*3.3. Arginine Citrullination*

Citrullination is another PTM that occurs to arginine residues. Instead of the addition of a functional group, the arginine side chain undergoes an oxidation (or deimination) reaction. In this reaction, peptidylarginine deiminases (PADs) catalyze the oxidation of an imine group (=NH), forming a ketone group (=O) [60,68]. This modification removes the positive charge, leaving a neutrally charged amino acid. Interestingly, the consensus site for PADs are the same RG/RGG motifs that are common to many RNA-binding and phase separating proteins [69]. Citrullination of FUS by PAD4 was shown to diminish FUS recruitment to SGs [69]. PAD4 knockout in mouse embryonic fibroblasts showed a greater amount of FUS sequestration into SGs than when PAD4 was overexpressed in these cells, suggesting citrullination hinders FUS phase separation. Cation-π interactions between arginine residues and the π orbitals of tyrosine residues modulate FUS phase separation [60], but when citrullination occurs, the positive charge of the arginine side chain is removed, disrupting cation-π interactions and disrupting FUS phase separation in vitro [60].

#### *3.4. Lysine Acetylation*

Similar to citrullination, acetylation neutralizes the positive charge of an amino acid. Lysine residues contain a positively charged amino head group that can be neutralized by addition of an acetyl group; this not only changes lysine's charge state but also increases its hydrophobicity [70]. Acetyl groups are enzymatically added via acetyltransferases and removed by deacetylases [71]. Acetylation has been shown to disrupt simple coacervation of DDX3X (dead box RNA helicase 3) in vitro [72]. DDX3X has two IDRs: at the C-terminus and the N-terminus. Analysis of an acetylome dataset identified several acetylated lysines in the N-terminal IDR that play a role in DDX3X incorporation into SGs [72]. DDX3X is a substrate of acetyltransferase CREB-binding protein (CBP) and histone deacetylase 6 (HDAC6). To better understand the role of acetylation of DDX3X and SG incorporation, acetyl mimetic (K→Q) constructs and acetyl-dead (K→R) were constructed and expressed in DDX3X knock-out cell lines. Expression of acetyl-dead DDX3X (or inhibition of CBP) increased SG volume, whereas expression of acetyl-mimetic mutant (or inhibition of HDAC6) decreased SG volume.

The formation of SG has been proposed as a two-step process. First a stable core structure is formed, which is followed by the recruitment of IDPs into an outer shell structure [73]. The increase in volume is an important step in SG maturation. Of significance to this growth mechanism, the interaction partners of the acetyl-dead and acetyl mimetic DDX3X mutants were different. The non-acetylated DDX3X interacts with numerous SG components, whereas the acetyl mimetic loses its capacity for interactions with SG proteins, thus showcasing how lysine acetylation can be used to regulate MLO maturation [72].

Another protein that is lysine acetylated is tau, which is of particular interest since its solid-phase aggregation in neurons is linked to Alzheimer's disease [74]. Tau is an IDP, and like DDX3X, its ability to phase separate is disrupted by lysine acetylation [75]. Ferreon et al. found that recombinant tau, when incubated with enzymatically-active p300 histone acetyltransferase (HAT), becomes hyperacetylated (ac-tau) [75]. This acetylation (removal of positive charges) was observed to disfavor LLPS, which is consistent with the previous observations of Wegmann et al. that phosphorylation (addition of negative charges) promotes tau phase separation [34]. Using mass spectrometry analysis, 15 acetylation sites were identified, 8 of which are located in IDRs. Tau readily undergoes LLPS in vitro in low-salt conditions, but ac-tau was unable to form droplets under the same conditions. The neutralization of charged residues was concluded to disrupt electrostatic interactions required for tau LLPS [52,75]. Interestingly, tau also phase separates into SGs [76]. Ukmar-Godec et al. showed that tau association into SGs is altered by the acetylation state of the lysines. Unmodified full length tau441 readily associated with SGs following proteasome inhibition by MG132. Consistent with the in vitro findings above, acetylation of tau strongly reduced the association of the protein with SGs in HeLa cells [77]. Lastly, Ac-tau also showed decreased solid-phase aggregation propensity and reduced thioflavin-t reactivity, which indicates less propensity to form amyloid-like solid aggregates. This suggests acetyltransferases and deacetylases are potential therapeutic targets for prevention of pathological tau aggregation [75].
