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
Abstract 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 PD1-3. 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 strategy4. A major barrier is the lack of high throughput platforms based on robust and accurate quantification of Parkin activity in 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 activity in 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.
De Cesare Virginia、Traynor Ryan、Merchant Kalpana、Davies Paul、Knebel Axel、Hastie C. James、Moran Jennifer、Stevens Michael、Muqit Miratul M. K.、Behrouz Bahareh
MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of DundeeMRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of DundeeVincere Biosciences IncMRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of DundeeMRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of DundeeMRC Protein Phosphorylation and Ubiquitylation Unit Reagents and Services, School of Life Sciences, University of DundeeMRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of DundeeMRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of DundeeMRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of DundeeVincere Biosciences Inc
医学研究方法神经病学、精神病学生物科学研究方法、生物科学研究技术
De Cesare Virginia,Traynor Ryan,Merchant Kalpana,Davies Paul,Knebel Axel,Hastie C. James,Moran Jennifer,Stevens Michael,Muqit Miratul M. K.,Behrouz Bahareh.Elaboration of a MALDI-TOF Mass Spectrometry-based Assay of Parkin Activity and High-Throughput screening platform for Parkin Activators[EB/OL].(2025-03-28)[2025-07-16].https://www.biorxiv.org/content/10.1101/2022.03.04.482851.点此复制
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