학술논문
Development and characterization of phospho-ubiquitin antibodies to monitor PINK1-PRKN signaling in cells and tissue.
Document Type
Academic Journal
Author
Watzlawik JO; Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.; Hou X; Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.; Richardson T; Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.; Lewicki SL; Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.; Siuda J; Department of Neurology, Faculty of Medical Sciences in Katowice, Medical University of Silesia, Katowice, Poland.; Wszolek ZK; Department of Neurology, Mayo Clinic, Jacksonville, FL, USA.; Cook CN; Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.; Neuroscience PhD Program, Mayo Clinic Graduate School of Biomedical Sciences, Jacksonville, FL, USA.; Petrucelli L; Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.; Neuroscience PhD Program, Mayo Clinic Graduate School of Biomedical Sciences, Jacksonville, FL, USA.; DeTure M; Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.; Dickson DW; Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.; Neuroscience PhD Program, Mayo Clinic Graduate School of Biomedical Sciences, Jacksonville, FL, USA.; Antico O; MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK.; Muqit MMK; MRC Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dundee, UK.; Fishman JB; 21st Century Biochemicals Inc, Marlborough, MA, USA.; Pirani K; ImmunoPrecise Antibodies Ltd, Victoria, BC, Canada.; Kumaran R; Abcam plc, Cambridge, UK.; Polinski NK; The Michael J. Fox Foundation for Parkinson's Research, New York, NY, USA.; Fiesel FC; Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.; Neuroscience PhD Program, Mayo Clinic Graduate School of Biomedical Sciences, Jacksonville, FL, USA.; Springer W; Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA.; Neuroscience PhD Program, Mayo Clinic Graduate School of Biomedical Sciences, Jacksonville, FL, USA.
Source
Publisher: Taylor & Francis Country of Publication: United States NLM ID: 101265188 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1554-8635 (Electronic) Linking ISSN: 15548627 NLM ISO Abbreviation: Autophagy Subsets: MEDLINE
Subject
Language
English
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.