Serotonylation

Protein serotonylation refers to the post-translational modification in which the monoamine serotonin is covalently attached to glutamine residues on substrate proteins via transamidation. Serotonylation is a type of monoaminylation, which itself refers to the overall class of post-translational modifications involving monoamines. However, monoaminylation reactions are further classified by the individual monoamine reactant they describe (ie., serotonylation, dopaminylation, histaminylation).

Serotonylation has been reported for both histone and non-histone protein substrates, and thus represents a distinct neuroepigenetic and neuroproteomic regulatory mechanism with various implications in health and disease.^[1] Since 2003, multiple studies have revealed the critical role of serotonylation in mediating a wide range of physiological processes, both in the nervous system and beyond.^[1][2][3] Serotonylation is known to contribute to several significant diseases, including neuropsychiatric disorders such as depression and schizophrenia, as well as a variety of cancers.^[1][4][5]

To date, notable protein serotonylation substrates include several metabolic enzymes (GAPDH, mTOR),^[6][7] Rab GTPases (Rab3a, Rab27a),^[8][9] Rho GTPases (RhoA, Rac1, Cdc42),^[10][11] proteins involved in muscle contractility (⍺-actinin, SERCA2a),^[12][13] extracellular matrix proteins (fibronectin),^[14][15] neural surface proteins,^[14] and Ras,^[16] as well as the histone protein H3.^[17][18]

Serotonylation has been reported in various cell types and tissues, including both serotonergic and dopaminergic neurons,^[19][3] enterochromaffin cells,^[7] cancer-associated fibroblasts,^[20] pancreatic 𝛽-cells,^[9] CD8+ T cells,^[6] pulmonary endothelial cells,^[21] platelets,^[22] neutrophils,^[18] mammary epithelial cells,^[23] vascular smooth muscle,^[12] and cells of the intestines.^[16] Serotonylation is known to influence both tumorigenesis and cancer metastasis, and has been implicated in several types of cancer, including colorectal cancer,^[7][24] neuroendocrine prostate cancer,^[18] pancreatic cancer,^[25] hepatocellular carcinoma,^[26][27] and ependymomas (brain cancer).^[28]

Protein monoaminylation was first discovered in 1957 by Heinrich Waelsch and colleagues at Columbia University. After demonstrating the incorporation of monoamines into proteins via transamidation at glutamine residues,^[29] the group went on to uncover the enzyme catalyzing these reactions, effectively naming it "transglutaminase" after its function.^[30][31]

Despite its discovery in the mid-twentieth century, protein monoaminylation was not investigated as a post-translational modification until 2003, when Diego Walther and colleagues at the Max-Planck-Institute for Molecular Genetics revealed that serotonylation of small GTPases mediates ⍺-granule release during the activation and aggregation of platelets.^[22]

Notably, monoaminylation itself was not uncovered as an epigenetic regulatory mechanism until 2019, when Lorna Farrelly and colleagues at the Icahn School of Medicine reported the H3Q5-serotonylation (H3Q5ser) modification for the first time.^[32] Thereafter, in 2021, Di Ye and colleagues at Sichuan University revealed mTOR-serotonylation as part of a novel feedback mechanism within the tryptophan pathway in colon cancer, demonstrating a link between protein serotonylation and cancer for the first time.^[7]

Serotonylation is catalyzed by transglutaminase 2 (TGM2) in a calcium-dependent manner, and relies upon the intracellular bioavailability of serotonin substrates.^[2][33] Generally, protein serotonylation occurs in the cytoplasm; however, histone serotonylation only occurs within the nucleus.^[1][2] Nevertheless, the mechanism for TGM2-catalyzed serotonylation is identical for both histone and non-histone proteins alike.^[1]

Structurally, Ca²⁺ binds directly to TGM2 itself and not to the substrate molecule.^[33] Once Ca²⁺ binds to TGM2, a 4 nm relaxation about the major axis of the protein exposes the active site to available substrates.^[33][34] The active site itself is composed of a well conserved catalytic triad (Cys277–His335–Asp358) situated within a substrate binding channel, which is bordered by two conserved residues (Trp241 and Trp332) that facilitate catalysis through stabilization of the transition state.^[33][35] Once intracellular Ca²⁺ binds to TGM2 and exposes the substrate binding channel, the glutamine residue of a substrate protein (ie., histone H3, RhoA) is free to enter the enzyme active site.^[1][33] As a transamidation reaction, the mechanism for protein serotonylation can be summarized in two parts: an initial thioester formation, followed by isopeptide bond formation.

When intracellular Ca²⁺ and serotonin concentrations are sufficient, TGM2-catalyzed serotonylation of substrate proteins can occur.^[33] First, the catalytic cysteine residue (Cys277) in the TGM2 active site nucleophilically attacks the 𝛾-carboxamido group of the glutamine residue in an acyl transfer reaction (Fig. 1A), forming a thioester intermediate and releasing one molecule of ammonia (NH₃) as a result.^[1][33] Next, the deprotonated primary amine of the serotonin molecule nucleophilically attacks the 𝛾-thioester group of the intermediate, forming a stable isopeptide bond and ultimately releasing the enzyme (Fig. 1B).^[1][33]

With the discovery of histone monoaminylation in 2019, monoaminylation thus entered into the complex and ever-growing field of epigenetics, posing as a novel set of dynamic regulatory mechanisms.^[1][16] To date, histone H3 is the only histone protein known to undergo monoaminylation modifications, and such modifications have only been reported for glutamine position 5 (Gln5) of histone H3 (hereafter referred to as H3Q5).^[1] Thus, histone monoaminylation currently refers to the covalent addition of monoamines to glutamine at position 5 (Gln5) of histone H3.^[1] Histone serotonylation remains the most widely reported histone monoaminylation modification to date, though both histone dopaminylation and histone histaminylation have also been reported.^[1]

Histone monoaminylation modifications are associated with a number of regulatory effects, no two of which appear to be the same. H3Q5-serotonylation (H3Q5ser) has been reported in a wide range of tissues and cell types, including serotonergic neurons of the dorsal raphe nucleus,^[17] astrocytes of the olfactory bulb,^[36][37] the inferior alveolar nerve (ie., of the lip and lower jaw),^[38] placenta,^[39] ependymomas (brain cancers),^[40] pancreatic ductal adenocarcinoma (PDAC) tissues,^[41] cancer-associated fibroblasts,^[20] hepatocellular carcinoma (HCC),^[42][43] and neutrophils.^[44] Combinatory effects between monoaminylation and other histone modifications have been reported.^[17] Herein, low levels of trimethylation and serotonylation of histone H3 at lysine position 4 (H3K4) and glutamine position 5 (H3Q5), respectively (ie., H3K4me3Q5ser), in the dorsal raphe nucleus led to depressive symptoms in both male and female mice exposed to chronic stress.^[17] Behavioral outcomes associated with H3K4me3Q5ser depletion were corrected by treatment with serotonin-associated antidepressants, thus evidencing such antidepressants as sufficient to attenuate stress-mediated gene expression and behavioral dysregulation.^[17] Interestingly, corresponding patterns of H3K4me3Q5ser depletion were observed in the brains of major depressive disorder (MDD) patients on vs. off antidepressants at their time of death, thus evidencing a neurotransmission-independent role for serotonin in mediating both stress-associated and anti-depressant-associated transcriptional plasticity and behavioral outcomes.^[17] Data as to the effects of H3Q5ser and H3K4me3Q5ser are displayed in detail within the table below:

• Monoaminylation
• Histone Monoaminylation
• Dopaminylation
• Histaminylation