Section On Molecular Transport, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, 9000 Rockville Pike, Bldg. 29B, Room 1G09, Bethesda, MD, 20892, USA.
Laboratory of Molecular Biophysics, Department of Physics, Universitat Jaume I, Castellón, 12071, Spain.
Cell Mol Life Sci. 2022 Jun 19;79(7):368. doi: 10.1007/s00018-022-04389-w.
Involvement of alpha-synuclein (αSyn) in Parkinson's disease (PD) is complicated and difficult to trace on cellular and molecular levels. Recently, we established that αSyn can regulate mitochondrial function by voltage-activated complexation with the voltage-dependent anion channel (VDAC) on the mitochondrial outer membrane. When complexed with αSyn, the VDAC pore is partially blocked, reducing the transport of ATP/ADP and other metabolites. Further, αSyn can translocate into the mitochondria through VDAC, where it interferes with mitochondrial respiration. Recruitment of αSyn to the VDAC-containing lipid membrane appears to be a crucial prerequisite for both the blockage and translocation processes. Here we report an inhibitory effect of HK2p, a small membrane-binding peptide from the mitochondria-targeting N-terminus of hexokinase 2, on αSyn membrane binding, and hence on αSyn complex formation with VDAC and translocation through it. In electrophysiology experiments, the addition of HK2p at micromolar concentrations to the same side of the membrane as αSyn results in a dramatic reduction of the frequency of blockage events in a concentration-dependent manner, reporting on complexation inhibition. Using two complementary methods of measuring protein-membrane binding, bilayer overtone analysis and fluorescence correlation spectroscopy, we found that HK2p induces detachment of αSyn from lipid membranes. Experiments with HeLa cells using proximity ligation assay confirmed that HK2p impedes αSyn entry into mitochondria. Our results demonstrate that it is possible to regulate αSyn-VDAC complexation by a rationally designed peptide, thus suggesting new avenues in the search for peptide therapeutics to alleviate αSyn mitochondrial toxicity in PD and other synucleinopathies.
α-突触核蛋白（αSyn）在帕金森病（PD）中的作用复杂，在细胞和分子水平上难以追踪。最近，我们发现αSyn 可以通过与线粒体外膜上的电压依赖性阴离子通道（VDAC）的电压激活复合物来调节线粒体功能。当与αSyn 复合物化时，VDAC 孔部分被阻断，减少 ATP/ADP 和其他代谢物的运输。此外，αSyn 可以通过 VDAC 易位进入线粒体，在那里它会干扰线粒体呼吸。αSyn 募集到包含 VDAC 的脂膜似乎是阻断和易位过程的关键前提。在这里，我们报告了来自己糖激酶 2 线粒体靶向 N 端的小膜结合肽 HK2p 对 αSyn 膜结合的抑制作用，从而抑制了 αSyn 与 VDAC 的复合物形成和通过它的易位。在电生理学实验中，以微摩尔浓度将 HK2p 添加到与 αSyn 相同的膜侧，以浓度依赖的方式导致阻断事件的频率急剧降低，报告了复合物抑制。使用两种互补的测量蛋白-膜结合的方法，双层泛音分析和荧光相关光谱法，我们发现 HK2p 诱导 αSyn 从脂质膜上脱离。使用邻近连接测定法对 HeLa 细胞进行的实验证实，HK2p 阻碍了 αSyn 进入线粒体。我们的结果表明，可以通过设计合理的肽来调节 αSyn-VDAC 复合物形成，从而为寻找缓解 PD 和其他突触核蛋白病中 αSyn 线粒体毒性的肽治疗方法提供了新的途径。
