电压依赖性阴离子通道亚型(VDAC1,VDAC2和VDAC3)是线粒体外膜(OMM)的相关成分,在代谢调节和生存途径中起着至关重要的作用。作为调节细胞代谢和凋亡的主要参与者,VDAC可以被认为是在两个广泛的病理家族之间的十字路口,即,癌症和神经变性,前者与糖酵解率升高和癌细胞凋亡抑制有关,后者以线粒体功能障碍和细胞死亡增加为特征。最近,我们报道了大鼠和人VDAC中甲硫氨酸和半胱氨酸的氧化模式的表征,表明这些蛋白质中的每个半胱氨酸都具有优选的氧化态。从还原到三氧化形式,并且这种氧化态在大鼠和人VDAC之间非常保守。然而,二硫键在VDAC中的存在和定位,它们结构表征的关键点,到目前为止还没有确定。本文通过nanoUHPLC/高分辨率nanoESI-MS/MS研究了大鼠VDAC2(rVDAC2)中分子内二硫键的位置,一种含有11个半胱氨酸的蛋白质。为此,提取,净化,和酶消化在微酸性或中性pH下进行,以使二硫键交换最小化。明确确定了六个二硫键的存在,包括连接两个相邻的半胱氨酸4和5的二硫键,连接半胱氨酸9和14的二硫键,以及在半胱氨酸48、77和104之间的可选二硫键。二硫键,非常抵抗还原,在半胱氨酸134和139之间也被检测到。除了之前的发现,这些结果显著地扩展了rVDAC2中半胱氨酸的氧化态的表征,并且表明它是高度复杂的并且呈现不寻常的特征。数据可通过ProteomeXchange获得,标识符为PXD044041。
Voltage-Dependent Anion Channel isoforms (VDAC1, VDAC2, and VDAC3) are relevant components of the outer mitochondrial membrane (OMM) and play a crucial role in regulation of metabolism and in survival pathways. As major players in the regulation of cellular metabolism and apoptosis, VDACs can be considered at the crossroads between two broad families of pathologies, namely, cancer and neurodegeneration, the former being associated with elevated glycolytic rate and suppression of apoptosis in cancer cells, the latter characterized by mitochondrial dysfunction and increased cell death. Recently, we reported the characterization of the oxidation pattern of methionine and cysteines in rat and human VDACs showing that each cysteine in these proteins is present with a preferred oxidation state, ranging from the reduced to the trioxidized form, and such an oxidation state is remarkably conserved between rat and human VDACs. However, the presence and localization of disulfide bonds in VDACs, a key point for their structural characterization, have so far remained undetermined. Herein we have investigated by nanoUHPLC/High-Resolution nanoESI-MS/MS the position of intramolecular disulfide bonds in rat VDAC2 (rVDAC2), a protein that contains 11 cysteines. To this purpose, extraction, purification, and enzymatic digestions were carried out at slightly acidic or neutral pH in order to minimize disulfide bond interchange. The presence of six disulfide bridges was unequivocally determined, including a disulfide bridge linking the two adjacent cysteines 4 and 5, a disulfide bridge linking cysteines 9 and 14, and the alternative disulfide bridges between cysteines 48, 77, and 104. A disulfide bond, which is very resistant to reduction, between cysteines 134 and 139 was also detected. In addition to the previous findings, these results significantly extend the characterization of the oxidation state of cysteines in rVDAC2 and show that it is highly complex and presents unusual features. Data are available via ProteomeXchange with the identifier PXD044041.