Center for Neurologic Diseases, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 2013 Mar 1;288(9):6371-85. doi: 10.1074/jbc.M112.403311. Epub 2013 Jan 14.
Aggregation of α-synuclein (αSyn) in neurons produces the hallmark cytopathology of Parkinson disease and related synucleinopathies. Since its discovery, αSyn has been thought to exist normally in cells as an unfolded monomer. We recently reported that αSyn can instead exist in cells as a helically folded tetramer that resists aggregation and binds lipid vesicles more avidly than unfolded recombinant monomers (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110). However, a subsequent study again concluded that cellular αSyn is an unfolded monomer (Fauvet, B., Mbefo, M. K., Fares, M. B., Desobry, C., Michael, S., Ardah, M. T., Tsika, E., Coune, P., Prudent, M., Lion, N., Eliezer, D., Moore, D. J., Schneider, B., Aebischer, P., El-Agnaf, O. M., Masliah, E., and Lashuel, H. A. (2012) J. Biol. Chem. 287, 15345-15364). Here we describe a simple in vivo cross-linking method that reveals a major ~60-kDa form of endogenous αSyn (monomer, 14.5 kDa) in intact cells and smaller amounts of ~80- and ~100-kDa forms with the same isoelectric point as the 60-kDa species. Controls indicate that the apparent 60-kDa tetramer exists normally and does not arise from pathological aggregation. The pattern of a major 60-kDa and minor 80- and 100-kDa species plus variable amounts of free monomers occurs endogenously in primary neurons and erythroid cells as well as neuroblastoma cells overexpressing αSyn. A similar pattern occurs for the homologue, β-synuclein, which does not undergo pathogenic aggregation. Cell lysis destabilizes the apparent 60-kDa tetramer, leaving mostly free monomers and some 80-kDa oligomer. However, lysis at high protein concentrations allows partial recovery of the 60-kDa tetramer. Together with our prior findings, these data suggest that endogenous αSyn exists principally as a 60-kDa tetramer in living cells but is lysis-sensitive, making the study of natural αSyn challenging outside of intact cells.
α-突触核蛋白（αSyn）在神经元中的聚集产生了帕金森病和相关突触核蛋白病的标志性细胞病理学。自发现以来，人们一直认为 αSyn 正常存在于细胞中，是未折叠的单体。我们最近报道称，αSyn 可以作为螺旋折叠的四聚体存在于细胞中，这种四聚体可以抵抗聚集，并且比未折叠的重组单体更强烈地结合脂质体（Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110）。然而，随后的一项研究再次得出结论，细胞内的 αSyn 是未折叠的单体（Fauvet, B., Mbefo, M. K., Fares, M. B., Desobry, C., Michael, S., Ardah, M. T., Tsika, E., Coune, P., Prudent, M., Lion, N., Eliezer, D., Moore, D. J., Schneider, B., Aebischer, P., El-Agnaf, O. M., Masliah, E., and Lashuel, H. A. (2012) J. Biol. Chem. 287, 15345-15364）。在这里，我们描述了一种简单的体内交联方法，该方法揭示了完整细胞中内源性 αSyn 的主要60-kDa 形式（单体，14.5 kDa）以及同电点与 60-kDa 物种相同的较小80-和~100-kDa 形式。对照表明，明显的 60-kDa 四聚体正常存在，并非源自病理性聚集。主要的 60-kDa 物种和较小的 80-和 100-kDa 物种以及不同量的游离单体的模式在原代神经元、红细胞和过表达 αSyn 的神经母细胞瘤中内源存在。同源物β-突触核蛋白也存在类似的模式，它不会发生致病聚集。细胞裂解使明显的 60-kDa 四聚体不稳定，只剩下游离单体和一些 80-kDa 寡聚物。然而，在高蛋白质浓度下裂解允许部分回收 60-kDa 四聚体。结合我们之前的发现，这些数据表明，内源性 αSyn 主要以 60-kDa 四聚体的形式存在于活细胞中，但对裂解敏感，这使得在完整细胞外研究天然 αSyn 具有挑战性。
