Kwon Jaeyul, Wang Aibing, Burke Devin J, Boudreau Howard E, Lekstrom Kristen J, Korzeniowska Agnieszka, Sugamata Ryuichi, Kim Yong-Soo, Yi Liang, Ersoy Ilker, Jaeger Stefan, Palaniappan Kannappan, Ambruso Daniel R, Jackson Sharon H, Leto Thomas L
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA.
Department of Medical Education, School of Medicine, Chungnam National University, Daejeon, 301-747, Korea.
Free Radic Biol Med. 2016 Jul;96:99-115. doi: 10.1016/j.freeradbiomed.2016.04.009. Epub 2016 Apr 14.
Nox1 is an abundant source of reactive oxygen species (ROS) in colon epithelium recently shown to function in wound healing and epithelial homeostasis. We identified Peroxiredoxin 6 (Prdx6) as a novel binding partner of Nox activator 1 (Noxa1) in yeast two-hybrid screening experiments using the Noxa1 SH3 domain as bait. Prdx6 is a unique member of the Prdx antioxidant enzyme family exhibiting both glutathione peroxidase and phospholipase A2 activities. We confirmed this interaction in cells overexpressing both proteins, showing Prdx6 binds to and stabilizes wild type Noxa1, but not the SH3 domain mutant form, Noxa1 W436R. We demonstrated in several cell models that Prdx6 knockdown suppresses Nox1 activity, whereas enhanced Prdx6 expression supports higher Nox1-derived superoxide production. Both peroxidase- and lipase-deficient mutant forms of Prdx6 (Prdx6 C47S and S32A, respectively) failed to bind to or stabilize Nox1 components or support Nox1-mediated superoxide generation. Furthermore, the transition-state substrate analogue inhibitor of Prdx6 phospholipase A2 activity (MJ-33) was shown to suppress Nox1 activity, suggesting Nox1 activity is regulated by the phospholipase activity of Prdx6. Finally, wild type Prdx6, but not lipase or peroxidase mutant forms, supports Nox1-mediated cell migration in the HCT-116 colon epithelial cell model of wound closure. These findings highlight a novel pathway in which this antioxidant enzyme positively regulates an oxidant-generating system to support cell migration and wound healing.
Nox1是结肠上皮细胞中活性氧（ROS）的丰富来源，最近研究表明其在伤口愈合和上皮稳态中发挥作用。在以Noxa1的SH3结构域为诱饵的酵母双杂交筛选实验中，我们鉴定出过氧化物酶体增殖物激活受体6（Prdx6）是Nox激活剂1（Noxa1）的新型结合伴侣。Prdx6是Prdx抗氧化酶家族的独特成员，兼具谷胱甘肽过氧化物酶和磷脂酶A2活性。我们在同时过表达这两种蛋白的细胞中证实了这种相互作用，结果显示Prdx6与野生型Noxa1结合并使其稳定，但不与SH3结构域突变体形式Noxa1 W436R结合。我们在多个细胞模型中证明，敲低Prdx6可抑制Nox1活性，而增强Prdx6表达则能支持更高水平的Nox1衍生超氧化物生成。Prdx6的过氧化物酶和脂肪酶缺陷突变体形式（分别为Prdx6 C47S和S32A）均无法与Nox1组分结合或使其稳定，也不能支持Nox1介导的超氧化物生成。此外，Prdx6磷脂酶A2活性的过渡态底物类似物抑制剂（MJ - 33）可抑制Nox1活性，这表明Nox1活性受Prdx6的磷脂酶活性调控。最后，在HCT - 116结肠上皮细胞伤口闭合模型中，野生型Prdx6而非脂肪酶或过氧化物酶突变体形式可支持Nox1介导的细胞迁移。这些发现突出了一条新途径，即这种抗氧化酶正向调节一个产氧化系统以支持细胞迁移和伤口愈合。
