ（197419）。在其他实验中，用LPS处理RAW 264.7细胞诱导了IKK γ免疫沉淀物中的瓜氨酸。体外瓜氨酸测定表明，在存在Ca 2+瓜氨酸化IKK γ的情况下，孵育纯化的PAD 2和IKK γ蛋白。这些结果表明PAD 2与IKK γ相互作用并抑制NF-κ B活性。
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 mu g/ml). Protein citrullination was suppressed by the intracellular calcium chelator BAPTA/AM (30 mu M). 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-kappa B activity, determined by NF-kappa B-driven luciferase activity. The effect of PAD2 on NF-kappa B activity was further examined by using HEK 293 cells transfected with NF-kappa B luciferase, I kappa B beta/gamma kinase (IKK beta/gamma) subunits, and PAD2. IKK beta increased NF-kappa B activity, but this increase was markedly suppressed when PAD2 was present in cells. IKK kappa-mediated NF-kappa B activation was further enhanced by IKK beta in the presence of calcium ionophore A23187. However, this stimulatory effect of IKK beta/gamma was abolished by PAD2. Coimmunoprecipitation of cell lysates showed that IKK gamma and PAD2 can coimmunoprecipitate in the presence of the Ca2+ ionophore. IKK gamma coimmunoprecipitated truncation mutants, PAD2(1-385) and PAD2(355-672). The substitution of Gln-358 (a putative ligand for Ca2+ binding) with an Ala abolished coimmunoprecipitation. Conversely, PAD2 coimmunoprecipitated truncation mutants IKK gamma(1-196) and IKK gamma(197419). In other experiments, treating RAW 264.7 cells with LPS induced citrullination in the immunoprecipitates of IKK gamma. In vitro citrullination assay showed that incubation of purified PAD2 and IKK gamma proteins in the presence of Ca2+ citrullinated IKK gamma. These results demonstrate that PAD2 interacts with IKK gamma and suppresses NF-kappa B activity.