TY - JOUR
T1 - Unbiased label-free quantitative proteomics of cells expressing amyotrophic lateral sclerosis (ALS) mutations in CCNF reveals activation of the apoptosis pathway
T2 - a workflow to screen pathogenic gene mutations
AU - Cheng, Flora
AU - Luca, Alana De
AU - Hogan, Alison L.
AU - Rayner, Stephanie L.
AU - Davidson, Jennilee M.
AU - Watchon, Maxinne
AU - Stevens, Claire H.
AU - Muñoz, Sonia Sanz
AU - Ooi, Lezanne
AU - Yerbury, Justin J.
AU - Don, Emily K.
AU - Fifita, Jennifer A.
AU - Villalva, Maria D.
AU - Suddull, Hannah
AU - Chapman, Tyler R.
AU - Hedl, Thomas J.
AU - Walker, Adam K.
AU - Yang, Shu
AU - Morsch, Marco
AU - Shi, Bingyang
AU - Blair, Ian P.
AU - Laird, Angela S.
AU - Chung, Roger S.
AU - Lee, Albert
N1 - Copyright the Author(s) 2021. Version archived for private and non-commercial use with the permission of the author/s and according to publisher conditions. For further rights please contact the publisher.
PY - 2021/4/27
Y1 - 2021/4/27
N2 - 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.
AB - 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.
KW - proteomics
KW - amyotrophic lateral sclerosis
KW - induced pluripotent stem cell
KW - proteogenomics
KW - cell death
KW - neurodegeneration
KW - proteostasis cell stress and aging
KW - zebrafish
UR - http://purl.org/au-research/grants/nhmrc/GNT1124005
UR - http://purl.org/au-research/grants/nhmrc/1095215
UR - http://purl.org/au-research/grants/nhmrc/1107644
UR - http://purl.org/au-research/grants/nhmrc/1146750
UR - http://purl.org/au-research/grants/nhmrc/1140386
UR - http://purl.org/au-research/grants/nhmrc/1135720
UR - http://www.scopus.com/inward/record.url?scp=85105653591&partnerID=8YFLogxK
U2 - 10.3389/fnmol.2021.627740
DO - 10.3389/fnmol.2021.627740
M3 - Article
C2 - 33986643
SN - 1662-5099
VL - 14
SP - 1
EP - 18
JO - Frontiers in Molecular Neuroscience
JF - Frontiers in Molecular Neuroscience
M1 - 627740
ER -