Unbiased Label-Free Quantitative Proteomics of Cells Expressing Amyotrophic Lateral Sclerosis (ALS) Mutations in CCNF Reveals Activation of the Apoptosis Pathway: A Workflow to Screen Pathogenic Gene Mutations.

Autor:	Cheng F; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., De Luca A; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., Hogan AL; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., Rayner SL; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., Davidson JM; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., Watchon M; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., Stevens CH; Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, NSW, Australia.; School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, NSW, Australia., Muñoz SS; Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, NSW, Australia.; School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, NSW, Australia., Ooi L; Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, NSW, Australia.; School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, NSW, Australia., Yerbury JJ; Illawarra Health and Medical Research Institute (IHMRI), University of Wollongong, Wollongong, NSW, Australia.; School of Chemistry and Molecular Bioscience and Molecular Horizons, University of Wollongong, Wollongong, NSW, Australia., Don EK; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., Fifita JA; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., Villalva MD; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., Suddull H; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., Chapman TR; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., Hedl TJ; Neurodegeneration Pathobiology Laboratory, Queensland Brain Institute, The University of Queensland, St Lucia, QLD, Australia., Walker AK; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia.; Neurodegeneration Pathobiology Laboratory, Queensland Brain Institute, The University of Queensland, St Lucia, QLD, Australia., Yang S; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., Morsch M; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., Shi B; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., Blair IP; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., Laird AS; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., Chung RS; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia., Lee A; Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine, Health, and Human Sciences, Macquarie University, North Ryde, NSW, Australia.
Jazyk:	angličtina
Zdroj:	Frontiers in molecular neuroscience [Front Mol Neurosci] 2021 Apr 27; Vol. 14, pp. 627740. Date of Electronic Publication: 2021 Apr 27 (Print Publication: 2021).
DOI:	10.3389/fnmol.2021.627740
Abstrakt:	The past decade has seen a rapid acceleration in the discovery of new genetic causes of ALS, with more than 20 putative ALS-causing genes now cited. These genes encode proteins that cover a diverse range of molecular functions, including free radical scavenging (e.g., SOD1), regulation of RNA homeostasis (e.g., TDP-43 and FUS), and protein degradation through the ubiquitin-proteasome system (e.g., ubiquilin-2 and cyclin F) and autophagy (TBK1 and sequestosome-1/p62). It is likely that the various initial triggers of disease (either genetic, environmental and/or gene-environment interaction) must converge upon a common set of molecular pathways that underlie ALS pathogenesis. Given the complexity, it is not surprising that a catalog of molecular pathways and proteostasis dysfunctions have been linked to ALS. One of the challenges in ALS research is determining, at the early stage of discovery, whether a new gene mutation is indeed disease-specific, and if it is linked to signaling pathways that trigger neuronal cell death. We have established a proof-of-concept proteogenomic workflow to assess new gene mutations, using CCNF (cyclin F) as an example, in cell culture models to screen whether potential gene candidates fit the criteria of activating apoptosis. This can provide an informative and time-efficient output that can be extended further for validation in a variety of in vitro and in vivo models and/or for mechanistic studies. As a proof-of-concept, we expressed cyclin F mutations (K97R, S195R, S509P, R574Q, S621G) in HEK293 cells for label-free quantitative proteomics that bioinformatically predicted activation of the neuronal cell death pathways, which was validated by immunoblot analysis. Proteomic analysis of induced pluripotent stem cells (iPSCs) derived from patient fibroblasts bearing the S621G mutation showed the same activation of these pathways providing compelling evidence for these candidate gene mutations to be strong candidates for further validation and mechanistic studies (such as E3 enzymatic activity assays, protein-protein and protein-substrate studies, and neuronal apoptosis and aberrant branching measurements in zebrafish). Our proteogenomics approach has great utility and provides a relatively high-throughput screening platform to explore candidate gene mutations for their propensity to cause neuronal cell death, which will guide a researcher for further experimental studies. Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. (Copyright © 2021 Cheng, De Luca, Hogan, Rayner, Davidson, Watchon, Stevens, Muñoz, Ooi, Yerbury, Don, Fifita, Villalva, Suddull, Chapman, Hedl, Walker, Yang, Morsch, Shi, Blair, Laird, Chung and Lee.)
Databáze:	MEDLINE
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