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. 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.

  • PAD4 enzyme modifies arginine in RNA-binding proteins to citrulline.
  • This makes them less likely to aggregate.
  • A genetic variant that lowers PAD4 expression associated with a slightly higher risk of ALS.

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 (Assohou-Luty et al., 2014; Guo et al., 2011). Matsuda and his group wanted to study the enzyme under more meaningful conditions.

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.]

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). 

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. 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.—Gwyneth Dickey Zakaib

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References

Paper Citations

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Further Reading

Primary Papers

  1. 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.

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