The 14-3-3 Protein Forms a Molecular Complex with Heat Shock Protein Hsp60 and Cellular Prion Protein
The 14-3-3 Protein Forms a Molecular Complex with Heat Shock Protein Hsp60 and Cellular Prion Protein
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DOI：
10.1097/01.jnen.0000182979.56612.08
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发表时间：
2005-10
影响因子：
3.2
通讯作者：
J. Satoh;H. Onoue;K. Arima;T. Yamamura
中科院分区：
文献类型：
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作者：
J. Satoh;H. Onoue;K. Arima;T. Yamamura
14-3-3蛋白家族由酸性30-kDa蛋白组成，其由7种亚型组成，在中枢神经系统（CNS）的神经元和神经胶质细胞中大量表达。在脑脊液中鉴定的14-3-3蛋白为克雅氏病的死前诊断提供了替代标记物，尽管14-3-3在朊病毒疾病的发病机制中的积极参与仍然未知。通过蛋白质覆盖和质谱分析的蛋白质提取物的NTera 2衍生的分化的神经元，我们确定热休克蛋白Hsp 60为14-3-3相互作用的蛋白质。14-3-3 β和γ亚型与Hsp 60相互作用，表明这种相互作用不是亚型特异性的。此外，在SK-N-SH神经母细胞瘤、U-373 MG星形细胞瘤和HeLa宫颈癌细胞中鉴定了相互作用。将细胞朊蛋白（PrPC）沿着Hsp 60与人脑蛋白提取物中的14-3-3共免疫沉淀。通过蛋白质重叠，14-3-3与重组人Hsp 60和大肠杆菌产生的PrPC相互作用，表明分子相互作用是磷酸化无关的。14-3-3-结合结构域位于Hsp 60的N-末端一半（NTF），跨越氨基酸残基27-287，以及PrPC的NTF，跨越氨基酸残基23-137。通过免疫组化染色，14-3-3蛋白Hsp 60和PrPC主要共定位于培养的人神经元祖细胞的线粒体中，并且在人脑中的神经元和反应性星形胶质细胞中共表达最显著。这些观察结果表明，14-3-3蛋白在生理条件下在人CNS中与Hsp 60和PrPC形成分子复合物，并表明该复合物可能在朊病毒疾病的病理过程中被分解。
The 14-3-3 protein family consists of acidic 30-kDa proteins composed of 7 isoforms expressed abundantly in neurons and glial cells of the central nervous system (CNS). The 14-3-3 protein identified in the cerebrospinal fluid provides a surrogate marker for premortem diagnosis of Creutzfeldt-Jakob disease, although an active involvement of 14-3-3 in the pathogenesis of prion diseases remains unknown. By protein overlay and mass spectrometric analysis of protein extract of NTera2-derived differentiated neurons, we identified heat shock protein Hsp60 as a 14-3-3-interacting protein. The 14-3-3ζ and γ isoforms interacted with Hsp60, suggesting that the interaction is not isoform-specific. Furthermore, the interaction was identified in SK-N-SH neuroblastoma, U-373MG astrocytoma, and HeLa cervical carcinoma cells. The cellular prion protein (PrPC) along with Hsp60 was coimmunoprecipitated with 14-3-3 in the human brain protein extract. By protein overlay, 14-3-3 interacted with both recombinant human Hsp60 and PrPC produced by Escherichia coli, indicating that the molecular interaction is phosphorylation-independent. The 14-3-3-binding domain was located in the N-terminal half (NTF) of Hsp60 spanning amino acid residues 27-287 and the NTF of PrPC spanning amino acid residues 23-137. By immunostaining, the 14-3-3 protein Hsp60 and PrPC were colocalized chiefly in the mitochondria of human neuronal progenitor cells in culture, and were coexpressed most prominently in neurons and reactive astrocytes in the human brain. These observations indicate that the 14-3-3 protein forms a molecular complex with Hsp60 and PrPC in the human CNS under physiological conditions and suggest that this complex might become disintegrated in the pathologic process of prion diseases.