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

TY - JOUR

T1 - Development and characterization of phospho-ubiquitin antibodies to monitor PINK1-PRKN signaling in cells and tissue

AU - Watzlawik, Jens O.

AU - Hou, Xu

AU - Richardson, Tyrique

AU - Lewicki, Szymon L.

AU - Siuda, Joanna

AU - Wszolek, Zbigniew K.

AU - Cook, Casey N.

AU - Petrucelli, Leonard

AU - DeTure, Michael

AU - Dickson, Dennis W.

AU - Antico, Odetta

AU - Muqit, Miratul M.K.

AU - Fishman, Jordan B.

AU - Pirani, Karima

AU - Kumaran, Ravindran

AU - Polinski, Nicole K.

AU - Fiesel, Fabienne C.

AU - Springer, Wolfdieter

N1 - Publisher Copyright: © 2024 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.

PY - 2024

Y1 - 2024

N2 - 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.

AB - 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.

KW - Autophagy

KW - mitochondria

KW - mitophagy

KW - Parkinson disease

KW - PINK1

KW - ubiquitin

UR - https://www.scopus.com/pages/publications/85194554339

U2 - 10.1080/15548627.2024.2356490

DO - 10.1080/15548627.2024.2356490

M3 - Article

C2 - 38802071

AN - SCOPUS:85194554339

SN - 1554-8627

VL - 20

SP - 2076

EP - 2091

JO - Autophagy

JF - Autophagy

IS - 9

ER -