Chemo-Enzymatic Modification of the 5' Cap Maintains Translation and Increases Immunogenic Properties of mRNA.

Eukaryotic mRNAs are emerging modalities for protein replacement therapy and vaccination. Their 5' cap is important for mRNA translation and immune response and can be naturally methylated at different positions by S-adenosyl-l-methionine (AdoMet)-dependent methyltransferases (MTases). We report on the cosubstrate scope of the MTase CAPAM responsible for methylation at the N6 -position of adenosine start nucleotides using synthetic AdoMet analogs. The chemo-enzymatic propargylation enabled production of site-specifically modified reporter-mRNAs. These cap-propargylated mRNAs were efficiently translated and showed ≈3-fold increased immune response in human cells. The same effects were observed when the receptor binding domain (RBD) of SARS-CoV-2-a currently tested epitope for mRNA vaccination-was used. Site-specific chemo-enzymatic modification of eukaryotic mRNA may thus be a suitable strategy to modulate translation and immune response of mRNAs for future therapeutic applications.

S-adenosylhomocysteine induces inflammation through NFkB: A possible role for EZH2 in endothelial cell activation.

S-adenosylhomocysteine (SAH) can induce endothelial dysfunction and activation, contributing to atherogenesis; however, its role in the activation of the inflammatory mediator NFkB has not been explored. Our aim was to determine the role of NFkB in SAH-induced activation of endothelial cells. Furthermore, we examined whether SAH, as a potent inhibitor of S-adenosylmethionine-dependent methyltransferases, suppresses the function of EZH2 methyltransferase to contribute to SAH-induced endothelial cell activation. We found that excess SAH increases the expression of adhesion molecules and cytokines in human coronary artery endothelial cells. Importantly, this up-regulation was suppressed in cells expressing a dominant negative form of the NFkB inhibitor, IkB. Moreover, SAH accumulation triggers the activation of both the canonical and non-canonical NFkB pathways, decreases EZH2, and reduces histone 3 lysine 27 trimethylation. EZH2 knockdown recapitulated the effects of excess SAH on endothelial activation, i.e., it induced NFkB activation and the subsequent up-regulation of adhesion molecules and cytokines. Our findings suggest that suppression of the epigenetic regulator EZH2 by excess SAH may contribute to NFkB activation and the consequent vascular inflammatory response. These studies unveil new targets of SAH regulation, demonstrating that EZH2 suppression and NFkB activation mediated by SAH accumulation may contribute to its adverse effects in the vasculature.

Metabolic control of the epigenome in systemic Lupus erythematosus.

Epigenetic mechanisms are proposed to underlie aberrant gene expression in systemic lupus erythematosus (SLE) that results in dysregulation of the immune system and loss of tolerance. Modifications of DNA and histones require substrates derived from diet and intermediary metabolism. DNA and histone methyltransferases depend on S-adenosylmethionine (SAM) as a methyl donor. SAM is generated from adenosine triphosphate (ATP) and methionine by methionine adenosyltransferase (MAT), a redox-sensitive enzyme in the SAM cycle. The availability of B vitamins and methionine regulate SAM generation. The DNA of SLE patients is hypomethylated, indicating dysfunction in the SAM cycle and methyltransferase activity. Acetyl-CoA, which is necessary for histone acetylation, is generated from citrate produced in mitochondria. Mitochondria are also responsible for de novo synthesis of flavin adenine dinucleotide (FAD) for histone demethylation. Mitochondrial oxidative phosphorylation is the dominant source of ATP. The depletion of ATP in lupus T cells may affect MAT activity as well as adenosine monophosphate (AMP) activated protein kinase (AMPK), which phosphorylates histones and inhibits mechanistic target of rapamycin (mTOR). In turn, mTOR can modify epigenetic pathways including methylation, demethylation, and histone phosphorylation and mediates enhanced T-cell activation in SLE. Beyond their role in metabolism, mitochondria are the main source of reactive oxygen intermediates (ROI), which activate mTOR and regulate the activity of histone and DNA modifying enzymes. In this review we will focus on the sources of metabolites required for epigenetic regulation and how the flux of the underlying metabolic pathways affects gene expression.

Elongator subunit 3 positively regulates plant immunity through its histone acetyltransferase and radical S-adenosylmethionine domains.

BACKGROUND: Pathogen infection triggers a large-scale transcriptional reprogramming in plants, and the speed of this reprogramming affects the outcome of the infection. Our understanding of this process has significantly benefited from mutants that display either delayed or accelerated defense gene induction. In our previous work we demonstrated that the Arabidopsis Elongator complex subunit 2 (AtELP2) plays an important role in both basal immunity and effector-triggered immunity (ETI), and more recently showed that AtELP2 is involved in dynamic changes in histone acetylation and DNA methylation at several defense genes. However, the function of other Elongator subunits in plant immunity has not been characterized. RESULTS: In the same genetic screen used to identify Atelp2, we found another Elongator mutant, Atelp3-10, which mimics Atelp2 in that it exhibits a delay in defense gene induction following salicylic acid treatment or pathogen infection. Similarly to AtELP2, AtELP3 is required for basal immunity and ETI, but not for systemic acquired resistance (SAR). Furthermore, we demonstrate that both the histone acetyltransferase and radical S-adenosylmethionine domains of AtELP3 are essential for its function in plant immunity. CONCLUSION: Our results indicate that the entire Elongator complex is involved in basal immunity and ETI, but not in SAR, and support that Elongator may play a role in facilitating the transcriptional induction of defense genes through alterations to their chromatin.

Lupus autoimmunity altered by cellular methylation metabolism.

Modifications of both DNA and protein by methylation are key factors in normal T and B cell immune responses as well as in the development of autoimmune disease. For example, the failure to maintain the methylation status of CpG dinucleotides in DNA triggers T cell autoreactivity. Methylated proteins are known targets of autoimmunity, including the symmetrical dimethylarginine residues of SmD1 and SmD3 in SLE. Herein, we demonstrate that altering the metabolism of S-adenosylmethionine (SAM), the major methyl donor for transmethylation reactions, can suppress T cell immunity. A by-product of SAM metabolism, 5'-deoxy-5'-methylthioadenosine (MTA), and an indirect inhibitor of methyltransferases, inhibits T cell responses including T cell activation markers, Th1/Th2 cytokines and TCR-related signaling events. Moreover, treatment of the lupus-prone MRL/lpr mouse with MTA markedly ameliorates splenomegaly, lymphadenopathy, autoantibody titers as well as IgG deposition and cellular infiltration in the kidney. Incubation of cells with SAM, which increases intracellular MTA levels, inhibits both TCR-mediated T cell proliferation and BCR (anti-IgM)-triggered B cell proliferation in a dose-dependent manner. These studies define the central role of MTA and SAM in immune responses and provide a simple approach to altering lymphocyte transmethylation and T cell mediated autoimmune syndromes.

Decreased DNA methyltransferase 3A and 3B mRNA expression in peripheral blood mononuclear cells and increased plasma SAH concentration in adult patients with idiopathic thrombocytopenic purpura.

OBJECTIVE: DNA methylation is known to play an important role in gene transcription and alterations of methylation contribute to the development of certain disorders such as cancer and immunodeficiency. Recent years have found an increasing interest in the role of epigenetic modifications in the etiology of human autoimmune diseases, such as systemic lupus erythromatosus (SLE) and rheumatoid arthritis (RA). DNA methyltransferases (DNMTs) are involved in the epigenetic control of DNA methylation processes. S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), as the substrate and product of essential cellular methyltransferase reactions, have important indicator action of cellular methylation status. The aim of this study is to explore if DNA methylation plays a role in the pathogenesis of idiopathic thrombocytopenic purpura (ITP). METHODS: DNMT1, DNMT3A, and DNMT3B mRNA expression in peripheral blood mononuclear cells (PBMCs) of adult ITP patients were analyzed by real-time quantitative polymerase chain reaction. Plasma SAM and SAH levels were assayed with reversed-phase high performance liquid chromatography (HPLC). RESULTS: DNMT3A and DNMT3B mRNA expressions were significantly lower in ITP patients than in healthy controls (p < 0.001), while DNMT1 mRNA expression was not significantly different between the two groups (p = 0.774). Plasma SAH concentration was significantly elevated in ITP patients than in healthy controls (p < 0.05), while the plasma SAM and SAM/SAH were not significantly different between the two groups (p = 0.133, p = 0.624 respectively). CONCLUSIONS: Our observations suggest that aberrant DNA methylation status reflected by increased plasma SAH concentration and decreased mRNA expression levels of DNMT3A and 3B are possibly involved in the pathogenesis of ITP although the precise mechanisms need further study.