Development and characterization of phospho-ubiquitin antibodies to monitor PINK1-PRKN signaling in cells and tissue

The selective removal of dysfunctional mitochondria, a process termed mitophagy, is critical for cellular health and impairments have been linked to aging, Parkinson disease, and other neurodegenerative conditions. A central mitophagy pathway is orchestrated by the ubiquitin (Ub) kinase PINK1 togeth...

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Published in:	Autophagy Vol. 20; no. 9; pp. 2076 - 2091
Main Authors:	Watzlawik, Jens O., Hou, Xu, Richardson, Tyrique, Lewicki, Szymon L., Siuda, Joanna, Wszolek, Zbigniew K., Cook, Casey N., Petrucelli, Leonard, DeTure, Michael, Dickson, Dennis W., Antico, Odetta, Muqit, Miratul M. K., Fishman, Jordan B., Pirani, Karima, Kumaran, Ravindran, Polinski, Nicole K., Fiesel, Fabienne C., Springer, Wolfdieter
Format:	Journal Article
Language:	English
Published:	United States Taylor & Francis 01-09-2024
Subjects:	Autophagy mitochondria mitophagy Parkinson disease PINK1 Resource ubiquitin Parkinson disease PINK1 mitophagy Autophagy mitochondria ubiquitin
Online Access:	Get full text
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Summary:	The selective removal of dysfunctional mitochondria, a process termed mitophagy, is critical for cellular health and impairments have been linked to aging, Parkinson disease, and other neurodegenerative conditions. A central mitophagy pathway is orchestrated by the ubiquitin (Ub) kinase PINK1 together with the E3 Ub ligase PRKN/Parkin. The decoration of damaged mitochondrial domains with phosphorylated Ub (p-S65-Ub) mediates their elimination though the autophagy system. As such p-S65-Ub has emerged as a highly specific and quantitative marker of mitochondrial damage with significant disease relevance. Existing p-S65-Ub antibodies have been successfully employed as research tools in a range of applications including western blot, immunocytochemistry, immunohistochemistry, and enzyme-linked immunosorbent assay. However, physiological levels of p-S65-Ub in the absence of exogenous stress are very low, therefore difficult to detect and require reliable and ultrasensitive methods. Here we generated and characterized a collection of novel recombinant, rabbit monoclonal p-S65-Ub antibodies with high specificity and affinity in certain applications that allow the field to better understand the molecular mechanisms and disease relevance of PINK1-PRKN signaling. These antibodies may also serve as novel diagnostic or prognostic tools to monitor mitochondrial damage in various clinical and pathological specimens. Abbreviations: AD: Alzheimer disease; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ELISA: enzyme-linked immunosorbent assay; HEK293E cell: human embryonic kidney E cell; ICC: immunocytochemistry; IHC: immunohistochemistry: KO: knockout; LoB: limit of blank; LoD: limit of detection; LoQ: limit of quantification; MEF: mouse embryonic fibroblast; MSD: Meso Scale Discovery; n.s.: non-significant; nonTg: non-transgenic; PBMC: peripheral blood mononuclear cell; PD: Parkinson disease; p-S65-PRKN: phosphorylated PRKN at serine 65; p-S65-Ub: phosphorylated Ub at serine 65; Ub: ubiquitin; WT: wild-type.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Contributed equally.
ISSN:	1554-8627 1554-8635 1554-8635
DOI:	10.1080/15548627.2024.2356490