Novel monoclonal antibodies demonstrate biochemical variation of brain parkin with age
Novel monoclonal antibodies demonstrate biochemical variation of brain parkin with age
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DOI：
10.1074/jbc.m306889200
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发表时间：
2003-11-28
影响因子：
4.8
通讯作者：
Lee, VMY
中科院分区：
文献类型：
--
作者：
Pawlyk, AC;Giasson, BI;Lee, VMY
常染色体隐性遗传性青少年帕金森综合征是一种与黑质多巴胺能神经元变性相关的运动障碍。由parkin基因突变引起的parkin功能丧失是青少年帕金森综合征最常见的单一原因。帕金蛋白已被证明有助于保护细胞免受内质网和氧化应激的影响，这可能是由于帕金蛋白的泛素连接酶活性靶向蛋白质进行蛋白酶体降解。然而，对parkin的研究一直受到阻碍，因为这种蛋白质的特异性试剂有限。在这里，我们报告的生成和表征的一组parkin特异性单克隆抗体。生物化学分析表明，帕金只存在于小鼠大脑的高盐可提取部分，而它存在于年轻人大脑的高盐可提取部分和抗RIPA，SDS可提取部分。帕金在高盐可提取部分中以降低的水平存在，而在来自老年人脑的SDS可提取部分中以增加的水平存在。衰老时帕金提取率的这种变化在人类中观察到，但在小鼠中未观察到，表明小鼠与人类帕金的生化特征存在物种特异性差异。最后，通过使用这些高度特异性的抗帕金单克隆抗体，不可能在α-突触核蛋白病中含有α-突触核蛋白的病变中检测到帕金，从而挑战了先前关于帕金在常染色体隐性青少年帕金森综合征以外的运动障碍中的作用的推断。
Autosomal recessive juvenile parkinsonism is a movement disorder associated with the degeneration of dopaminergic neurons in substantia nigra pars compacta. The loss of functional parkin caused by parkin gene mutations is the most common single cause of juvenile parkinsonism. Parkin has been shown to aid in protecting cells from endoplasmic reticulum and oxidative stressors presumably due to ubiquitin ligase activity of parkin that targets proteins for proteasomal degradation. However, studies on parkin have been impeded because of limited reagents specific for this protein. Here we report the generation and characterization of a panel of parkin-specific monoclonal antibodies. Biochemical analyses indicate that parkin is present only in the high salt-extractable fraction of mouse brain, whereas it is present in both the high salt-extractable and RIPA-resistant, SDS-extractable fraction in young human brain. Parkin is present at decreased levels in the high salt-extractable fraction and at increased levels in the SDS-extractable fraction from aged human brain. This shift in the extractability of parkin upon aging is seen in humans but not in mice, demonstrating species-specific differences in the biochemical characteristics of murine versus human parkin. Finally, by using these highly specific anti-parkin monoclonal antibodies, it was not possible to detect parkin in alpha-synuclein-containing lesions in alpha-synucleinopathies, thereby challenging prior inferences about the role of parkin in movement disorders other than autosomal recessive juvenile parkinsonism.