Lek Sovanarak, Vargas-Medrano Javier, Villanueva Ernesto, Marcus Brian, Godfrey Wesley, Perez Ruth G
Department of Biomedical Sciences, Center of Emphasis in Neurosciences, Graduate School of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso.
Department of Biomedical Sciences, Center of Emphasis in Neurosciences, Graduate School of Biomedical Sciences, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso;
J Vis Exp. 2017 Aug 13(126):55361. doi: 10.3791/55361.
α-Synuclein (aSyn), β-Synuclein (bSyn), and γ-Synuclein (gSyn) are members of a conserved family of chaperone-like proteins that are highly expressed in vertebrate neuronal tissues. Of the three synucleins, only aSyn has been strongly implicated in neurodegenerative disorders such as Parkinson's disease, Dementia with Lewy Bodies, and Multiple System Atrophy. In studying normal aSyn function, data indicate that aSyn stimulates the activity of the catalytic subunit of an abundantly expressed dephosphorylating enzyme, PP2Ac in vitro and in vivo. Prior data show that aSyn aggregation in human brain reduces PP2Ac activity in regions with Lewy body pathology, where soluble aSyn has become insoluble. However, because all three synucleins have considerable homology in the amino acid sequences, experiments were designed to test if all can modulate PP2Ac activity. Using recombinant synucleins and recombinant PP2Ac protein, activity was assessed by malachite green colorimetric assay. Data revealed that all three recombinant synucleins stimulated PP2Ac activity in cell-free assays, raising the possibility that the conserved homology between synucleins may endow all three homologs with the ability to bind to and activate the PP2Ac. Co-immunoprecipitation data, however, suggest that PP2Ac modulation likely occurs through endogenous interactions between aSyn and PP2Ac in vivo.
α-突触核蛋白(aSyn)、β-突触核蛋白(bSyn)和γ-突触核蛋白(gSyn)是一类保守的伴侣样蛋白家族成员,在脊椎动物神经组织中高度表达。在这三种突触核蛋白中,只有aSyn与帕金森病、路易体痴呆和多系统萎缩等神经退行性疾病密切相关。在研究正常aSyn功能时,数据表明aSyn在体外和体内均可刺激一种大量表达的去磷酸化酶PP2Ac催化亚基的活性。先前的数据显示,人脑中aSyn的聚集会降低路易体病理区域的PP2Ac活性,在这些区域中,可溶性aSyn已变为不可溶性。然而,由于所有三种突触核蛋白在氨基酸序列上具有相当高的同源性,因此设计了实验来测试它们是否都能调节PP2Ac活性。使用重组突触核蛋白和重组PP2Ac蛋白,通过孔雀石绿比色法评估活性。数据显示,在无细胞实验中,所有三种重组突触核蛋白均刺激了PP2Ac活性,这增加了突触核蛋白之间保守的同源性可能赋予所有三种同源物结合并激活PP2Ac能力的可能性。然而,免疫共沉淀数据表明,PP2Ac的调节可能是通过体内aSyn与PP2Ac之间的内源性相互作用发生的。