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Effects of mutations in the novel FTD‐ALS gene, CYLD , on SFPQ protein
Author(s) -
Boccanfuso Lauren,
Lee Albert,
Chung Roger S,
Oyston Lisa J,
Kwok John B,
DobsonStone Carol
Publication year - 2020
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1002/alz.047382
Subject(s) - deubiquitinating enzyme , microbiology and biotechnology , hek 293 cells , western blot , blot , mutant , nuclear protein , stress granule , frontotemporal dementia , biology , gene , ubiquitin , genetics , medicine , messenger rna , transcription factor , dementia , disease , translation (biology)
Background Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative disorders that share similar clinical and pathological hallmarks, as well as overlapping genetic aetiologies. Previously, we identified that mutations in CYLD can cause FTD‐ALS. CYLD is a lysine‐63 deubiquitinase, and the FTD‐ALS causal mutation CYLD M719V shows significantly increased deubiquitinase activity [Dobson‐Stone et al . 2020, Brain 143:783‐799]. CYLD is known to interact with the protein products of at least three established FTD and ALS genes. We sought to determine whether other FTD‐ALS proteins were influenced by modulation of CYLD. Method We performed mass spectrometry quantitative proteomic analysis of HEK293 lysates overexpressing wild‐type and mutant CYLD (CYLD WT & CYLD M719V ), followed by western blots of total cell lysates from SH‐SY5Y and HEK293 cells overexpressing CYLD WT , CYLD M719V and the deubiquitinase‐inactive mutation CYLD D681G . Densitometry analysis was performed in Image Lab (Bio‐Rad, USA) and normalised to total protein. Result Mass spectrometry analysis indicated significant differences in expression of TIA1, MATR3, VCP and SFPQ when CYLD was overexpressed. Validation by western blot showed that overexpression of CYLD increased relative protein expression of SFPQ (2.7 fold increase for CYLD WT versus empty vector, p = 0.0048) in SH‐SY5Y cells. This is likely due to deubiquitinase activity, as CYLD D681G did not show such an increase. SFPQ is a nucleic acid binding protein that has been shown to mislocalise from the nucleus to the cytoplasm in FTD and ALS. We are currently examining whether CYLD affects subcellular localisation of SFPQ by western blot and immunofluorescent microscopy analyses. Conclusion Our study indicates that CYLD overexpression increases SFPQ protein expression via its deubiquitinase activity. However, it remains to be shown whether this mechanism is essential to the pathogenesis of FTD and ALS driven by CYLD.