Human HemK2/KMT9/N6AMT1 is an active protein methyltransferase, but does not act on DNA in vitro, in the presence of Trm112.
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ABSTRACT:
SUBMITTER: Woodcock CB
PROVIDER: S-EPMC6796829 | biostudies-literature | 2019
REPOSITORIES: biostudies-literature
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Human HemK2/KMT9/N6AMT1 is an active protein methyltransferase, but does not act on DNA in vitro, in the presence of Trm112.
Woodcock Clayton B CB Yu Dan D Zhang Xing X Cheng Xiaodong X
Cell discovery 20190910
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Project description:DNA methylation is an important epigenetic modification in many organisms and can occur on cytosine or adenine. N6-methyladenine (6mA) exists widespreadly in bacterial genomes, which plays a vital role in the bacterial restriction-modification system. Recently, 6mA has also been reported to exist in the genomes of a variety of eukaryotes from unicellular organisms to metazoans. There were controversial reports on whether human N6amt1, which was originally reported as a glutamine MTase for eRF1, is a putative 6mA DNA MTase. We report here the crystal structure of human N6amt1-Trm112 in complex with cofactor SAM. Structural analysis shows that Trm112 binds to a hydrophobic surface of N6amt1 to stabilize its structure but does not directly contribute to substrate binding and catalysis. The active site and potential substrate-binding site of N6amt1 are dominantly negatively charged and thus are unsuitable for DNA binding. The biochemical data confirm that the complex cannot bind DNA and has no MTase activity for DNA, but exhibits activity for the methylation of Gln185 of eRF1. Our structural and biochemical data together demonstrate that N6amt1 is a bona fide protein MTase rather than a DNA MTase.
| S-EPMC6796863 | biostudies-literature
Project description:We previously identified Bud23 as the methyltransferase that methylates G1575 of rRNA in the P-site of the small (40S) ribosomal subunit. In this paper, we show that Bud23 requires the methyltransferase adaptor protein Trm112 for stability in vivo. Deletion of Trm112 results in a bud23Δ-like mutant phenotype. Thus Trm112 is required for efficient small-subunit biogenesis. Genetic analysis suggests the slow growth of a trm112Δ mutant is due primarily to the loss of Bud23. Surprisingly, suppression of the bud23Δ-dependent 40S defect revealed a large (60S) biogenesis defect in a trm112Δ mutant. Using sucrose gradient sedimentation analysis and coimmunoprecipitation, we show that Trm112 is also involved in 60S subunit biogenesis. The 60S defect may be dependent on Nop2 and Rcm1, two additional Trm112 interactors that we identify. Our work extends the known range of Trm112 function from modification of tRNAs and translation factors to both ribosomal subunits, showing that its effects span all aspects of the translation machinery. Although Trm112 is required for Bud23 stability, our results suggest that Trm112 is not maintained in a stable complex with Bud23. We suggest that Trm112 stabilizes its free methyltransferase partners not engaged with substrate and/or helps to deliver its methyltransferase partners to their substrates.
| S-EPMC3484107 | biostudies-literature
Project description:BACKGROUND: In humans, inorganic arsenic is metabolized to methylated metabolites mainly by arsenic (+3 oxidation state) methyltransferase (AS3MT). AS3MT polymorphisms are associated with arsenic metabolism efficiency. Recently, a putative N-6-adenine-specific DNA methyltransferase 1 (N6AMT1) was found to methylate arsenic in vitro. OBJECTIVE: We evaluated the role of N6AMT1 polymorphisms in arsenic methylation efficiency in humans. METHODS: We assessed arsenic methylation efficiency in 188 women exposed to arsenic via drinking water (~ 200 µg/L) in the Argentinean Andes by measuring the relative concentrations of arsenic metabolites in urine [inorganic arsenic, methylarsonic acid (MMA), and dimethylarsinic acid] by high-performance liquid chromatography coupled with hydride generation and inductively coupled plasma mass spectrometry. We performed genotyping for N6AMT1 and AS3MT polymorphisms by Taqman assays, and gene expression (in blood; n = 63) with Illumina HumanHT-12 v4.0. RESULTS: Five N6AMT1 single nucleotide polymorphisms (SNPs; rs1997605, rs2205449, rs2705671, rs16983411, and rs1048546) and two N6AMT1 haplotypes were significantly associated with the percentage of MMA (%MMA) in urine, even after adjusting for AS3MT haplotype. %MMA increased monotonically according to the number of alleles for each SNP (e.g., for rs1048546, mean %MMA was 7.5% for GG, 8.8% for GT, and 9.7% for TT carriers). Three SNPs were in linkage disequilibrium (R2 > 0.8). Estimated associations for joint effects of N6AMT1 (haplotype 1) and AS3MT (haplotype 2) were generally consistent with expectations for additive effects of each haplotype on %MMA. Carriers of N6AMT1 genotypes associated with lower %MMA showed the lowest N6AMT1 expression, but associations were monotonic according to copy number for only one genotype and one haplotype. CONCLUSIONS: N6AMT1 polymorphisms were associated with arsenic methylation in Andean women, independent of AS3MT. N6AMT1 polymorphisms may be susceptibility markers for arsenic-related toxic effects.
| S-EPMC3702000 | biostudies-literature
Project description:Post-transcriptional and post-translational modifications of factors involved in translation are very important for the control and accuracy of protein biosynthesis. Among these factors, tRNAs harbor the largest variety of grafted chemical structures, which participate in tRNA stability or mRNA decoding. Here, we focused on Trm112 protein, which associates with four different eukaryotic methyltransferases modifying tRNAs (Trm9 and Trm11) but also 18S-rRNA (Bud23) and translation termination factor eRF1 (Mtq2). In particular, we have investigated the role of Trm112 in the Trm11-Trm112 complex, which forms 2-methylguanosine at position 10 on several tRNAs and thereby is assumed to stabilize tRNA structure. We show that Trm112 is important for Trm11 enzymatic activity by influencing S-adenosyl-L-methionine binding and by contributing to tRNA binding. Using hydrogen-deuterium eXchange coupled to mass spectrometry, we obtained experimental evidences that the Trm11-Trm112 interaction relies on the same molecular bases as those described for other Trm112-methyltransferases complexes. Hence, all Trm112-dependent methyltransferases compete to interact with this partner.
| S-EPMC5389515 | biostudies-literature
Project description:Inhibition of epigenetic regulators by small molecules is an attractive strategy for cancer treatment. Recently, we characterised the role of lysine methyltransferase 9 (KMT9) in prostate, lung, and colon cancer. Our observation that the enzymatic activity was required for tumour cell proliferation identified KMT9 as a potential therapeutic target. Here, we report the development of a potent and selective KMT9 inhibitor (compound 4, KMI169) with cellular activity through structure-based drug design. KMI169 functions as a bi-substrate inhibitor targeting the SAM and substrate binding pockets of KMT9 and exhibits high potency, selectivity, and cellular target engagement. KMT9 inhibition selectively downregulates target genes involved in cell cycle regulation and impairs proliferation of tumours cells including castration- and enzalutamide-resistant prostate cancer cells. KMI169 represents a valuable tool to probe cellular KMT9 functions and paves the way for the development of clinical candidate inhibitors as therapeutic options to treat malignancies such as therapy-resistant prostate cancer.
| S-EPMC10762027 | biostudies-literature
Project description:The methyltransferase N6AMT1 has been associated with the progression of different pathological conditions, such as tumours and neurological malfunctions, but the underlying mechanism is not fully understood. Analysis of N6AMT1-depleted cells revealed that N6AMT1 is involved in the cell cycle and cell proliferation. In N6AMT1-depleted cells, the cell doubling time was increased, and cell progression out of mitosis and the G0/G1 and S phases was disrupted. It was discovered that in N6AMT1-depleted cells, the transcription of cyclin E was downregulated, which indicates that N6AMT1 is involved in the regulation of cyclin E transcription. Understanding the functions and importance of N6AMT1 in cell proliferation and cell cycle regulation is essential for developing treatments and strategies to control diseases that are associated with N6AMT1.
| S-EPMC10889616 | biostudies-literature
Project description:Protein synthesis is a complex and highly coordinated process requiring many different protein factors as well as various types of nucleic acids. All translation machinery components require multiple maturation events to be functional. These include post-transcriptional and post-translational modification steps and methylations are the most frequent among these events. In eukaryotes, Trm112, a small protein (COG2835) conserved in all three domains of life, interacts and activates four methyltransferases (Bud23, Trm9, Trm11 and Mtq2) that target different components of the translation machinery (rRNA, tRNAs, release factors). To clarify the function of Trm112 in archaea, we have characterized functionally and structurally its interaction network using Haloferax volcanii as model system. This led us to unravel that methyltransferases are also privileged Trm112 partners in archaea and that this Trm112 network is much more complex than anticipated from eukaryotic studies. Interestingly, among the identified enzymes, some are functionally orthologous to eukaryotic Trm112 partners, emphasizing again the similarity between eukaryotic and archaeal translation machineries. Other partners display some similarities with bacterial methyltransferases, suggesting that Trm112 is a general partner for methyltransferases in all living organisms.
| S-EPMC6144793 | biostudies-literature
Project description:Inter-individual differences in arsenic metabolism have been linked to arsenic-related disease risks. Arsenic (+3) methyltransferase (AS3MT) is the primary enzyme involved in arsenic metabolism, and we previously demonstrated in vitro that N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) also methylates the toxic inorganic arsenic (iAs) metabolite, monomethylarsonous acid (MMA), to the less toxic dimethylarsonic acid (DMA). Here, we evaluated whether AS3MT and N6AMT1 gene polymorphisms alter arsenic methylation and impact iAs-related cancer risks. We assessed AS3MT and N6AMT1 polymorphisms and urinary arsenic metabolites (%iAs, %MMA, %DMA) in 722 subjects from an arsenic-cancer case-control study in a uniquely exposed area in northern Chile. Polymorphisms were genotyped using a custom designed multiplex, ligation-dependent probe amplification (MLPA) assay for 6 AS3MT SNPs and 14 tag SNPs in the N6AMT1 gene. We found several AS3MT polymorphisms associated with both urinary arsenic metabolite profiles and cancer risk. For example, compared to wildtypes, individuals carrying minor alleles in AS3MT rs3740393 had lower %MMA (mean difference = -1.9%, 95% CI: -3.3, -0.4), higher %DMA (mean difference = 4.0%, 95% CI: 1.5, 6.5), and lower odds ratios for bladder (OR = 0.3; 95% CI: 0.1-0.6) and lung cancer (OR = 0.6; 95% CI: 0.2-1.1). Evidence of interaction was also observed for both lung and bladder cancer between these polymorphisms and elevated historical arsenic exposures. Clear associations were not seen for N6AMT1. These results are the first to demonstrate a direct association between AS3MT polymorphisms and arsenic-related internal cancer risk. This research could help identify subpopulations that are particularly vulnerable to arsenic-related disease. Environ. Mol. Mutagen. 58:411-422, 2017. © 2017 Wiley Periodicals, Inc.
| S-EPMC5515250 | biostudies-literature
Project description: Not available
| S-EPMC6928010 | biostudies-literature
Project description:BackgroundLung cancer is the leading cause of cancer related death worldwide. Over the past 15 years no major improvement of survival rates could be accomplished. The recently discovered histone methyltransferase KMT9 that acts as epigenetic regulator of prostate tumor growth has now raised hopes of enabling new cancer therapies. In this study, we aimed to identify the function of KMT9 in lung cancer.MethodsWe unraveled the KMT9 transcriptome and proteome in A549 lung adenocarcinoma cells using RNA-Seq and mass spectrometry and linked them with functional cell culture, real-time proliferation and flow cytometry assays.ResultsWe show that KMT9α and -β subunits of KMT9 are expressed in lung cancer tissue and cell lines. Importantly, high levels of KMT9α correlate with poor patient survival. We identified 460 genes that are deregulated at the RNA and protein level upon knock-down of KMT9α in A549 cells. These genes cluster with proliferation, cell cycle and cell death gene sets as well as with subcellular organelles in gene ontology analysis. Knock-down of KMT9α inhibits lung cancer cell proliferation and induces non-apoptotic cell death in A549 cells.ConclusionsThe novel histone methyltransferase KMT9 is crucial for proliferation and survival of lung cancer cells harboring various mutations. Small molecule inhibitors targeting KMT9 therefore should be further examined as potential milestones in modern epigenetic lung cancer therapy.
| S-EPMC7027090 | biostudies-literature