The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity.
The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity.
复制标题
DOI：
10.1186/s13024-017-0174-z
复制
发表时间：
2017-04-24
影响因子：
15.1
通讯作者：
Springer W
中科院分区：
文献类型：
--
作者：
Ando M;Fiesel FC;Hudec R;Caulfield TR;Ogaki K;Górka-Skoczylas P;Koziorowski D;Friedman A;Chen L;Dawson VL;Dawson TM;Bu G;Ross OA;Wszolek ZK;Springer W
PINK 1和PARKIN的突变是隐性早发性帕金森病（EOPD）的最常见原因。线粒体泛素（Ub）激酶PINK 1和细胞溶质E3 Ub连接酶PARKIN共同指导复杂的调节，连续的线粒体质量控制。因此，受损的线粒体被鉴定并靶向降解，以防止它们的积累和最终的细胞死亡。任一基因功能的纯合或复合杂合缺失破坏了这种保护途径，尽管是在不同的步骤和通过不同的机制。虽然PARKIN变体的结构和功能已经得到了很好的研究，但PINK 1突变仍然缺乏特征，特别是在内源性条件下。更好地理解致病性背后的确切分子致病机制对于未来合理的药物设计至关重要。在这里，我们在患者的成纤维细胞和基于细胞的生化测定中，在临床和遗传以及结构和功能水平上表征了PINK 1 p.I368N突变的致病性。在内源性条件下，PINK 1 p.I368N在健康线粒体中表达、输入和N-末端加工，类似于PINK 1野生型（WT）。然而，在线粒体损伤时，全长PINK 1 p.I368N在外线粒体膜（OMM）上不充分稳定，导致线粒体质量控制的丧失。我们发现PINK 1 p.I368N与辅助分子伴侣复合物HSP 90/CDC 37的结合减少，并且消除了应激诱导的与线粒体蛋白输入机器的TOM 40的相互作用。结构PINK 1 p.I368N模型的分析还表明，Ub激酶活性受损，因为发现ATP结合口袋变形，底物Ub在激酶的活性位点内轻微错位。功能测定证实缺乏Ub激酶活性。在这里，我们证明了突变体PINK 1 p.I368N在线粒体应激时不能稳定在OMM上，并且由于活性位点的构象变化而不能对Ub发挥激酶活性。在患者的成纤维细胞中，通过生物化学测定和结构分析，我们揭示了导致p.I368N突变后功能丧失的两种病理机制，并强调了未来药物开发的潜在策略。本文的在线版本（doi：10.1186/s13024-017-0174-z）包含补充材料，可供授权用户使用。
Mutations in PINK1 and PARKIN are the most common causes of recessive early-onset Parkinson’s disease (EOPD). Together, the mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PARKIN direct a complex regulated, sequential mitochondrial quality control. Thereby, damaged mitochondria are identified and targeted to degradation in order to prevent their accumulation and eventually cell death. Homozygous or compound heterozygous loss of either gene function disrupts this protective pathway, though at different steps and by distinct mechanisms. While structure and function of PARKIN variants have been well studied, PINK1 mutations remain poorly characterized, in particular under endogenous conditions. A better understanding of the exact molecular pathogenic mechanisms underlying the pathogenicity is crucial for rational drug design in the future. Here, we characterized the pathogenicity of the PINK1 p.I368N mutation on the clinical and genetic as well as on the structural and functional level in patients’ fibroblasts and in cell-based, biochemical assays. Under endogenous conditions, PINK1 p.I368N is expressed, imported, and N-terminally processed in healthy mitochondria similar to PINK1 wild type (WT). Upon mitochondrial damage, however, full-length PINK1 p.I368N is not sufficiently stabilized on the outer mitochondrial membrane (OMM) resulting in loss of mitochondrial quality control. We found that binding of PINK1 p.I368N to the co-chaperone complex HSP90/CDC37 is reduced and stress-induced interaction with TOM40 of the mitochondrial protein import machinery is abolished. Analysis of a structural PINK1 p.I368N model additionally suggested impairments of Ub kinase activity as the ATP-binding pocket was found deformed and the substrate Ub was slightly misaligned within the active site of the kinase. Functional assays confirmed the lack of Ub kinase activity. Here we demonstrated that mutant PINK1 p.I368N can not be stabilized on the OMM upon mitochondrial stress and due to conformational changes in the active site does not exert kinase activity towards Ub. In patients’ fibroblasts, biochemical assays and by structural analyses, we unraveled two pathomechanisms that lead to loss of function upon mutation of p.I368N and highlight potential strategies for future drug development. The online version of this article (doi:10.1186/s13024-017-0174-z) contains supplementary material, which is available to authorized users.