Elaboration of a MALDI-TOF Mass Spectrometry-based Assay of Parkin Activity and High-Throughput screening platform for Parkin Activators
Elaboration of a MALDI-TOF Mass Spectrometry-based Assay of Parkin Activity and High-Throughput screening platform for Parkin Activators
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阐述基于 MALDI-TOF 质谱的 Parkin 活性测定和 Parkin 激活剂的高通量筛选平台
DOI：
10.1101/2022.03.04.482851
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Traynor R
中科院分区：
文献类型：
--
作者：
Traynor R
帕金森病（PD）是一种进行性神经系统疾病，临床表现为运动改变（运动迟缓、姿势不稳定、平衡丧失和静息性震颤）以及多种非运动症状，包括但不限于认知和自主神经异常。线粒体功能障碍与散发性帕金森病有关，编码泛素E3连接酶Parkin和蛋白激酶pten诱导的激酶1 （PINK1）的基因的功能丧失突变是家族性和青少年性帕金森病的原因。在探索治疗或改善PD患者预后的几种治疗方法中，使用能够恢复或增强Parkin活性的小分子代表了一种潜在的药物治疗策略。一个主要的障碍是缺乏基于体外Parkin活性的稳健和准确定量的高通量平台。在这里，我们提出了两种不同的和互补的基于基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）的方法来定量体外Parkin E3连接酶的活性。这些方法概括了泛素偶联的不同方面：帕金自泛素化和帕金催化的赖氨酸残基放电。这两种方法都是可扩展的高通量初级筛选，以促进帕金调节剂的鉴定。
Parkinson’s disease (PD) is a progressive neurological disorder that manifests clinically as alterations in movement (bradykinesia, postural instability, loss of balance, and resting tremors) as well as multiple non-motor symptoms including but not limited to cognitive and autonomic abnormalities. Mitochondrial dysfunction has been linked to sporadic PD and loss-of-function mutations in genes encoding the ubiquitin E3 ligase Parkin and protein kinase, PTEN-induced kinase 1 (PINK1), that regulate mitophagy, are causal for familial and juvenile PD-. Among several therapeutic approaches being explored to treat or improve PD patient’s prognosis, the use of small molecules able to reinstate or boost Parkin activity represents a potential pharmacological treatment strategy. A major barrier is the lack of high throughput platforms based on robust and accurate quantification of Parkin activityin vitro. Here we present two different and complementary Matrix Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (MALDI-TOF MS) based approaches for the quantification of Parkin E3 ligase activityin vitro. These methods recapitulate distinct aspects of ubiquitin conjugation: Parkin auto-ubiquitylation and Parkin-catalysed discharge on lysine residues. Both approaches are scalable for high-throughput primary screening to facilitate the identification of Parkin modulators.
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影响因子：
11.4
作者：
Wauer, Tobias;Komander, David
通讯作者：
Komander, David
DOI：
10.15252/embj.201592337
发表时间：
2015-10-14
期刊：
The EMBO journal
影响因子：
--
作者：
Kumar A;Aguirre JD;Condos TE;Martinez-Torres RJ;Chaugule VK;Toth R;Sundaramoorthy R;Mercier P;Knebel A;Spratt DE;Barber KR;Shaw GS;Walden H
通讯作者：
Walden H
DOI：
10.15252/embj.201592237
发表时间：
2015-10-14
期刊：
The EMBO journal
影响因子：
--
作者：
Sauvé V;Lilov A;Seirafi M;Vranas M;Rasool S;Kozlov G;Sprules T;Wang J;Trempe JF;Gehring K
通讯作者：
Gehring K
影响因子：
64.8
作者：
Kitada, T;Asakawa, S;Shimizu, N
通讯作者：
Shimizu, N