Mapping of a N-terminal a-helix domain required for human PINK1 stabilisation, Serine228 autophosphorylation and activation in cells
Mapping of a N-terminal a-helix domain required for human PINK1 stabilisation, Serine228 autophosphorylation and activation in cells
复制标题
绘制细胞中人 PINK1 稳定、丝氨酸 228 自磷酸化和激活所需的 N 端 a 螺旋结构域
DOI：
10.1101/2021.09.06.459138
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Kakade P
中科院分区：
文献类型：
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作者：
Kakade P
人类常染色体隐性突变的PINK1基因是帕金森病(PD)的病因。PINK1编码一种线粒体定位蛋白激酶，它是线粒体质量控制途径的主调节因子。到目前为止，结构研究已经详细阐述了位于激酶域的突变如何破坏PINK1功能的机制，然而，位于激酶域的上游和下游的PINK1突变的分子机制尚不清楚。我们利用细胞中人PINK1的突变研究来确定PINK1的最小区域，从残基Ile111开始，这是最佳泛素磷酸化所必需的。对从Ile111到激活域的区域的生物信息学分析和对AlphaFold人PINK1结构模型的检查预测，该区域内存在保守的N端α-螺旋结构域延伸(NTE域)，这一结果得到了重组昆虫PINK1蛋白的氢/氢交换质谱学的证实。AlphaFold结构还预测NTE结构域与C末端延伸(CTE)形成分子内相互作用。基于细胞的分析表明，位于NTE：CTE界面内的PD相关突变(例如Q126P)显著抑制PINK1的稳定、丝氨酸228(Ser228)的自动磷酸化和泛素丝氨酸65(Ser65)的磷酸化。此外，我们提供的证据表明，NTE结构域突变不会影响内在催化激酶的活性，但会破坏外膜线粒体易位酶(TOM)复合体中PINK1的稳定。由于PINK1 Q126P纯合突变导致的早发性帕金森病患者的人成纤维细胞中内源性PINK1的稳定和激活存在缺陷，这支持了我们的发现的临床相关性。总之，我们定义了NTE：CTE界面对PINK1稳定和激活的功能作用，并表明NTE：CTE相互作用的丧失是PINK1相关突变与帕金森病相关的主要机制。
Human autosomal recessive mutations in the PINK1 gene are causal for Parkinson’s disease (PD). PINK1 encodes a mitochondrial localised protein kinase that is a master-regulator of mitochondrial quality control pathways. Structural studies to date have elaborated the mechanism of how mutations located within the kinase domain disrupt PINK1 function, however, the molecular mechanism of PINK1 mutations located upstream and downstream of the kinase domain are unknown. We have employed mutagenesis studies of human PINK1 in cells to define the minimal region of PINK1, required for optimal ubiquitin phosphorylation, beginning at residue Ile111. Bioinformatic analysis of the region spanning Ile111 to the kinase domain and inspection of the AlphaFold human PINK1 structure model predicts a conserved N-terminal α-helical domain extension (NTE domain) within this region corroborated by hydrogen/deuterium exchange mass spectrometry (HDX-MS) of recombinant insect PINK1 protein. The AlphaFold structure also predicts the NTE domain forms an intramolecular interaction with the C-terminal extension (CTE). Cell-based analysis of human PINK1 reveals that PD-associated mutations (eg Q126P), located within the NTE: CTE interface, markedly inhibit stabilization of PINK1; autophosphorylation at Serine228 (Ser228); and Ubiquitin Serine65 (Ser65) phosphorylation. Furthermore, we provide evidence that NTE domain mutants do not affect intrinsic catalytic kinase activity but do disrupt PINK1 stabilisation at the mitochondrial Translocase of outer membrane (TOM) complex. The clinical relevance of our findings is supported by the demonstration of defective stabilization and activation of endogenous PINK1 in human fibroblasts of a patient with early-onset PD due to homozygous PINK1 Q126P mutations. Overall, we define a functional role of the NTE: CTE interface towards PINK1 stabilisation and activation and show that loss of NTE: CTE interactions is a major mechanism of PINK1-associated mutations linked to PD.