Themis2 regulates natural killer cell memory function and formation.

Immunological memory is a hallmark of the adaptive immune system. Although natural killer (NK) cells are innate immune cells important for the immediate host defence, they can differentiate into memory NK cells. The molecular mechanisms controlling this differentiation are yet to be fully elucidated. Here we identify the scaffold protein Themis2 as a critical regulator of memory NK cell differentiation and function. Themis2-deficient NK cells expressing Ly49H, an activating NK receptor for the mouse cytomegalovirus (MCMV) antigen m157, show enhanced differentiation into memory NK cells and augment host protection against MCMV infection. Themis2 inhibits the effector function of NK cells after stimulation of Ly49H and multiple activating NK receptors, though not specific to memory NK cells. Mechanistically, Themis2 suppresses Ly49H signalling by attenuating ZAP70/Syk phosphorylation, and it also translocates to the nucleus, where it promotes Zfp740-mediated repression to regulate the persistence of memory NK cells. Zfp740 deficiency increases the number of memory NK cells and enhances the effector function of memory NK cells, which further supports the relevance of the Themis2-Zfp740 pathway. In conclusion, our study shows that Themis2 quantitatively and qualitatively regulates NK cell memory formation.

Histidine Nτ-methylation identified as a new posttranslational modification in histone H2A at His-82 and H3 at His-39.

Histone posttranslational modifications play critical roles in a variety of eukaryotic cellular processes. In particular, methylation at lysine and arginine residues is an epigenetic mark that determines the chromatin state. In addition, histone "histidine" methylation was initially reported over 50 years ago; however, further studies in this area were not conducted, leaving a gap in our understanding. Here, we aimed to investigate the occurrence of histidine methylation in histone proteins using highly sensitive mass spectrometry. We found that acid hydrolysates of whole histone fraction from calf thymus contained Nτ-methylhistidine, but not Nπ-methylhistidine. Both core and linker histones carried a Nτ-methylhistidine modification, and methylation levels were relatively high in histone H3. Furthermore, through MALDI-TOF MS, we identified two histidine methylation sites at His-82 in the structured globular domain of histone H2A and His-39 in the N-terminal tail of histones H3. Importantly, these histidine methylation signals were also detected in histones purified from a human cell line HEK293T. Moreover, we revealed the overall methylation status of histone H3, suggesting that methylation is enriched primarily at lysine residues and to a lesser extent at arginine and histidine residues. Thus, our findings established histidine Nτ-methylation as a new histone modification, which may serve as a chemical flag that mediates the epigenetic mark of adjacent residues of the N-terminal tail and the conformational properties of the globular domain.

γ-enolase (ENO2) is methylated at the Nτ position of His-190 among enolase isozymes.

Protein methylation is mainly observed in lysine, arginine and histidine residues. Histidine methylation occurs at one of two different nitrogen atoms of the imidazole ring, producing Nτ-methylhistidine and Nπ-methylhistidine, and it has recently attracted attention with the identification of SETD3, METTL18 and METTL9 as catalytic enzymes in mammals. Although accumulating evidence had suggested the presence of more than 100 proteins containing methylated histidine residues in cells, much less information has been known regarding histidine-methylated proteins than lysine- and arginine-methylated ones, because no method has been developed to identify substrates for histidine methylation. Here, we established a method to screen novel target proteins for histidine methylation, using biochemical protein fractionation combined with the quantification of methylhistidine by LC-MS/MS. Interestingly, the differential distribution pattern of Nτ-methylated proteins was found between the brain and skeletal muscle, and identified γ-enolase where the His-190 at the Nτ position is methylated in mouse brain. Finally, in silico structural prediction and biochemical analysis showed that the His-190 in γ-enolase is involved in the intermolecular homodimeric formation and enzymatic activity. In the present study, we provide a new methodology to find histidine-methylated proteins in vivo and suggest an insight into the importance of histidine methylation.

Mechanism underlying retinoic acid-dependent metamorphosis in the starfish.

The evolution of the biphasic life cycle in marine invertebrates has attracted considerable interest in zoology. We recently provided evidence that retinoic acid (RA) is involved in the regulation of metamorphosis in starfish. It also functions in life cycle transitions of jellyfish (cnidaria). Thus, documenting the evolutionarily conserved role of RA in such transitions will help to trace the life cycle evolution of bilaterians and cnidarians. In this study, we examined the molecular mechanisms by which RA signaling is involved in the commencement of metamorphosis in starfish. First, we measured RA levels during the larval and metamorphosis stages by liquid chromatography-tandem mass spectrometry. We found that all-trans RA levels in the larval body are high before larvae acquire competence for metamorphosis, suggesting that the commencement of metamorphosis is not controlled by increased RA synthesis. Furthermore, the suppression of rar gene expression by TALEN-mediated gene knockout revealed that RA receptor (RAR) is essential for metamorphosis. These observations suggest that the initiation of metamorphosis is regulated at the level of synthesized RA to activate RAR. We discuss the divergence of ligand molecules and receptors during the evolution of life cycle regulation.

The molecular and neural regulation of ultraviolet light phototaxis and its food-associated learning behavioral plasticity in C. elegans.

Ultraviolet light is quite toxic to all the animals and evoke the avoidance behavior of UV. The soil nematode Caenorhabditis elegans senses UV and is known to avoid UV by using four sensory neurons. However, it is not clear what signaling molecules act for UV avoidance in the neuronal pathway constituted of four sensory neurons. In addition, it is not clear whether this harmful environmental signal can be associated with other benefit signals such as food. In this study, by using newly developed assay system, we found that C. elegans can associate UV and food and changes behavioral strategy against harmful UV signal. This is the first indication that C. elegans shows associate learning with UV and food. Using our assay system, we also found that glutamate is used as a transmitter in both the UV avoidance and UV associate learning neural circuits. However, one sensory neuron showed a significant role for associative learning, compared to a complimentary role in four sensory neurons for direct associative learning, and different sets of glutamate receptors seemed to be acting for UV avoidance and UV associate learning. These findings suggest that a distinct neuronal network is used for UV learning compared to that for direct avoidance behavior of UV.

siRNA screening identifies METTL9 as a histidine Nπ-methyltransferase that targets the proinflammatory protein S100A9.

Protein methylation is one of the most common post-translational modifications observed in basic amino acid residues, including lysine, arginine, and histidine. Histidine methylation occurs on the distal or proximal nitrogen atom of its imidazole ring, producing two isomers: Nτ-methylhistidine or Nπ-methylhistidine. However, the biological significance of protein histidine methylation remains largely unclear owing in part to the very limited knowledge about its contributing enzymes. Here, we identified mammalian seven-ß-strand methyltransferase METTL9 as a histidine Nπ-methyltransferase by siRNA screening coupled with methylhistidine analysis using LC-tandem MS. We demonstrated that METTL9 catalyzes Nπ-methylhistidine formation in the proinflammatory protein S100A9, but not that of myosin light chain kinase MYLK2, in vivo and in vitro. METTL9 does not affect the heterodimer formation of S100A9 and S100A8, although Nπ-methylation of S100A9 at His-107 overlaps with a zinc-binding site, attenuating its affinity for zinc. Given that S100A9 exerts an antimicrobial activity, probably by chelation of zinc essential for the growth of bacteria and fungi, METTL9-mediated S100A9 methylation might be involved in the innate immune response to bacterial and fungal infection. Thus, our findings suggest a functional consequence for protein histidine Nπ-methylation and may add a new layer of complexity to the regulatory mechanisms of post-translational methylation.

Structural Revision of Natural Cyclic Depsipeptide MA026 Established by Total Synthesis and Biosynthetic Gene Cluster Analysis.

A revised structure of natural 14-mer cyclic depsipeptide MA026, isolated from Pseudomonas sp. RtlB026 in 2002 was established by physicochemical analysis with HPLC, MS/MS, and NMR and confirmed by total solid-phase synthesis. The revised structure differs from that previously reported in that two amino acid residues, assigned in error, have been replaced. Synthesized MA026 with the revised structure showed a tight junction (TJ) opening activity like that of the natural one in a cell-based TJ opening assay. Bioinformatic analysis of the putative MA026 biosynthetic gene cluster (BGC) of RtIB026 demonstrated that the stereochemistry of each amino acid residue in the revised structure can be reasonably explained. Phylogenetic analysis with xantholysin BGC indicates an exceptionally high homology (ca. 90 %) between xantholysin and MA026. The TJ opening activity of MA026 when binding to claudin-1 is a key to new avenues for transdermal administration of large hydrophilic biologics.

Histamine receptor agonist alleviates severe cardiorenal damages by eliciting anti-inflammatory programming.

Heart failure and chronic kidney disease are major causes of morbidity and mortality internationally. Although these dysfunctions are common and frequently coexist, the factors involved in their relationship in cardiorenal regulation are still largely unknown, mainly due to a lack of detailed molecular targets. Here, we found the increased plasma histamine in a preclinical mouse model of severe cardiac dysfunction, that had been cotreated with angiotensin II (Ang II), nephrectomy, and salt (ANS). The ANS mice exhibited impaired renal function accompanied with heart failure, and histamine depletion, by the genetic inactivation of histidine decarboxylase in mice, exacerbated the ANS-induced cardiac and renal abnormalities, including the reduction of left ventricular fractional shortening and renal glomerular and tubular injuries. Interestingly, while the pharmacological inhibition of the histamine receptor H3 facilitated heart failure and kidney injury in ANS mice, administration of the H3 agonist immethridine (Imm) was protective against cardiorenal damages. Transcriptome analysis of the kidney and biochemical examinations using blood samples illustrated that the increased inflammation in ANS mice was alleviated by Imm. Our results extend the pharmacological use of H3 agonists beyond the initial purposes of its drug development for neurogenerative diseases and have implications for therapeutic potential of H3 agonists that invoke the anti-inflammatory gene expression programming against cardiorenal damages.

Tricarboxylic acid cycle activity suppresses acetylation of mitochondrial proteins during early embryonic development in Caenorhabditis elegans.

The tricarboxylic acid (TCA) cycle (or citric acid cycle) is responsible for the complete oxidation of acetyl-CoA and formation of intermediates required for ATP production and other anabolic pathways, such as amino acid synthesis. Here, we uncovered an additional mechanism that may help explain the essential role of the TCA cycle in the early embryogenesis of Caenorhabditis elegans. We found that knockdown of citrate synthase (cts-1), the initial and rate-limiting enzyme of the TCA cycle, results in early embryonic arrest, but that this phenotype is not because of ATP and amino acid depletions. As a possible alternative mechanism explaining this developmental deficiency, we observed that cts-1 RNAi embryos had elevated levels of intracellular acetyl-CoA, the starting metabolite of the TCA cycle. Of note, we further discovered that these embryos exhibit hyperacetylation of mitochondrial proteins. We found that supplementation with acetylase-inhibiting polyamines, including spermidine and putrescine, counteracted the protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Contrary to the hypothesis that spermidine acts as an acetyl sink for elevated acetyl-CoA, the levels of three forms of acetylspermidine, N1-acetylspermidine, N8-acetylspermidine, and N1,N8-diacetylspermidine, were not significantly increased in embryos treated with exogenous spermidine. Instead, we demonstrated that the mitochondrial deacetylase sirtuin 4 (encoded by the sir-2.2 gene) is required for spermidine's suppression of protein hyperacetylation and developmental arrest in the cts-1 RNAi embryos. Taken together, these results suggest the possibility that during early embryogenesis, acetyl-CoA consumption by the TCA cycle in C. elegans prevents protein hyperacetylation and thereby protects mitochondrial function.

Emerging impacts of biological methylation on genetic information.

The central dogma of molecular biology explains the fundamental flow of genetic information for life. Although genome sequence (DNA) itself is a static chemical signature, it includes multiple layers of information composed of mRNA, tRNA, rRNA and small RNAs, all of which are involved in protein synthesis and is passing from parents to offspring via DNA. Methylation is a biologically important modification, because DNA, RNAs and proteins, components of the central dogma, are methylated by a set of methyltransferases. Recent works focused on understanding a variety of biological methylation have shed light on new regulation of cellular functions. In this review, we briefly discuss some of those recent findings of methylation, including DNA, RNAs and proteins.

Gestational changes in PRMT1 expression of murine placentas.

INTRODUCTION: In mammals, the placenta is an organ that is required to maintain the development of fetus during pregnancy. Although the proper formation of placenta is in part regulated by the post-translational modifications of proteins, little is known regarding protein arginine methylation during placental development. Here, we characterized developmental expression of protein arginine methyltransferase 1 (PRMT1) in mouse placentas. METHODS: Expression levels of PRMT1 mRNA and protein in placentas were investigated using the real-time quantitative PCR and Western blot, respectively. Next, the localization of PRMT1 was determined by immunohistochemistry and immunofluorescence analyses. In addition, the levels of methylarginines of placental proteins were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: PRMT1 mRNA and its protein were expressed at highest levels in mid-gestation stages, and their expression showed stepwise decrease in the late gestation. At embryonic (E) day 9, PRMT1 was observed in several different trophoblast cell (TC) subtypes. Furthermore, PRMT1 was mainly expressed in the labyrinth zone of TCs at E13. Finally, total methylarginines of proteins were significantly reduced in late gestation of placentas compared with mid-gestation stages. DISCUSSION: In this study, we found developmental changes in the placental expression of PRMT1 and in protein arginine methylation status during pregnancy. These findings provide fundamental information regarding placental PRMT1-mediated arginine methylation during the development.

rRNA adenine methylation requires T07A9.8 gene as rram-1 in Caenorhabditis elegans.

RNAs are post-transcriptionally modified in all kingdoms of life. Of these modifications, base methylations are highly conserved in eukaryote ribosomal RNA (rRNA). Recently, rRNA processing protein 8 (Rrp8) and nucleomethylin (NML) were identified as factors of N1-methyladenosine (m1A) modification in yeast 25 S and mammalian 28 S rRNA, respectively. However, m1A modification of rRNA is still poorly understood in Caenorhabditis elegans (C. elegans). Here, using the liquid chromatography/tandem mass spectrometry analysis and RNA immunoprecipitation assay, we have identified that the m1A modification is located around position 674 (A674) of 26 S rRNA in C. elegans. Furthermore, quantitative PCR-based analysis revealed that T07A9.8, a C. elegans homolog of yeast Rrp8 and human NML, is responsible for m1A modification at A674 of 26 S rRNA. This m1A modification site in C. elegans corresponds to those in yeast 25 S rRNA and human 28 S rRNA. Intriguingly, T07A9.8 is not associated with pre-rRNA transcription under normal nutrient conditions. Since the m1A modification of 26 S rRNA requires T07A9.8 in C. elegans, we designated the gene as rRNA adenine methyltransferase-1 (rram-1).

The GATA transcription factor ELT-2 modulates both the expression and methyltransferase activity of PRMT-1 in Caenorhabditis elegans.

Protein arginine methyltransferase 1 (PRMT1) catalyzes asymmetric arginine dimethylation of cellular proteins and thus modulates various biological processes, including gene regulation, RNA metabolism, cell signaling and DNA repair. Since prmt-1 null mutant completely abolishes asymmetric dimethylarginine in C. elegans, PRMT-1 is thought to play a crucial role in determining levels of asymmetric arginine dimethylation. However, the mechanism underlying the regulation of PRMT-1 activity remains largely unknown. Here, we explored for transcription factors that induce the expression of PRMT-1 by an RNAi screen using transgenic C. elegans harbouring prmt-1 promoter upstream of gfp. Of 529 clones, we identify a GATA transcription factor elt-2 as a positive regulator of Pprmt-1:: gfp expression and show that elt-2 RNAi decreases endogenous PRMT-1 expression at mRNA and protein levels. Nevertheless, surprisingly arginine methylation levels are increased when elt-2 is silenced, implying that erythroid-like transcription factor (ELT)-2 may also have ability to inhibit methyltransferase activity of PRMT-1. Supporting this idea, GST pull-down and co-immunoprecipitation assays demonstrate the interaction between ELT-2 and PRMT-1. Furthermore, we find that ELT-2 interferes with PRMT-1-induced arginine methylation in a dose-dependent manner. Collectively, our results illustrate the two modes of PRMT-1 regulation, which could determine the levels of asymmetric arginine dimethylation in C. elegans.

Simultaneous ablation of prmt-1 and prmt-5 abolishes asymmetric and symmetric arginine dimethylations in Caenorhabditis elegans.

Protein arginine methyltransferases (PRMTs) catalyze the transfer of a methyl group from S-adenosylmethionine to arginine residues and are classified into two types: type I producing asymmetric dimethylarginine (ADMA) and type II producing symmetric dimethylarginine (SDMA). PRMTs have been shown to regulate many cellular processes, including signal transduction, transcriptional regulation and RNA processing. Since the loss-of-function mutation of PRMT1 and PRMT5, each of which is the predominant type I and II, respectively, causes embryonic lethality in mice, their physiological significance at the whole-body level remains largely unknown. Here, we show the morphological and functional phenotypes of single or double null alleles of prmt-1 and prmt-5 in Caenorhabditis elegans. The prmt-1;prmt-5 double mutants are viable, and exhibit short body length and small brood size compared to N2 and each of the single mutants. The liquid chromatography-tandem mass spectrometry analysis demonstrated that the levels of ADMA and SDMA were abolished in the prmt-1;prmt-5 double mutants. Both prmt-1 and prmt-5 were required for resistance to heat and oxidative stresses, whereas prmt-5 is not involved in lifespan regulation even when prmt-1 is ablated. This mutant strain would be a useful model animal for investigating the role of asymmetric and symmetric arginine dimethylation in vivo.

PRMT-5 converts monomethylarginines into symmetrical dimethylarginines in Caenorhabditis elegans.

The transmethylation to arginine residues of proteins is catalyzed by protein arginine methyltransferases (PRMTs) that form monomethylarginine (MMA), asymmetric (ADMA) and symmetric dimethylarginines (SDMA). Although we previously demonstrated that the generation of ADMA residues in whole proteins is driven by PRMT-1 in Caenorhabditis elegans, much less is known about MMA and SDMA in vivo. In this study, we measured the amounts of different methylarginines in whole protein extracts made from wild-type (N2) C. elegans and from prmt-1 and prmt-5 null mutants using liquid chromatography-tandem mass spectrometry. Interestingly, we found that the amounts of MMA and SDMA are about fourfold higher than those of ADMA in N2 protein lysates using acid hydrolysis. We were unable to detect SDMA residues in the prmt-5 null mutant. In comparison with N2, an increase in SDMA and decrease in MMA were observed in prmt-1 mutant worms with no ADMA, but ADMA and MMA levels were unchanged in prmt-5 mutant worms. These results suggest that PRMT-1 contributes, at least in part, to MMA production, but that PRMT-5 catalyzes the symmetric dimethylation of substrates containing MMA residues in vivo.

Hydralazine is involved in tele-methylhistamine metabolism by inhibiting monoamine oxidase B in pregnancy-associated hypertensive mice.

Hypertensive disorders of pregnancy globally affect 6-8% of gestation and remain a major cause of both foetal and maternal morbidity and mortality. However, the antihypertensive medications for the patients of this disease are strictly limited due to the teratogenic potentials. Here, we found that tele-methylhistamine (tMH) increased in response to the administration of hydralazine (Hdz), a vasodilative agent, in the pregnancy-associated hypertensive (PAH) mice. Hdz abrogated the degradation of tMH catalyzed by monoamine oxidase B (MAO-B) in vitro. These results suggested that Hdz inhibited the MAO-B activity and consequently tMH increased in the maternal circulation of PAH mice.

Asymmetric Arginine Dimethylation Modulates Mitochondrial Energy Metabolism and Homeostasis in Caenorhabditis elegans.

Protein arginine methyltransferase 1 (PRMT-1) catalyzes asymmetric arginine dimethylation on cellular proteins and modulates various aspects of biological processes, such as signal transduction, DNA repair, and transcriptional regulation. We have previously reported that the null mutant of prmt-1 in Caenorhabditis elegans exhibits a slightly shortened life span, but the physiological significance of PRMT-1 remains largely unclear. Here we explored the role of PRMT-1 in mitochondrial function as hinted by a two-dimensional Western blot-based proteomic study. Subcellular fractionation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that PRMT-1 is almost entirely responsible for asymmetric arginine dimethylation on mitochondrial proteins. Importantly, isolated mitochondria from prmt-1 mutants represent compromised ATP synthesis in vitro, and whole-worm respiration in prmt-1 mutants is decreased in vivo Transgenic rescue experiments demonstrate that PRMT-1-dependent asymmetric arginine dimethylation is required to prevent mitochondrial reactive oxygen species (ROS) production, which consequently causes the activation of the mitochondrial unfolded-protein response. Furthermore, the loss of enzymatic activity of prmt-1 induces food avoidance behavior due to mitochondrial dysfunction, but treatment with the antioxidant N-acetylcysteine significantly ameliorates this phenotype. These findings add a new layer of complexity to the posttranslational regulation of mitochondrial function and provide clues for understanding the physiological roles of PRMT-1 in multicellular organisms.

NML-mediated rRNA base methylation links ribosomal subunit formation to cell proliferation in a p53-dependent manner.

Ribosomal RNAs (rRNAs) act as scaffolds and ribozymes in ribosomes, and these functions are modulated by post-transcriptional modifications. However, the biological role of base methylation, a well-conserved modification of rRNA, is poorly understood. Here, we demonstrate that a nucleolar factor, nucleomethylin (NML; also known as RRP8), is required for the N(1)-methyladenosine (m(1)A) modification in 28S rRNAs of human and mouse cells. NML also contributes to 60S ribosomal subunit formation. Intriguingly, NML depletion increases 60S ribosomal protein L11 (RPL11) levels in the ribosome-free fraction and protein levels of p53 through an RPL11-MDM2 complex, which activates the p53 pathway. Consequently, the growth of NML-depleted cells is suppressed in a p53-dependent manner. These observations reveal a new biological function of rRNA base methylation, which links ribosomal subunit formation to p53-dependent inhibition of cell proliferation in mammalian cells.

Pterosin B has multiple targets in gluconeogenic programs, including coenzyme Q in RORα-SRC2 signaling.

Hepatic gluconeogenic programs are regulated by a variety of signaling cascades. Glucagon-cAMP signaling is the main initiator of the gluconeogenic programs, including glucose-6-phosphatase catalytic subunit (G6pc) gene expression. Pterosin B, an ingredient in Pteridium aquilinum, inhibits salt-inducible kinase 3 signaling that represses cAMP-response element-binding protein regulated transcription coactivator 2, an inducer of gluconeogenic programs. As the results, pterosin B promotes G6pc expression even in the absence of cAMP. In this work, however, we noticed that once cAMP signaling was initiated, pterosin B became a strong repressor of G6pc expression. The search for associated transcription factors for pterosin B actions revealed that retinoic acid receptor-related orphan receptor alpha-steroid receptor coactivator 2 (RORα-SRC2) complex on the G6pc promoter was the target. Meanwhile, pterosin B impaired the oxidation-reduction cycle of coenzyme Q in mitochondrial oxidative phosphorylation (OXPHOS); and antimycin A, an inhibitor of coenzyme Q: cytochrome c-oxidoreductase (termed mitochondrial complex III), also mimicked pterosin B actions on RORα-SRC2 signaling. Although other respiratory toxins (rotenone and oligomycin) also suppressed G6pc expression accompanied by lowered ATP levels following the activation of AMP-activated kinase, minimal or no effect of these other toxins on RORα-SRC2 activity was observed. These results suggested that individual components in OXPHOS differentially linked to different transcriptional machineries for hepatic gluconeogenic programs, and the RORα-SRC2 complex acted as a sensor for oxidation-reduction cycle of coenzyme Q and regulated G6Pc expression. This was a site disrupted by pterosin B in gluconeogenic programs.