Naja naja oxiana Cobra Venom Cytotoxins CTI and CTII Disrupt Mitochondrial Membrane Integrity: Implications for Basic Three-Fingered Cytotoxins
Naja naja oxiana Cobra Venom Cytotoxins CTI and CTII Disrupt Mitochondrial Membrane Integrity: Implications for Basic Three-Fingered Cytotoxins
复制标题
DOI：
10.1371/journal.pone.0129248
复制
发表时间：
2015-06-19
期刊：
影响因子：
3.7
通讯作者：
Dagda, Ruben K.
中科院分区：
文献类型：
--
作者：
Gasanov, Sardar E.;Shrivastava, Indira H.;Dagda, Ruben K.
眼镜蛇毒液的细胞毒素是基本的三指，两性，非酶的蛋白质，构成了眼镜蛇毒液的主要部分。虽然细胞毒素在不同类型的细胞中引起线粒体功能障碍，但细胞毒素与线粒体结合的机制尚不清楚。我们分析了Naja Naja xiana的CTI和CTII， s型和p型细胞毒素，分别与分离的线粒体部分或模型膜相关联的能力，通过使用无数的生物物理技术来模拟线粒体脂质环境。磷-31核磁共振（P-31-NMR）波谱数据表明，这两种细胞毒素与分离的线粒体部分结合，并促进异常非双层结构的形成。然后我们假设CTI和CTII结合心磷脂（CL）破坏线粒体膜。p -31核磁共振、电子顺磁共振（EPR）、质子核磁共振（h -1核磁共振）、氘核磁共振（h -2核磁共振）光谱、差示扫描量热法和红血球荧光分析表明，CTI和CTII与CL结合产生非双层结构，促进富含CL的大单层磷脂酰胆碱（PC）脂体的渗透、脱水和融合。另一方面，CTII而非CTI引起富含磷脂酰丝氨酸（PS）的大单层PC脂质体的生物物理改变。在机制上，单分子对接模拟确定了CTI和CTII中假定的CL、PS和PC结合位点。虽然预测的PS和PC结合位点在CTI和CTII中具有大量的相互作用氨基酸残基，但CTII和CTI中的CL结合位点由于含有额外的相互作用氨基酸残基而更加分散。总的来说，我们的数据表明细胞毒素通过与CL结合破坏线粒体结构完整性而与线粒体膜发生物理关联。
Cobra venom cytotoxins are basic three-fingered, amphipathic, non-enzymatic proteins that constitute a major fraction of cobra venom. While cytotoxins cause mitochondrial dysfunction in different cell types, the mechanisms by which cytotoxins bind to mitochondria remain unknown. We analyzed the abilities of CTI and CTII, S-type and P-type cytotoxins from Naja naja oxiana respectively, to associate with isolated mitochondrial fractions or with model membranes that simulate the mitochondrial lipid environment by using a myriad of biophysical techniques. Phosphorus-31 nuclear magnetic resonance (P-31-NMR) spectroscopy data suggest that both cytotoxins bind to isolated mitochondrial fractions and promote the formation of aberrant non-bilayer structures. We then hypothesized that CTI and CTII bind to cardiolipin (CL) to disrupt mitochondrial membranes. Collectively, P-31-NMR, electron paramagnetic resonance (EPR), proton NMR (H-1-NMR), deuterium NMR (H-2-NMR) spectroscopy, differential scanning calorimetry, and erythrosine phosphorescence assays suggest that CTI and CTII bind to CL to generate non-bilayer structures and promote the permeabilization, dehydration and fusion of large unilamellar phosphatidylcholine (PC) liposomes enriched with CL. On the other hand, CTII but not CTI caused biophysical alterations of large unilamellar PC liposomes enriched with phosphatidylserine (PS). Mechanistically, single molecule docking simulations identified putative CL, PS and PC binding sites in CTI and CTII. While the predicted binding sites for PS and PC share a high number of interactive amino acid residues in CTI and CTII, the CL biding sites in CTII and CTI are more divergent as it contains additional interactive amino acid residues. Overall, our data suggest that cytotoxins physically associate with mitochondrial membranes by binding to CL to disrupt mitochondrial structural integrity.