帕金森病相关突变对DJ-1稳定性和折叠的差异影响
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Gorner K, Holtorf E, Odoy S, Nuscher B, Yamamoto A, Regula JT, Beyer K, Haass C, Kahle PJ
帕金森病相关突变对DJ-1稳定性和折叠的差异影响
中国生物化学杂志，2004年2月20日;279(8):6943-51。Epub 2003年11月7日
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14607841 (PubMed视图］
- 摘要
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PARK7/DJ-1基因突变导致常染色体隐性帕金森病。在一些患者中，该基因被删除。点突变患者疾病的分子基础不太明显。我们研究了[L166P]DJ-1及其新变体[E64D]DJ-1的分子性质。当转染非神经元和神经元细胞系时，[L166P]DJ-1的稳态表达水平显著低于野生型[WT]DJ-1和[E64D]DJ-1。环己亚胺和脉冲追踪实验表明，[L166P]DJ-1表达水平下降是由于蛋白质周转加速所致。蛋白酶体降解并不是DJ-1分解的主要途径，因为蛋白酶体抑制剂MG-132的处理只导致极少的DJ-1积累，即使是非常不稳定的[L166P]DJ-1突变体。由于DJ-1与细菌半胱氨酸蛋白酶在结构上的相似性，我们考虑了其自身蛋白水解机制。然而，无论是药物抑制还是对假定活性位点残基Cys-106的定向突变都无法稳定DJ-1。为了进一步了解DJ-1突变体的结构缺陷，将人[WT]DJ-1和两种突变体在大肠杆菌中表达。 As in eukaryotic cells, expression levels of [L166P]DJ-1 were dramatically reduced compared with [WT]DJ-1 and [E64D]DJ-1. Circular dichroism spectrometry revealed that the solution structures of [WT]DJ-1 and [E64D]DJ-1 are rich in beta-strand and alpha-helix conformation. Alpha-helices were more susceptible to thermal denaturation than the beta-sheet, and [WT]DJ-1 was more flexible in this regard than [E64D]DJ-1. Thus, structural defects of [E64D]DJ-1 only become apparent upon denaturing conditions, whereas the L166P mutation causes a drastic defect that leads to excessive degradation.