Citrullination, Anyone? New Gene Implicated in ALS

Scientists in Japan may have come across a new ALS gene, according to the February 6 Cell Reports. Koichi Matsuda, University of Tokyo, and colleagues found that the enzyme peptidylarginine deiminase 4—a newcomer to neurodegeneration research—modifies protein motifs containing arginine/glycine (Tanigawa et al., 2018). These are commonly found in the floppy C-terminal of ALS-related RNA-binding proteins. PAD4 converts these select arginines to citrullines. Citrullination renders the proteins less likely to aggregate, the authors report. What’s more, people are slightly more prone to ALS if they carry a genetic variant that reduces PAD4 expression.

A Fluid Situation. RNA-binding proteins such as TAF15 and FUS contain RGG motifs. These can be citrullinated by PAD4, making them more soluble, or methylated by protein arginine methyltransferases (PRMTs), making them stickier. [Courtesy of Cell Reports, Tanikawa et al., 2018]

During citrullination, PADs replace a positively charged ketamine group on an arginine residue in a protein with a neutral ketone, changing the conformation of the protein (Anzilotti et al., 2010). Citrullination has been linked to some neurological disorders, including Alzheimer’s disease and traumatic brain injury (Ishigami et al., 2005; Lazarus et al., 2015). PAD4 belongs to a family of five peptidylarginine deiminases, and is the only one that acts in the nucleus.

PAD4 was previously shown to regulate apoptosis, gene activity, and pluripotency in cells, though most of those studies relied on high calcium concentrations or ionophore treatment (Christophorou et al., 2014; Assohou-Luty et al., 2014; Guo et al., 2011). Matsuda and his group wanted to study the enzyme under more meaningful conditions.

First author Chizu Tanikawa and colleagues transfected human kidney cells (HEK293T) with a plasmid encoding wild-type PAD4, an enzyme-dead version, or no PAD4. They then figured out which proteins were citrullinated using liquid chromatography-mass spectrometry. Wild-type PAD4 citrullinated 159 proteins. All were involved in governing the biology of RNA: its processing, splicing, or metabolism. About a fifth of the 159 were citrullinated at arginine-glycine (RG/RGG) motifs commonly found in such RNA-binding proteins as hnRNPs, FUS, EWS, and TAF15, the last three being part of the FET family. Aggregation of FET proteins has been implicated in ALS (Kapeli et al., 2017).

RGG Proteins. [Courtesy of Molecular Cell, Thandapani et al., 2013]

Taking TAF15 as one example, the researchers reported that PAD4 citrullinated seven arginines, six of which were in RG/RGG motifs. Citrullination competed for methylation, a different post-translational modification that can take place at these same sites. The researchers found similar results for other FET proteins.

In the presence of PAD4, a protein called survival of motor neuron (SMN), which normally binds to methylated arginine residues, stopped interacting with FETs and hnRNPA1 (see March 2017 news; Rodriguez-Muela et al., 2017). Importantly, citrullination by PAD4 also kept these proteins from forming insoluble aggregates. What’s more, hnRNP proteins stopped forming complexes with TDP-43, which is notorious for forming toxic inclusions in ALS. All told, the data suggest that PAD4 curbs protein binding and aggregation.

Additionally, PAD4 seemed to prevent some proteins from entering stress granules, which entrap nuclear RNA-binding proteins when cells are under duress. In mouse embryonic fibroblasts lacking PAD4, TAF15 and FUS turned up more frequently in these cytosolic granules than in cells that expressed PAD4 normally.

Does any of this matter? Tanikawa and colleagues pored through genetic data from 1,955 ALS cases and 28,244 controls in Japan, and found a single nucleotide polymorphism in PAD4 whose minor allele made carriers 7 percent likelier to develop ALS. In this SNP, a cytosine replaces an adenine at rs2240335, which roughly halves PAD4 expression in the brain. Being homozygous for the allele meant a two-year earlier onset of ALS, the scientists found. The same SNP had been previously associated with rheumatoid arthritis, and trended toward association with ALS risk in a European population (Freudenberg et al., 2011; Fogh et al., 2014).

Adding indirect support for a role of rs2240335 in ALS risk, Tanikawa found that carriers of a C9ORF72 hexanucleotide expansion expressed about 70 percent less PAD4 in the frontal cortex than did healthy controls. The C9ORF72 GGCCC repeat is translated to yield five different dipeptide repeat proteins, and one of those—poly glycine/arginine—might be a PAD4 substrate, the authors note. Low expression of PAD4 could result in more aggregation of these dipeptide repeats, hastening disease onset among C9ORF72 mutation carriers, they suggested.

All in all, the authors suspect that their results hint that PAD4 citrullination influences neurodegenerative diseases such as ALS by counteracting aggregation of certain proteins. Ekaterina Rogaeva, University of Toronto, wrote to Alzforum that it will be essential to investigate this further. “Currently, it is not clear if ALS-causing mutations in FUS are linked to citrullination,” she added. Though it is intriguing that an SNP in PAD4 might associate with ALS in the Japanese population, this finding will require independent replication, too, she said.

Featured Paper

Tanikawa C, Ueda K, Suzuki A, Iida A, Nakamura R, Atsuta N, Tohnai G, Sobue G, Saichi N, Momozawa Y, Kamatani Y, Kubo M, Yamamoto K, Nakamura Y, Matsuda K. Citrullination of RGG Motifs in FET Proteins by PAD4 Regulates Protein Aggregation and ALS Susceptibility. Cell Rep. 2018 Feb 6;22(6):1473-1483. PubMed.


Anzilotti C, Pratesi F, Tommasi C, Migliorini P. Peptidylarginine deiminase 4 and citrullination in health and disease. Autoimmun Rev. 2010 Jan;9(3):158-60. Epub 2009 Jun 18 PubMed.

Ishigami A, Ohsawa T, Hiratsuka M, Taguchi H, Kobayashi S, Saito Y, Murayama S, Asaga H, Toda T, Kimura N, Maruyama N. Abnormal accumulation of citrullinated proteins catalyzed by peptidylarginine deiminase in hippocampal extracts from patients with Alzheimer’s disease. J Neurosci Res. 2005 Apr 1;80(1):120-8. PubMed.

Lazarus RC, Buonora JE, Flora MN, Freedy JG, Holstein GR, Martinelli GP, Jacobowitz DM, Mueller GP. Protein Citrullination: A Proposed Mechanism for Pathology in Traumatic Brain Injury. Front Neurol. 2015;6:204. Epub 2015 Sep 22 PubMed.

Assohou-Luty C, Raijmakers R, Benckhuijsen WE, Stammen-Vogelzangs J, de Ru A, van Veelen PA, Franken KL, Drijfhout JW, Pruijn GJ. The human peptidylarginine deiminases type 2 and type 4 have distinct substrate specificities. Biochim Biophys Acta. 2014 Apr;1844(4):829-36. Epub 2014 Mar 2 PubMed.

Guo Q, Bedford MT, Fast W. Discovery of peptidylarginine deiminase-4 substrates by protein array: antagonistic citrullination and methylation of human ribosomal protein S2. Mol Biosyst. 2011 Jul;7(7):2286-95. Epub 2011 May 16 PubMed.

Kapeli K, Martinez FJ, Yeo GW. Genetic mutations in RNA-binding proteins and their roles in ALS. Hum Genet. 2017 Sep;136(9):1193-1214. Epub 2017 Jul 31 PubMed.

Freudenberg J, Lee HS, Han BG, Shin HD, Kang YM, Sung YK, Shim SC, Choi CB, Lee AT, Gregersen PK, Bae SC. Genome-wide association study of rheumatoid arthritis in Koreans: population-specific loci as well as overlap with European susceptibility loci. Arthritis Rheum. 2011 Apr;63(4):884-93. PubMed.

Fogh I, Ratti A, Gellera C, Lin K, Tiloca C, Moskvina V, Corrado L, Sorarù G, Cereda C, Corti S, Gentilini D, Calini D, Castellotti B, Mazzini L, Querin G, Gagliardi S, Del Bo R, Conforti FL, Siciliano G, Inghilleri M, Saccà F, Bongioanni P, Penco S, Corbo M, Sorbi S, Filosto M, Ferlini A, Di Blasio AM, Signorini S, Shatunov A, Jones A, Shaw PJ, Morrison KE, Farmer AE, Van Damme P, Robberecht W, Chiò A, Traynor BJ, Sendtner M, Melki J, Meininger V, Hardiman O, Andersen PM, Leigh NP, Glass JD, Overste D, Diekstra FP, Veldink JH, van Es MA, Shaw CE, Weale ME, Lewis CM, Williams J, Brown RH, Landers JE, Ticozzi N, Ceroni M, Pegoraro E, Comi GP, D’Alfonso S, van den Berg LH, Taroni F, Al-Chalabi A, Powell J, Silani V, SLAGEN Consortium and Collaborators. A genome-wide association meta-analysis identifies a novel locus at 17q11.2 associated with sporadic amyotrophic lateral sclerosis. Hum Mol Genet. 2014 Apr 15;23(8):2220-31. Epub 2013 Nov 20 PubMed.

Further Reading

Christophorou MA, Castelo-Branco G, Halley-Stott RP, Oliveira CS, Loos R, Radzisheuskaya A, Mowen KA, Bertone P, Silva JC, Zernicka-Goetz M, Nielsen ML, Gurdon JB, Kouzarides T. Citrullination regulates pluripotency and histone H1 binding to chromatin. Nature. 2014 Mar 6;507(7490):104-8. PubMed.

Rodriguez-Muela N, Litterman NK, Norabuena EM, Mull JL, Galazo MJ, Sun C, Ng SY, Makhortova NR, White A, Lynes MM, Chung WK, Davidow LS, Macklis JD, Rubin LL. Single-Cell Analysis of SMN Reveals Its Broader Role in Neuromuscular Disease. Cell Rep. 2017 Feb 7;18(6):1484-1498. PubMed.

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citrullination disease-als disease-ftd FET genetic risk PAD4 RGG motif RNA binding protein SMA1 topic-genetics topic-preclinical
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