Pathogenic mutation in the ALS/FTD gene, <Emphasis Type="Italic">CCNF,</Emphasis> causes elevated Lys48-linked ubiquitylation and defective autophagy |
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Authors: | Albert Lee Stephanie L Rayner Serene S L Gwee Alana De Luca Hamideh Shahheydari Vinod Sundaramoorthy Audrey Ragagnin Marco Morsch Rowan Radford Jasmin Galper Sarah Freckleton Bingyang Shi Adam K Walker Emily K Don Nicholas J Cole Shu Yang Kelly L Williams Justin J Yerbury Ian P Blair Julie D Atkin Mark P Molloy Roger S Chung |
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Institution: | 1.Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences,Macquarie University,North Ryde,Australia;2.Australian Proteome Analysis Facility,Macquarie University,North Ryde,Australia;3.Department of Chemistry and Biomolecular Sciences, Faculty of Science and Engineering,Macquarie University,North Ryde,Australia;4.Illawarra Health and Medical Research Institute, School of Biological Sciences,University of Wollongong,Wollongong,Australia;5.Department of Biochemistry and Genetics,La Trobe Institute for Molecular Science,Melbourne,Australia |
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Abstract: | Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin–protein ligase (SCFcyclin F) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin–proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin FS621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin FWT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal–lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin FS621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis. |
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