Dingeldein Artur Peter Günther, Pokorná Šárka, Lidman Martin, Sparrman Tobias, Šachl Radek, Hof Martin, Gröbner Gerhard
Department of Chemistry, Umeå University, Umeå, Sweden.
J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Biophys J. 2017 May 23;112(10):2147-2158. doi: 10.1016/j.bpj.2017.04.019.
Mitochondria are crucial compartments of eukaryotic cells because they function as the cellular power plant and play a central role in the early stages of programmed cell death (apoptosis). To avoid undesired cell death, this apoptotic pathway is tightly regulated by members of the Bcl-2 protein family, which interact on the external surface of the mitochondria, i.e., the mitochondrial outer membrane (MOM), and modulate its permeability to apoptotic factors, controlling their release into the cytosol. A growing body of evidence suggests that the MOM lipids play active roles in this permeabilization process. In particular, oxidized phospholipids (OxPls) formed under intracellular stress seem to directly induce apoptotic activity at the MOM. Here we show that the process of MOM pore formation is sensitive to the type of OxPls species that are generated. We created MOM-mimicking liposome systems, which resemble the cellular situation before apoptosis and upon triggering of oxidative stress conditions. These vesicles were studied using P solid-state magic-angle-spinning nuclear magnetic resonance spectroscopy and differential scanning calorimetry, together with dye leakage assays. Direct polarization and cross-polarization nuclear magnetic resonance experiments enabled us to probe the heterogeneity of these membranes and their associated molecular dynamics. The addition of apoptotic Bax protein to OxPls-containing vesicles drastically changed the membranes' dynamic behavior, almost completely negating the previously observed effect of temperature on the lipids' molecular dynamics and inducing an ordering effect that led to more cooperative membrane melting. Our results support the hypothesis that the mitochondrion-specific lipid cardiolipin functions as a first contact site for Bax during its translocation to the MOM in the onset of apoptosis. In addition, dye leakage assays revealed that different OxPls species in the MOM-mimicking vesicles can have opposing effects on Bax pore formation.
线粒体是真核细胞的关键细胞器，因为它们起着细胞动力工厂的作用，并在程序性细胞死亡（凋亡）的早期阶段发挥核心作用。为避免意外的细胞死亡，这种凋亡途径受到Bcl-2蛋白家族成员的严格调控，这些成员在线粒体的外表面即线粒体外膜（MOM）上相互作用，并调节其对凋亡因子的通透性，控制它们释放到细胞质中。越来越多的证据表明，MOM脂质在这种通透性过程中发挥着积极作用。特别是，细胞内应激下形成的氧化磷脂（OxPls）似乎直接在MOM处诱导凋亡活性。在这里，我们表明MOM孔形成过程对所产生的OxPls种类敏感。我们创建了模仿MOM的脂质体系统，其类似于凋亡前和触发氧化应激条件时的细胞情况。使用P固态魔角旋转核磁共振光谱和差示扫描量热法以及染料泄漏试验对这些囊泡进行了研究。直接极化和交叉极化核磁共振实验使我们能够探测这些膜的异质性及其相关的分子动力学。将凋亡的Bax蛋白添加到含有OxPls的囊泡中，极大地改变了膜的动态行为，几乎完全消除了先前观察到的温度对脂质分子动力学的影响，并诱导了一种有序效应，导致更协同的膜熔化。我们的结果支持这样的假设，即线粒体特异性脂质心磷脂在凋亡开始时Bax转运到MOM的过程中作为其第一个接触位点。此外，染料泄漏试验表明，模仿MOM的囊泡中的不同OxPls种类对Bax孔形成可能有相反的影响。
