VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications.
VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications.
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DOI：
10.18632/oncotarget.6850
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发表时间：
2016-01-19
期刊：
影响因子：
--
通讯作者：
De Pinto V
中科院分区：
文献类型：
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作者：
Reina S;Checchetto V;Saletti R;Gupta A;Chaturvedi D;Guardiani C;Guarino F;Scorciapino MA;Magrì A;Foti S;Ceccarelli M;Messina AA;Mahalakshmi R;Szabo I;De Pinto V
电压依赖性阴离子选择通道（VDAC）是形成孔的线粒体外膜蛋白。在哺乳动物中，VDAC3是最不具特征的同种异构体，呈现出一组预计暴露于膜间隙的半胱氨酸。我们发现VDAC3中的半胱氨酸可以停留在不同的氧化态。这是初步观察到的，在我们的实验条件下，完全缺乏任何还原剂，VDAC3呈现出稍微不同的电泳迁移率模式。这一观察结果适用于大鼠肝脏线粒体VDAC3和重组和折叠的人类VDAC3。质谱分析表明，半胱氨酸2和8可以在天然VDAC3中形成二硫桥。半胱氨酸2、8和122的单一或联合定点突变表明，SDS-PAGE中蛋白质的流动性受到半胱氨酸存在和氧化还原状态的影响。此外，电生理、互补分析和化学物理表征表明，半胱氨酸2、8和122参与了孔的稳定性控制。此外，发现突变体的孔导率与它们补充无孔酵母突变体细胞生长的能力呈正相关。我们的工作为线粒体蛋白的复杂氧化模式提供了证据，该模式不直接参与电子传递。这种行为最可能的生物学意义是缓冲ROS负荷并跟踪膜间空间的氧化还原水平，最终通过构象变化发出信号。
Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes.