N‐terminal phosphorylation of Fused in Sarcoma (FUS) protein results in distinct changes to the cellular proteome that differ from the P525L model of pathology

Abstract

AbstractBackgroundFrontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) share many genetic and neuropathological markers of diseases (http://doi.org/10.1016/j.neuron.2013.07.033). One such neuropathological marker is the abnormal cytoplasmic aggregation of the Fused in Sarcoma (FUS) protein. FUS is a RNA/DNA binding protein involved in gene transcription, mRNA splicing, DNA‐repair pathways, and mRNA transport. To accomplish its many roles in the cell, FUS shuttles between the nucleus and cytoplasm carrying mRNA transcripts to distinct cellular locations. However, in FTD and ALS with FUS pathology, nuclear/cytoplasmic shuttling of FUS is disrupted resulting in its mislocalization into insoluble cytoplasmic inclusions (http://doi.org/10.1080/15476286.2016.1211225). Most pathological genetic mutations (including P525L) do not cause FTD (http://doi.org/10.1038/nn.3584). Therefore, non‐genetic factors such as post‐translational modifications may trigger FTD‐FUS pathology. Our lab discovered that double‐strand DNA damage induces phosphorylation of FUS (p‐FUS) at multiple N‐terminal sites leading to an accumulation of p‐FUS in the cytoplasm, yet the consequence of this cytoplasmic p‐FUS remain unknown (http://doi.org/10.1523/JNEUROSCI.0172‐14.2014). We set out to use an unbiased proximity labeling approach termed APEX2 to interrogate the WT FUS, P525L FUS, and p‐FUS proteomes.MethodWe and others have mapped the major N‐terminal phosphorylation sites, enabling the creation of a phosphomimetic variant of FUS (PM FUS) that mimics the phosphorylation of FUS caused by double‐stranded DNA damage. We then fused WT FUS, PM FUS, or P525L FUS to the engineered peroxidase, APEX2, and used proximity‐dependent biotin tagging to enrich for all proteins near the APEX2 complex. Biotin‐tagged proteins were identified and quantified using label‐free quantitative proteomics. Intensity readings were then corrected against controls using SAINT (Significance Analysis of INTeractome), a series of software tools that assign confidence values to observed protein‐protein interactions (http://doi.org/10.1038/nmeth.1541).ResultWe identified 3,313 differentially expressed proteins that were enriched in PM FUS, P525L FUS, and/or WT FUS over background. As expected, PM FUS enriched for cytoplasmic proteins as compared to WT FUS. Interestingly, the protein interactomes for PM FUS and P525L FUS separated independently of each other, suggesting distinct pathological mechanisms.ConclusionFindings from these studies will inform whether phosphorylation is a mechanism that could be targeted to shift FUS localization in FTD with FUS pathology.