Dynamics of PARKIN-Dependent Mitochondrial Ubiquitylation in Induced Neurons and Model Systems Revealed by Digital Snapshot Proteomics.
Dynamics of PARKIN-Dependent Mitochondrial Ubiquitylation in Induced Neurons and Model Systems Revealed by Digital Snapshot Proteomics.
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DOI：
10.1016/j.molcel.2018.03.012
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发表时间：
2018-04-19
期刊：
影响因子：
16
通讯作者：
Harper JW
中科院分区：
文献类型：
--
作者：
Ordureau A;Paulo JA;Zhang W;Ahfeldt T;Zhang J;Cohn EF;Hou Z;Heo JM;Rubin LL;Sidhu SS;Gygi SP;Harper JW
通过激酶和泛素驱动的信号系统的通量取决于通路内的修饰动力学、化学计量学、主要位点特异性和靶标丰度，但我们很少了解这些参数及其在细胞内的空间组织。在这里，我们开发了胚胎干细胞来源的神经元线粒体外膜上泛素信号的时间数字快照，并通过泛素化和磷酸化事件的蛋白质组学计数来模拟PINK1激酶和PARKIN泛素连接酶激活时的HeLa细胞系统。我们定义了PARKIN依赖性靶泛素化的动力学和位点特异性，并证明了这种方法在量化途径调节剂和机制上定义PARKIN UBL磷酸化在诱导神经元通路激活中的作用的能力。最后，通过对线粒体上的pS65-Ub的调节，我们证明了Ub的超磷酸化抑制了线粒体自噬受体的募集，这表明体内pS65-Ub的化学配比优化了，通过pS65-Ub和线粒体自噬受体通过未磷酸化链协调PARKIN募集。PARKIN泛素连接酶通过PINK1激酶在受损线粒体上激活，使一系列蛋白质泛素化。Ordureau等人开发了一种定量蛋白质组学方法来测量神经元细胞中parkin依赖性底物泛素化的动力学、位点特异性和化学计量学，为该途径提供了定量分析。
Flux through kinase and ubiquitin-driven signaling systems depends upon the modification kinetics, stoichiometry, primary site-specificity, and target abundance within the pathway, yet we rarely understand these parameters and their spatial organization within cells. Here, we develop temporal digital snapshots of ubiquitin signaling on the mitochondrial outer membrane in embryonic stem cell-derived neurons and model HeLa cell systems upon activation of the PINK1 kinase and PARKIN ubiquitin ligase by proteomic counting of ubiquitylation and phosphorylation events. We define the kinetics and site-specificity of PARKIN-dependent target ubiquitylation and demonstrate the power of this approach to quantify pathway modulators and to mechanistically define the role of PARKIN UBL phosphorylation in pathway activation in induced neurons. Finally, through modulation of pS65-Ub on mitochondria, we demonstrate that Ub hyper-phosphorylation is inhibitory to mitophagy receptor recruitment, indicating that pS65-Ub stoichiometry in vivo is optimized to coordinate PARKIN recruitment via pS65-Ub and mitophagy receptors via unphosphorylated chains. The PARKIN ubiquitin ligase is activated on damaged mitochondria via the PINK1 kinase, where it ubiquitylates an array of proteins. Ordureau et al. develop a quantitative proteomics approach to measure the dynamics, site-specificity and stoichiometry of PARKIN-dependent substrate ubiquitylation in neuronal cells, providing a quantitative analysis of the pathway.
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影响因子：
7.7
作者：
Kazlauskaite A;Martínez-Torres RJ;Wilkie S;Kumar A;Peltier J;Gonzalez A;Johnson C;Zhang J;Hope AG;Peggie M;Trost M;van Aalten DM;Alessi DR;Prescott AR;Knebel A;Walden H;Muqit MM
通讯作者：
Muqit MM
影响因子：
64.5
作者：
Huttlin EL;Jedrychowski MP;Elias JE;Goswami T;Rad R;Beausoleil SA;Villén J;Haas W;Sowa ME;Gygi SP
通讯作者：
Gygi SP
影响因子：
5.8
作者：
Kazlauskaite A;Kelly V;Johnson C;Baillie C;Hastie CJ;Peggie M;Macartney T;Woodroof HI;Alessi DR;Pedrioli PG;Muqit MM
通讯作者：
Muqit MM
影响因子：
7.7
作者：
Hung V;Lam SS;Udeshi ND;Svinkina T;Guzman G;Mootha VK;Carr SA;Ting AY
通讯作者：
Ting AY
DOI：
10.1083/jcb.201210111
发表时间：
2013-01-21
期刊：
The Journal of cell biology
影响因子：
--
作者：
Lazarou M;Narendra DP;Jin SM;Tekle E;Banerjee S;Youle RJ
通讯作者：
Youle RJ