Ubiquitination of alpha-synuclein.
Ubiquitination of alpha-synuclein.
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发表时间：
2005
期刊：
影响因子：
2.9
通讯作者：
T. Nonaka;T. Iwatsubo;M. Hasegawa
中科院分区：
文献类型：
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作者：
T. Nonaka;T. Iwatsubo;M. Hasegawa
细丝α-突触核蛋白沉积是神经退行性疾病子集的定义标志，包括帕金森病（PD）、路易体痴呆和多系统萎缩。我们先前报道了这些脑中的α-突触核蛋白在Ser 129处被广泛磷酸化[Fujiwara等（2002）Nat. Cell Biol.4，160-164]，并且还被部分泛素化[Hasegawa等（2002）J.Biol.Chem.277，49071-49076]。在这里，我们研究了α-突触核蛋白在体外和体内的泛素化，并报告了泛素化位点和家族性PD连锁突变、磷酸化和原纤维形成对泛素化的影响。蛋白质序列分析表明，赖氨酸21，赖氨酸23，赖氨酸32，赖氨酸34内的重复氨基末端的一半，易于在体外泛素化。一项定点诱变研究证实，这些是主要的泛素化位点。A53 T和A30 P突变对泛素化没有显著影响。类似地，α-突触核蛋白在Ser 129的磷酸化不影响泛素化。值得注意的是，我们表明，组装，丝状α-突触核蛋白是较低的泛素化比可溶性形式和主要的泛素化位点定位于赖氨酸6，赖氨酸10，赖氨酸12在丝状α-突触核蛋白的氨基末端区域。此外，我们成功地检测到泛素化的α-突触核蛋白在293 T细胞与α-突触核蛋白和泛素共转染。发现体内泛素化位点与丝状α-突触核蛋白中的那些相同。PD连锁突变和Ser 129的磷酸化对体内α-突触核蛋白的泛素化没有影响。这些数据可能对α-突触核蛋白病大脑中α-突触核蛋白沉积物的形成机制有影响。
Filamentous alpha-synuclein depositions are the defining hallmarks of a subset of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. We previously reported that alpha-synuclein in those brains are extensively phosphorylated at Ser129 [Fujiwara et al. (2002) Nat. Cell Biol. 4, 160-164] and also partially ubiquitinated [Hasegawa et al. (2002) J. Biol. Chem. 277, 49071-49076]. Here, we investigate ubiquitination of alpha-synuclein in vitro and in vivo and report the ubiquitination sites and the effects of familial PD-linked mutations, phosphorylation, and fibril formation on ubiquitination. Protein-sequence analysis revealed that Lys21, Lys23, Lys32, and Lys34 within the repeats in the amino-terminal half are liable to ubiquitination in vitro. A site-directed mutagensis study confirmed that these are the major ubiquitination sites. A53T and A30P mutations had no significant effect on ubiquitination. Similarly, phosphorylation of alpha-synuclein at Ser129 did not affect ubiquitination. Notably, we show that assembled, filamentous alpha-synuclein is less ubiquitinated than the soluble form and that the major ubiquitination sites are localized to Lys6, Lys10, and Lys12 at the amino-terminal region of filamentous alpha-synuclein. Furthermore, we successfully detected ubiquitination of alpha-synuclein in 293T cells by cotransfection with alpha-synuclein and ubiquitin. The in vivo ubiquitination sites were found to be identical to those in filamentous alpha-synuclein. PD-linked mutations and phosphorylation at Ser129 had no effects on ubiquitination of alpha-synuclein in vivo. These data may have implications for the mechanisms of the formation of alpha-synuclein deposits in alpha-synucleinopathy brains.