Department of Neuroscience, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan.
Division of Malaria Research, Proteo-Science Center, Ehime University, Matsuyama, Japan.
Front Cell Infect Microbiol. 2022 Sep 9;12:992198. doi: 10.3389/fcimb.2022.992198. eCollection 2022.
Irgb6 is a member of interferon γ-induced immunity related GTPase (IRG), and one of twenty "effector" IRGs, which coordinately attack parasitophorous vacuole membrane (PVM), causing death of intracellular pathogen. Although Irgb6 plays a pivotal role as a pioneer in the process of PVM disruption, the direct effect of Irgb6 on membrane remained to be elucidated. Here, we utilized artificial lipid membranes to reconstitute Irgb6-membrane interaction , and revealed that Irgb6 directly deformed the membranes. Liposomes incubated with recombinant Irgb6 were drastically deformed generating massive tubular protrusions in the absence of guanine nucleotide, or with GMP-PNP. Liposome deformation was abolished by incubating with Irgb6-K275A/R371A, point mutations at membrane targeting residues. The membrane tubules generated by Irgb6 were mostly disappeared by the addition of GTP or GDP, which are caused by detachment of Irgb6 from membrane. Binding of Irgb6 to the membrane, which was reconstituted using lipid monolayer, was stimulated at GTP-bound state. Irgb6 GTPase activity was stimulated by the presence of liposomes more than eightfold. Irgb6 GTPase activity in the absence of membrane was also slightly stimulated, by lowering ionic strength, or by increasing protein concentration, indicating synergistic stimulation of the GTPase activity. These results suggest that membrane targeting of Irgb6 and resulting membrane deformation does not require GTP, but converting into GTP-bound state is crucial for detaching Irgb6 from the membrane, which might coincident with local membrane disruption.
Irgb6 是干扰素 γ 诱导的免疫相关 GTPase（IRG）家族的成员，也是二十种“效应”IRG 之一，它们协同攻击寄生泡膜（PVM），导致细胞内病原体死亡。虽然 Irgb6 在破坏 PVM 的过程中发挥了关键作用，但 Irgb6 对膜的直接作用仍有待阐明。在这里，我们利用人工脂质膜重建了 Irgb6-膜相互作用，揭示了 Irgb6 直接使膜变形。在没有鸟嘌呤核苷酸或 GMP-PNP 的情况下，与重组 Irgb6 孵育的脂质体严重变形，产生大量管状突起。在膜靶向残基点突变 Irgb6-K275A/R371A 的情况下，脂质体变形被消除。添加 GTP 或 GDP 会使 Irgb6 产生的膜管大部分消失，这是由于 Irgb6 从膜上脱离。使用脂质单层重建的膜，Irgb6 与膜的结合在 GTP 结合状态下受到刺激。脂质体的存在使 Irgb6 的 GTPase 活性增加了超过八倍。在没有膜的情况下，Irgb6 GTPase 活性也略有增加，这是通过降低离子强度或增加蛋白质浓度实现的，这表明 GTPase 活性的协同刺激。这些结果表明，Irgb6 的膜靶向和由此产生的膜变形不需要 GTP，但转化为 GTP 结合状态对于 Irgb6 从膜上脱离至关重要，这可能与局部膜破坏一致。
