Citrullination of RGG Motifs in FET Proteins by PAD4 Regulates Protein Aggregation and ALS Susceptibility

Abstract

Recent proteome analyses have provided a comprehensive overview of various posttranslational modifications (PTMs); however, PTMs involving protein citrullination remain unclear. We performed a proteomic analysis of citrullinated proteins, and we identified more than 100 PAD4 (peptidyl arginine deiminase 4) substrates. Approximately one-fifth of the PAD4 substrates contained an RG/RGG motif, and PAD4 competitively inhibited the methylation of the RGG motif in FET proteins (FUS, EWS, and TAF15) and hnRNPA1, which are causative genes for ALS (amyotrophic lateral sclerosis). PAD4-mediated citrullination significantly inhibited the aggregation of FET proteins, a frequently observed feature in neurodegenerative diseases. FUS protein levels in arsenic-induced stress granules were significantly increased in Padi4 mouse embryonic fibroblasts (MEFs). Moreover, rs2240335 was associated with low expression of PADI4 in the brain and a high risk of ALS (p = 0.0381 and odds ratio of 1.072). Our findings suggest that PAD4-mediated RGG citrullination plays a key role in protein solubility and ALS pathogenesis.

Arginine citrullination at the C-terminal domain controls RNA polymerase II transcription

“…Depletion of the methyltransferases responsible for R1810 dimethylation, CARM1 and PRMT5, did not affect the expression of PADI2 dependent genes, and depletion of PADI2 did not change the expression of a broad range of snRNAs, the targets of CARM1. As PADI2 can not act on merthylate R1810, and citrullination will preclude methylation by arginine methyltrasferases, its seems that these are alternative types of modifications influencing different stages of transcription, as observed in other arginine residues that can undergo methylation and citrullination (Tanikawa et al 2018;Cuthbert et al, 2004). This implies the dynamic nature of R1810 modifications, that change the docking surface for regulatory protein complexes to control various phases of transcription.…”

“…Citrullination is known to modulate functional protein-protein interactions (Tanikawa et al, 2018;Tanikawa et al, 2012;Vossenaar et al, 2003). We wondered whether the Cit1810 influences the interaction of RNAP2 with the components of P-TEFb complex CDK9 and CCNTI (Ccylin T1), which are required for RNAP2 pause release and productive elongation (Jonkers et al 2015;Gressel et al, 2017).…”

“…The functional significance of dynamic post-translational deimination of arginine residues in pathophysiological conditions (Slade et al, 2014;Tanikawa et al, 2018) prompted us to investigate whether this modification occurs at R1810 of RNAP2-CTD and its possible implication in transcription regulation.…”

“…Citrullination leads to a reduction in hydrogen-bond formation, affects histone-DNA interactions and influences the chromatin organization. Citrullination also increases the hydrophobicity of proteins that affect the protein folding ability and therefore the functional activity of proteins (Vossenaar et al, 2003;Tanikawa et al, 2018). This reaction is catalyzed by enzymes called peptidyl arginine deiminases (PADIs), which have been associated with diverse disease conditions such as thrombosis, prion disease, neurological disorders, autoimmune disease and cancer (Witalison et al, 2015;Gyorgy et al, 2006;Vossenaar et al, 2003;Baka et al, 2012).…”

Androgen signaling connects short isoform production to breakpoint formation at Ewing sarcoma breakpoint region 1 via an R-loop-dependent mechanism

“…Aside from cancer, all three FET family members are mutated in ALS and in frontotemporal dementia (FTD) (Couthouis et al, 2012;Kapeli et al, 2017). FET proteins have been demonstrated to form higher order structures that cause liquid demixing and formation of membraneless organelles through aggregation in the PrLD (Altmeyer et al, 2015;Qamar et al, 2018;Tanikawa et al, 2018) or RNA binding (Maharana et al, 2018). When reversible, these "droplets" facilitate normal cellular processes such as cytoplasmic stress granule formation but when irreversible may form pathological plaques in disease (Patel et al, 2015).…”