Evidence for a superoxide permeability pathway in endosomal membranes
Evidence for a superoxide permeability pathway in endosomal membranes
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DOI：
10.1128/mcb.02038-07
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发表时间：
2008-06-01
影响因子：
5.3
通讯作者：
Engelhardt, John F.
中科院分区：
文献类型：
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作者：
Mumbengegwi, Davis R.;Li, Qiang;Engelhardt, John F.
内体NADPH氧化酶区域化生产超氧化物（中心点O-2(-)）在白细胞介素1 β (IL-10)刺激后nf - κ B的氧化还原依赖性激活中是重要的。目前尚不清楚核内体中产生的中心点O-2（-）如何促进细胞质中氧化还原依赖的信号事件。我们评估了中心点O-2（-）从IL-1 β刺激的核内体中移出，以及核内体表面的SOD1是否介导nf - κ B激活所需的氧化还原信号事件。利用膜透性（发光氨和荧光素）和-不透性（异亮醇）发光探针对中心点O-2（-）进行了nadph依赖性中心点O-2（-）的相对外透性评估。在这些研究中，大约60%的中心点O-2（-）流出核内体被两种阴离子通道阻滞剂(4'-二异硫氰酸-2,2'-二磺酸二苯乙烯（DIDS）或尼氟酸（NFA）中的任何一种抑制。此外，对膜蛋白脂质体的放射性同位素电扩散通量测定表明，O-2（-）和Cl-通过相同的dids敏感通道运输。基于rab5的免疫亲和分离IL-1 β刺激的早期核内体显示SOD1向含有IL-1受体的核内体募集。最后，发现sod1缺陷细胞在IL-1刺激后激活nf - κ B的能力存在缺陷。总之，这些结果表明，中心点O-2（-）通过dids敏感的氯离子通道进入核内体，sod1介导的核内体表面中心点O-2（-）的突变可能产生NF-kappa B氧化还原激活所需的局部H2O2。
The compartmentalized production of superoxide (center dot O-2(-)) by endosomal NADPH oxidase is important in the redox-dependent activation of NF-kappa B following interleuldn 1 beta (IL-10) stimulation. It remains unclear how center dot O-2(-) produced within endosomes facilitates redox-dependent signaling events in the cytoplasm. We evaluated center dot O-2(-) movement out of IL-1 beta-stimulated endosomes and whether SOD1 at the endosomal surface mediates redox-signaling events required for NF-kappa B activation. The relative outward permeability of NADPH-dependent center dot O-2(-) from fractionated endosomes was assessed using membrane-permeable (luminol and lucigenin) and -impermeable (isoluminol) luminescent probes for center dot O-2(-). In these studies, similar to 60% of center dot O-2(-) efflux out of endosomes was inhibited by treatment with either of two anion channel blockers, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) or niflumic acid (NFA). Furthermore, radioisotopic electrodiffusion flux assays on endomembrane proteoliposomes suggested that O-2(-) and Cl- are transported through the same DIDS-sensitive channel(s). Rab5-based immunoaffinity isolation of IL-1 beta-stimulated early endosomes demonstrated SOD1 recruitment to endosomes harboring the IL-1 receptor. Finally, SOD1-deficient cells were found to be defective in their ability to activate NF-kappa B following IL-1 beta stimulation. Together, these results suggest that center dot O-2(-) exits endosomes through a DIDS-sensitive chloride channel(s) and that SOD1-mediated dismutation of center dot O-2(-) at the endosomal surface may produce the localized H2O2 required for redox-activation of NF-kappa B.