Institute of Clinical Biochemistry, Hannover Medical School (MHH), Carl-Neuberg-Straße 1, 30625 Hannover, Germany; Institute of Experimental Diabetes Research, Hannover Medical School (MHH), Carl-Neuberg-Straße 1, 30625 Hannover, Germany.
Institute of Clinical Biochemistry, Hannover Medical School (MHH), Carl-Neuberg-Straße 1, 30625 Hannover, Germany.
Biochim Biophys Acta Biomembr. 2020 Feb 1;1862(2):183096. doi: 10.1016/j.bbamem.2019.183096. Epub 2019 Oct 28.
Hydrogen peroxide (HO) plays a central role in redox signalling and in oxidative stress-mediated cell death. It is generated through multiple mechanisms at various intracellular sites. Due to its chemical stability it can reach distant sites of action. However, its hydrophilicity can hamper lipid membrane passage. We therefore studied the kinetics of HO diffusion through subcellular membranes employing the HO biosensor HyPer in insulin-producing RINm5F cells. Plasma- and ER-membrane-bound HyPer sensors facing the cytosolic compartment reacted twice as fast to HO compared to sensors expressed in peroxisomes and mitochondria. Overexpression of the HO-inactivating enzyme catalase in the ER-lumen and in the peroxisomes retarded the reaction time of HyPer, both localised within the peroxisomes as well as at the cytosolic surface of the ER. The unsaturated fatty acid oleic acid did not affect the reaction of the peroxisomal HyPer sensor to HO, while the saturated fatty acid palmitic acid accelerated its reaction time to HO in this organelle. The results show that the plasma-, peroxisomal, and mitochondrial membrane of insulin-producing RINm5F cells are permeable for HO. Nonetheless, the organelle membranes retard HO diffusion due to a barrier function of the lipid membrane, as documented by retarded reaction times of the intraorganellar sensors. Accelerated decomposition of HO by catalase, expressed in the peroxisomes or the ER, further retarded the HyPer sensor reaction time. The results show that redox signalling and oxidative stress-mediated toxicity are crucially dependent on physicochemical membrane properties and antioxidative defence mechanisms in health and disease.
过氧化氢（HO）在氧化还原信号转导和氧化应激介导的细胞死亡中起着核心作用。它通过多种机制在细胞内的不同部位产生。由于其化学稳定性，它可以到达作用的遥远部位。然而，其亲水性会阻碍脂质膜的穿透。因此，我们使用 HO 生物传感器 HyPer 研究了 HO 通过亚细胞膜扩散的动力学，该传感器在胰岛素产生的 RINm5F 细胞中表达。与在过氧化物酶体和线粒体中表达的传感器相比，面向细胞质区室的质膜和内质网膜结合的 HyPer 传感器对 HO 的反应速度快两倍。过氧化物酶体和内质网腔中 HO 失活酶过氧化氢酶的过表达会延迟 HyPer 的反应时间，无论是在过氧化物酶体中还是在内质网的细胞质表面表达。不饱和脂肪酸油酸不会影响过氧化物酶体 HyPer 传感器对 HO 的反应，而饱和脂肪酸棕榈酸会加速该细胞器中 HO 的反应时间。结果表明，胰岛素产生的 RINm5F 细胞的质膜、过氧化物酶体膜和线粒体膜对 HO 是可渗透的。尽管如此，由于脂质膜的屏障功能，细胞器膜会延迟 HO 的扩散，这可以通过细胞器内传感器的反应时间延迟来证明。过氧化物酶体或内质网中表达的过氧化氢酶加速 HO 的分解，进一步延迟了 HyPer 传感器的反应时间。结果表明，氧化还原信号转导和氧化应激介导的毒性在健康和疾病中都极大地依赖于物理化学膜性质和抗氧化防御机制。
