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

ABSTRACT

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.