Raw mass spectrometry data for publication "Vertebrate cellular endolysosome modulating pore-forming protein is negatively regulated by its homologue under environmental oxidative conditions"

Abstract: Endolysosomes are key players in cell physiology, including material exchange, immunity and environmental adaptation etc. Bacterial pore-forming toxin aerolysin-like proteins (ALPs) are widely distributed in animals and plants. βγ-CAT is a complex of an ALP (BmALP1) and a trefoil factor (BmTFF3) in the frog Bombina maxima. It is the first example that a secreted endogenous pore-forming protein modulates the biochemical properties of endolysosomes via pore formation in these vesicles. Here, we report the identification of BmALP3, a homologue of BmALP1 that lacks membrane pore formation capacity. Both BmALP3 and BmALP1 contain a conserved cysteine in their C-terminal regions. BmALP3 was readily oxidized to disulfide bond linked homodimer, and the homodimer could then oxidize BmALP1 via disulfide bond exchange, resulting in the dissociation of βγ-CAT subunits and elimination of its biological activity. Consistent with its behavior in vitro, BmALP3 senses environmental oxygen tension in vivo, leading to modulation of βγ-CAT activity. Interestingly, this C-terminal cysteine site is well conserved in numerous vertebrate ALPs. These findings, for the first time, uncovered the existence of a regulatory ALP (BmALP3) and its modulating action on a cell executive ALP (BmALP1) in a redox-dependent manner, which is completely different from that of bacterial toxin aerolysins.

摘要：内体溶酶体（endolysosomes）是细胞生理活动的核心参与者，参与物质交换、免疫调控及环境适应等多种生命过程。细菌孔道形成毒素类气单胞菌溶素蛋白（aerolysin-like proteins，ALPs）广泛分布于动植物体内。βγ-CAT是大蹼铃蟾（Bombina maxima）体内由类气单胞菌溶素蛋白BmALP1与三叶因子BmTFF3组成的复合物，也是首个被证实可通过在囊泡中形成孔道来调控内体溶酶体生化特性的分泌型内源性孔道形成蛋白。本研究鉴定得到BmALP3——一种缺乏膜孔形成能力的BmALP1同源蛋白。BmALP3与BmALP1的C端区域均含有保守的半胱氨酸残基。BmALP3可自发氧化形成二硫键连接的同源二聚体，该二聚体可通过二硫键交换介导BmALP1的氧化，进而导致βγ-CAT的亚基解离并使其生物学活性丧失。与体外实验结果一致，BmALP3在活体中可感知环境氧分压，从而调控βγ-CAT的活性。值得注意的是，该C端半胱氨酸位点在众多脊椎动物的类气单胞菌溶素蛋白中均高度保守。本研究首次揭示了一类调控型类气单胞菌溶素蛋白BmALP3的存在，及其以氧化还原依赖方式对细胞执行型类气单胞菌溶素蛋白BmALP1的调控作用，这一机制与细菌毒素类气单胞菌溶素的作用模式完全不同。