Development of a Series of Pyrrolopyridone MAT2A Inhibitors.

The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by relative binding free energy calculations, resulted in AZ'9567 (21), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.

Discovery of novel MAT2A inhibitors by an allosteric site-compatible fragment growing approach.

The methionine adenosyltransferase MAT2A catalyzes the synthesis ofthe methyl donor S-adenosylmethionine (SAM) and thereby regulates critical aspects of metabolism and transcription. Aberrant MAT2A function can lead to metabolic and transcriptional reprogramming of cancer cells, and MAT2A has been shown to promote survival of MTAP-deficient tumors, a genetic alteration that occurs in âˆ¼ 13 % of all tumors. Thus, MAT2A holds great promise as a novel anticancer target. Here, we report a novel series of MAT2A inhibitors generated by a fragment growing approach from AZ-28, a low-molecular weight MAT2A inhibitor with promising pre-clinical properties. X-ray co-crystal structure revealed that compound 7 fully occupies the allosteric pocket of MAT2A as a single molecule mimicking MAT2B. By introducing additional backbone interactions and rigidifying the requisite linker extensions, we generated compound 8, which exhibited single digit nanomolar enzymatic and sub-micromolar cellular inhibitory potency for MAT2A.

Profiling the compendium of changes in Saccharomyces cerevisiae due to mutations that alter availability of the main methyl donor S-Adenosylmethionine.

The SAM1 and SAM2 genes encode for S-Adenosylmethionine (AdoMet) synthetase enzymes, with AdoMet serving as the main cellular methyl donor. We have previously shown that independent deletion of these genes alters chromosome stability and AdoMet concentrations in opposite ways in Saccharomyces cerevisiae. To characterize other changes occurring in these mutants, we grew wildtype, sam1Δ/sam1Δ, and sam2Δ/sam2Δ strains in 15 different Phenotypic Microarray plates with different components and measured growth variations. RNA-Sequencing was also carried out on these strains and differential gene expression determined for each mutant. We explored how the phenotypic growth differences are linked to the altered gene expression, and hypothesize mechanisms by which loss of the SAM genes and subsequent AdoMet level changes, impact pathways and processes. We present 6 stories, discussing changes in sensitivity or resistance to azoles, cisplatin, oxidative stress, arginine biosynthesis perturbations, DNA synthesis inhibitors, and tamoxifen, to demonstrate the power of this novel methodology to broadly profile changes due to gene mutations. The large number of conditions that result in altered growth, as well as the large number of differentially expressed genes with wide-ranging functionality, speaks to the broad array of impacts that altering methyl donor abundance can impart. Our findings demonstrate that some cellular changes are directly related to AdoMet-dependent methyltransferases and AdoMet availability, some are directly linked to the methyl cycle and its role in production of several important cellular components, and others reveal impacts of SAM gene mutations on previously unconnected pathways.

Identification of a Sonically Activated Degrader of Methionine Adenosyltransferase 2A by an in Silico Approach Assisted with the Hole-Electron Analysis.

Small molecules capable of modulating methionine adenosyltransferase 2A (MAT2A) are of significant interest in precise cancer therapeutics. Herein, we raised the hole-electron Coulombic attraction as a reliable molecular descriptor for predicting the reactive oxygen generation capacity of MAT2A inhibitors, based on which we discovered compound H3 as a sonically activated degrader of MAT2A. Upon sonication, H3 can generate reactive oxygen species to specifically degrade cellular MAT2A via rapid oxidative reactions. Combination of H3 and sonication induced 87% MAT2A depletion in human colon cancer cells, thus elevating its antiproliferation effects by 8-folds. In vivo, H3 had a favorable pharmacokinetic profile (bioavailability = 77%) and ADME properties. Owing to the MAT2A degradation merits, H3 at a dosage of 10 mg/kg induced 31% tumor regression in xenograft colon tumor models. The significantly boosted antitumor potency can potentially alleviate the toxicity of high-dose MAT2A inhibitors to normal cells and tissues, especially to the liver.

Blocking methionine catabolism induces senescence and confers vulnerability to GSK3 inhibition in liver cancer.

Availability of the essential amino acid methionine affects cellular metabolism and growth, and dietary methionine restriction has been implicated as a cancer therapeutic strategy. Nevertheless, how liver cancer cells respond to methionine deprivation and underlying mechanisms remain unclear. Here we find that human liver cancer cells undergo irreversible cell cycle arrest upon methionine deprivation in vitro. Blocking methionine adenosyl transferase 2A (MAT2A)-dependent methionine catabolism induces cell cycle arrest and DNA damage in liver cancer cells, resulting in cellular senescence. A pharmacological screen further identified GSK3 inhibitors as senolytics that selectively kill MAT2A-inhibited senescent liver cancer cells. Importantly, combined treatment with MAT2A and GSK3 inhibitors therapeutically blunts liver tumor growth in vitro and in vivo across multiple models. Together, methionine catabolism is essential for liver tumor growth, and its inhibition can be exploited as an improved pro-senescence strategy for combination with senolytic agents to treat liver cancer.

Combined inhibition of MTAP and MAT2a mimics synthetic lethality in tumor models via PRMT5 inhibition.

Homozygous 5'-methylthioadenosine phosphorylase (MTAP) deletions occur in approximately 15% of human cancers. Co-deletion of MTAP and methionine adenosyltransferase 2 alpha (MAT2a) induces a synthetic lethal phenotype involving protein arginine methyltransferase 5 (PRMT5) inhibition. MAT2a inhibitors are now in clinical trials for genotypic MTAP-/- cancers, however the MTAP-/- genotype represents fewer than 2% of human colorectal cancers (CRCs), limiting the utility of MAT2a inhibitors in these and other MTAP+/+ cancers. Methylthio-DADMe-immucillin-A (MTDIA) is a picomolar transition state analog inhibitor of MTAP that renders cells enzymatically MTAP-deficient to induce the MTAP-/- phenotype. Here, we demonstrate that MTDIA and MAT2a inhibitor AG-270 combination therapy mimics synthetic lethality in MTAP+/+ CRC cell lines with similar effects in mouse xenografts and without adverse histology on normal tissues. Combination treatment is synergistic with a 104-fold increase in drug potency for inhibition of CRC cell growth in culture. Combined MTDIA and AG-270 decreases S-adenosyl-L-methionine and increases 5'-methylthioadenosine in cells. The increased intracellular methylthioadenosine:S-adenosyl-L-methionine ratio inhibits PRMT5 activity, leading to cellular arrest and apoptotic cell death by causing MDM4 alternative splicing and p53 activation. Combination MTDIA and AG-270 treatment differs from direct inhibition of PRMT5 by GSK3326595 by avoiding toxicity caused by cell death in the normal gut epithelium induced by the PRMT5 inhibitor. The combination of MTAP and MAT2a inhibitors expands this synthetic lethal approach to include MTAP+/+ cancers, especially the remaining 98% of CRCs without the MTAP-/- genotype.

S-adenosylmethionine biosynthesis is a targetable metabolic vulnerability in multiple myeloma.

Multiple myeloma (MM) is the second most prevalent hematologic malignancy and is incurable because of the inevitable development of drug resistance. Methionine adenosyltransferase 2α (MAT2A) is the primary producer of the methyl donor S-adenosylmethionine (SAM) and several studies have documented MAT2A deregulation in different solid cancers. As the role of MAT2A in MM has not been investigated yet, the aim of this study was to clarify the potential role and underlying molecular mechanisms of MAT2A in MM, exploring new therapeutic options to overcome drug resistance. By analyzing publicly available gene expression profiling data, MAT2A was found to be more highly expressed in patient-derived myeloma cells than in normal bone marrow plasma cells. The expression of MAT2A correlated with an unfavorable prognosis in relapsed patients. MAT2A inhibition in MM cells led to a reduction in intracellular SAM levels, which resulted in impaired cell viability and proliferation, and induction of apoptosis. Further mechanistic investigation demonstrated that MAT2A inhibition inactivated the mTOR-4EBP1 pathway, accompanied by a decrease in protein synthesis. MAT2A targeting in vivo with the small molecule compound FIDAS-5 was able to significantly reduce tumor burden in the 5TGM1 model. Finally, we found that MAT2A inhibition can synergistically enhance the anti-MM effect of the standard-of-care agent bortezomib on both MM cell lines and primary human CD138+ MM cells. In summary, we demonstrate that MAT2A inhibition reduces MM cell proliferation and survival by inhibiting mTOR-mediated protein synthesis. Moreover, our findings suggest that the MAT2A inhibitor FIDAS-5 could be a novel compound to improve bortezomib-based treatment of MM.

A growth-based assay using fluorescent protein emission to screen for S-adenosylmethionine synthetase inhibitors.

The use of cell growth-based assays to identify inhibitory compounds is straightforward and inexpensive, but is also inherently insensitive and somewhat nonspecific. To overcome these limitations and develop a sensitive, specific cell-based assay, two different approaches were combined. To address the sensitivity limitation, different fluorescent proteins have been introduced into a bacterial expression system to serve as growth reporters. To overcome the lack of specificity, these protein reporters have been incorporated into a plasmid in which they are paired with different orthologs of an essential target enzyme, in this case l-methionine S-adenosyltransferase (MAT, AdoMet synthetase). Screening compounds that serve as specific inhibitors will reduce the growth of only a subset of strains, because these strains are identical, except for which target ortholog they carry. Screening several such strains in parallel not only reveals potential inhibitors but the strains also serve as specificity controls for one another. The present study makes use of an existing Escherichia coli strain that carries a deletion of metK, the gene for MAT. Transformation with these plasmids leads to a complemented strain that no longer requires externally supplied S-adenosylmethionine for growth, but its growth is now dependent on the activity of the introduced MAT ortholog. The resulting fluorescent strains provide a platform to screen chemical compound libraries and identify species-selective inhibitors of AdoMet synthetases. A pilot study of several chemical libraries using this platform identified new lead compounds that are ortholog-selective inhibitors of this enzyme family, some of which target the protozoal human pathogen Cryptosporidium parvum.

Hepatic prohibitin 1 and methionine adenosyltransferase α1 defend against primary and secondary liver cancer metastasis.

BACKGROUND & AIMS: The liver is a common site of cancer metastasis, most commonly from colorectal cancer, and primary liver cancers that have metastasized are associated with poor outcomes. The underlying mechanisms by which the liver defends against these processes are largely unknown. Prohibitin 1 (PHB1) and methionine adenosyltransferase 1A (MAT1A) are highly expressed in the liver. They positively regulate each other and their deletion results in primary liver cancer. Here we investigated their roles in primary and secondary liver cancer metastasis. METHODS: We identified common target genes of PHB1 and MAT1A using a metastasis array, and measured promoter activity and transcription factor binding using luciferase reporter assays and chromatin immunoprecipitation, respectively. We examined how PHB1 or MAT1A loss promotes liver cancer metastasis and whether their loss sensitizes to colorectal liver metastasis (CRLM). RESULTS: Matrix metalloproteinase-7 (MMP-7) is a common target of MAT1A and PHB1 and its induction is responsible for increased migration and invasion when MAT1A or PHB1 is silenced. Mechanistically, PHB1 and MAT1A negatively regulate MMP7 promoter activity via an AP-1 site by repressing the MAFG-FOSB complex. Loss of MAT1A or PHB1 also increased MMP-7 in extracellular vesicles, which were internalized by colon and pancreatic cancer cells to enhance their oncogenicity. Low hepatic MAT1A or PHB1 expression sensitized to CRLM, but not if endogenous hepatic MMP-7 was knocked down first, which lowered CD4+ T cells while increasing CD8+ T cells in the tumor microenvironment. Hepatocytes co-cultured with colorectal cancer cells express less MAT1A/PHB1 but more MMP-7. Consistently, CRLM raised distant hepatocytes' MMP-7 expression in mice and humans. CONCLUSION: We have identified a PHB1/MAT1A-MAFG/FOSB-MMP-7 axis that controls primary liver cancer metastasis and sensitization to CRLM. IMPACT AND IMPLICATIONS: Primary and secondary liver cancer metastasis is associated with poor outcomes but whether the liver has underlying defense mechanism(s) against metastasis is unknown. Here we examined the hypothesis that hepatic prohibitin 1 (PHB1) and methionine adenosyltransferase 1A (MAT1A) cooperate to defend the liver against metastasis. Our studies found PHB1 and MAT1A form a complex that suppresses matrix metalloproteinase-7 (MMP-7) at the transcriptional level and loss of either PHB1 or MAT1A sensitizes the liver to metastasis via MMP-7 induction. Strategies that target the PHB1/MAT1A-MMP-7 axis may be a promising approach for the treatment of primary and secondary liver cancer metastasis.

The SMYD3-dependent H3K4me3 status of IGF2 intensifies local Th2 differentiation in CRSwNP via positive feedback.

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a heterogeneous and common upper airway disease divided into various inflammatory endotypes. Recent epidemiological findings showed a T helper 2 (Th2)-skewed dominance in CRSwNP patients. Histone modification alterations can regulate transcriptional and translational expression, resulting in abnormal pathogenic changes and the occurrence of diseases. Trimethylation of histone H3 lysine 4 (H3K4me3) is considered an activator of gene expression through modulation of accessibility for transcription, which is closely related to CRSwNP. H3K4me3 levels in the human nasal epithelium may change under Th2-biased inflammatory conditions, resulting in exaggerated local nasal Th2 responses via the regulation of naïve CD4+ T-cell differentiation. Here, we revealed that the level of SET and MYND domain-containing protein 3 (SMYD3)-mediated H3K4me3 was increased in NPs from Th2 CRSwNP patients compared with those from healthy controls. We demonstrated that SMYD3-mediated H3K4me3 is increased in human nasal epithelial cells under Th2-biased inflammatory conditions via S-adenosyl-L-methionine (SAM) production and further found that the H3K4me3high status of insulin-like growth factor 2 (IGF2) produced in primary human nasal epithelial cells could promote naïve CD4+ T-cell differentiation into Th2 cells. Moreover, we found that SAM production was dependent on the c-Myc/methionine adenosyltransferase 2A (MAT2A) axis in the nasal epithelium. Understanding histone modifications in the nasal epithelium has immense potential utility in the development of novel classes of therapeutics targeting Th2 polarization in Th2 CRSwNP. Video Abstract.

Positive Regulation of S-Adenosylmethionine on Chondrocytic Differentiation via Stimulation of Polyamine Production and the Gene Expression of Chondrogenic Differentiation Factors.

S-adenosylmethionine (SAM) is considered to be a useful therapeutic agent for degenerative cartilage diseases, although its mechanism is not clear. We previously found that polyamines stimulate the expression of differentiated phenotype of chondrocytes. We also found that the cellular communication network factor 2 (CCN2) played a huge role in the proliferation and differentiation of chondrocytes. Therefore, we hypothesized that polyamines and CCN2 could be involved in the chondroprotective action of SAM. In this study, we initially found that exogenous SAM enhanced proteoglycan production but not cell proliferation in human chondrocyte-like cell line-2/8 (HCS-2/8) cells. Moreover, SAM enhanced gene expression of cartilage-specific matrix (aggrecan and type II collagen), Sry-Box transcription factor 9 (SOX9), CCN2, and chondroitin sulfate biosynthetic enzymes. The blockade of the methionine adenosyltransferase 2A (MAT2A) enzyme catalyzing intracellular SAM biosynthesis restrained the effect of SAM on chondrocytes. The polyamine level in chondrocytes was higher in SAM-treated culture than control culture. Additionally, Alcian blue staining and RT-qPCR indicated that the effects of SAM on the production and gene expression of aggrecan were reduced by the inhibition of polyamine synthesis. These results suggest that the stimulation of polyamine synthesis and gene expression of chondrogenic differentiation factors, such as CCN2, account for the mechanism underlying the action of SAM on chondrocytes.

Knockdown of TPI in human dermal microvascular endothelial cells and its impact on angiogenesis in vitro.

INTRODUCTION: Angiogenic behaviour has been shown as highly versatile among Endothelial cells (ECs) causing problems of in vitro assays of angiogenesis considering their reproducibility. It is indispensable to investigate influencing factors of the angiogenic potency of ECs. OBJECTIVE: The present study aimed to analyse the impact of knocking down triosephosphate isomerase (TPI) on in vitro angiogenesis and simultaneously on vimentin (VIM) and adenosylmethionine synthetase isoform type 2 (MAT2A) expression. Furthermore, native expression profiles of TPI, VIM and MAT2A in the course of angiogenesis in vitro were examined. METHODS: Two batches of human dermal microvascular ECs were cultivated over 50 days and stimulated to undergo angiogenesis. A shRNA-mediated knockdown of TPI was performed. During cultivation, time-dependant morphological changes were detected and applied for EC-staging as prerequisite for quantifying in vitro angiogenesis. Additionally, mRNA and protein levels of all proteins were monitored. RESULTS: Opposed to native cells, knockdown cells were not able to enter late stages of angiogenesis and primarily displayed a downregulation of VIM and an uprise in MAT2A expression. Native cells increased their TPI expression and decreased their VIM expression during the course of angiogenesis in vitro. For MAT2A, highest expression was observed to be in the beginning and at the end of angiogenesis. CONCLUSION: Knocking down TPI provoked expressional changes in VIM and MAT2A and a deceleration of in vitro angiogenesis, indicating that TPI represents an angiogenic protein. Native expression profiles lead to the assumption of VIM being predominantly relevant in beginning stages, MAT2A in beginning and late stages and TPI during the whole course of angiogenesis in vitro.

Formaldehyde regulates S-adenosylmethionine biosynthesis and one-carbon metabolism.

One-carbon metabolism is an essential branch of cellular metabolism that intersects with epigenetic regulation. In this work, we show how formaldehyde (FA), a one-carbon unit derived from both endogenous sources and environmental exposure, regulates one-carbon metabolism by inhibiting the biosynthesis of S-adenosylmethionine (SAM), the major methyl donor in cells. FA reacts with privileged, hyperreactive cysteine sites in the proteome, including Cys120 in S-adenosylmethionine synthase isoform type-1 (MAT1A). FA exposure inhibited MAT1A activity and decreased SAM production with MAT-isoform specificity. A genetic mouse model of chronic FA overload showed a decrease n SAM and in methylation on selected histones and genes. Epigenetic and transcriptional regulation of Mat1a and related genes function as compensatory mechanisms for FA-dependent SAM depletion, revealing a biochemical feedback cycle between FA and SAM one-carbon units.

Structural and Biochemical Studies on Product Inhibition of S-Adenosylmethionine Synthetase from Corynebacterium glutamicum.

S-Adenosylmethionine (SAM) acts as a methyl donor in living organisms, and S-adenosylmethionine synthetase (MetK) is an essential enzyme for cells, as it synthesizes SAM from methionine and adenosine triphosphate (ATP). This study determined the crystal structures of the apo form and adenosine/triphosphate complex form of MetK from Corynebacterium glutamicum (CgMetK). Results showed that CgMetK has an allosteric inhibitor binding site for the SAM product in the vicinity of the active site and is inhibited by SAM both competitively and noncompetitively. Through structure-guided protein engineering, the CgMetKE68A variant was developed that exhibited an almost complete release of inhibition by SAM with rather enhanced enzyme activity. The crystal structure of the CgMetKE68A variant revealed that the formation of a new hydrogen bond between Tyr66 and Glu102 by the E68A mutation disrupted the allosteric SAM binding site and also improved the protein thermal stability by strengthening the tetramerization of the enzyme.

Hepatic levels of S-adenosylmethionine regulate the adaptive response to fasting.

There has been an intense focus to uncover the molecular mechanisms by which fasting triggers the adaptive cellular responses in the major organs of the body. Here, we show that in mice, hepatic S-adenosylmethionine (SAMe)-the principal methyl donor-acts as a metabolic sensor of nutrition to fine-tune the catabolic-fasting response by modulating phosphatidylethanolamine N-methyltransferase (PEMT) activity, endoplasmic reticulum-mitochondria contacts, ß-oxidation, and ATP production in the liver, together with FGF21-mediated lipolysis and thermogenesis in adipose tissues. Notably, we show that glucagon induces the expression of the hepatic SAMe-synthesizing enzyme methionine adenosyltransferase α1 (MAT1A), which translocates to mitochondria-associated membranes. This leads to the production of this metabolite at these sites, which acts as a brake to prevent excessive ß-oxidation and mitochondrial ATP synthesis and thereby endoplasmic reticulum stress and liver injury. This work provides important insights into the previously undescribed function of SAMe as a new arm of the metabolic adaptation to fasting.

Translocation of Methionine Adenosyl Transferase MAT2A and Its Prognostic Relevance for Liver Hepatocellular Carcinoma.

Methionine adenosyl transferases (MATs) catalyze the synthesis of the biological methyl donor adenosylmethionine (SAM). Dysregulation of MATs has been associated with carcinogenesis in humans. We previously found that downregulation of the MAT1A gene enriches the protein-associated translation process and worsens liver hepatocellular carcinoma (LIHC) prognosis. We also discovered that subcellular localization of the MAT2A protein has independently prognostic relevance in breast cancer patients. The present study aimed to examined the clinical relevance of MAT2A translocation in human LIHC. Essential methionine cycle gene expressions in TCGA LIHC datasets were analyzed using Gene Expression Profiling Interactive Analysis 2 (GEPIA2). The protein expression pattern of MAT2A was determined in the tissue array of our own LIHC cohort (n = 261) using immuno-histochemistry, and the prognostic relevance of MAT2A protein's subcellular localization expression was examined using Kaplan-Meier survival curves. LIHC patients with higher MAT2A mRNA expression had a worse survival rate (p = 0.0083). MAT2A protein immunoreactivity was observed in both cytoplasm and nucleus fractions in the tissue array. Tumor tissues had elevated MAT2A protein expression in both cytoplasm and nucleus compared to their adjacent normal tissues. A higher cytoplasmic to nuclear MAT2A protein expression ratio (C/N) was found in female LIHC patients compared to that of male patients (p = 0.047). Kaplan-Meier survival curves showed that a lower MAT2A C/N correlated with poor overall survival in female LIHC patients (10-year survival rate: 29.2% vs. 68.8%, C/N ≤ 1.0 vs. C/N > 1.0, log-rank p = 0.004). Moreover, we found that specificity protein 1 (SP1) may have a potential interaction with nuclear MAT2A protein, using protein-protein interaction; this we found using the GeneMANIA algorithm. We explored the possible protective effects of the estrogen axis in LIHC using the Human Protein Atlas (HPA), and found evidence supporting a possible protective effect of estrogen-related protein ESSRG in LIHC. The localization of SP1 and MAT2 appeared to be inversely associated with ESRRG expression in LIHC. The present study demonstrated the translocation of MAT2A and its prognostic relevance in female LIHC patients. Our findings suggest the potential of estrogen in SP1 regulation and localization of MAT2A, as therapeutic modalities against in female LIHC patients.

MAT1A Suppression by the CTBP1/HDAC1/HDAC2 Transcriptional Complex Induces Immune Escape and Reduces Ferroptosis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) remains a significant health burden globally due to its high prevalence and morbidity. C-terminal-binding protein 1 (CTBP1) is a transcriptional corepressor that modulates gene transcription by interacting with transcription factors or chromatin-modifying enzymes. High CTBP1 expression has been associated with the progression of various human cancers. In this study, bioinformatics analysis suggested the existence of a CTBP1/histone deacetylase 1 (HDAC1)/HDAC2 transcriptional complex that regulates the expression of methionine adenosyltransferase 1A (MAT1A), whose loss has been associated with ferroptosis suppression and HCC development. Thus, this study aims to investigate the interactions between the CTBP1/HDAC1/HDAC2 complex and MAT1A and their roles in HCC progression. First, high expression of CTBP1 was observed in HCC tissues and cells, where it promoted HCC cell proliferation and mobility while inhibiting cell apoptosis. CTBP1 interacted with HDAC1 and HDAC2 to suppress the MAT1A transcription, and silencing of either HDAC1 or HDAC2 or overexpression of MAT1A led to the inhibition of cancer cell malignancy. In addition, MAT1A overexpression resulted in increased S-adenosylmethionine levels, which promoted ferroptosis of HCC cells directly or indirectly by increasing CD8+ T-cell cytotoxicity and interferon-γ production. In vivo, MAT1A overexpression suppressed growth of CTBP1-induced xenograft tumors in mice while enhancing immune activity and inducing ferroptosis. However, treatment with ferrostatin-1, a ferroptosis inhibitor, blocked the tumor-suppressive effects of MAT1A. Collectively, this study reveals that the CTBP1/HDAC1/HDAC2 complex-induced MAT1A suppression is liked to immune escape and reduced ferroptosis of HCC cells.

A role for protein arginine methyltransferase 7 in repetitive and mild traumatic brain injury.

Mild traumatic brain injury affects the largest proportion of individuals in the United States and world-wide. Pre-clinical studies of repetitive and mild traumatic brain injury (rmTBI) have been limited in their ability to recapitulate human pathology (i.e. diffuse rotational injury). We used the closed-head impact model of engineered rotation acceleration (CHIMERA) to simulate rotational injury observed in patients and to study the pathological outcomes post-rmTBI using C57BL/6J mice. Enhanced cytokine production was observed in both the cortex and hippocampus to suggest neuroinflammation. Furthermore, microglia were assessed via enhanced iba1 protein levels and morphological changes using immunofluorescence. In addition, LC/MS analyses revealed excess glutamate production, as well as diffuse axonal injury via Bielschowsky's silver stain kit. Moreover, the heterogeneous nature of rmTBI has made it challenging to identify drug therapies that address rmTBI, therefore we sought to identify novel targets in the concurrent rmTBI pathology. The pathophysiological findings correlated with a time-dependent decrease in protein arginine methyltransferase 7 (PRMT7) protein expression and activity post-rmTBI along with dysregulation of PRMT upstream mediators s-adenosylmethionine and methionine adenosyltransferase 2 (MAT2) in vivo. In addition, inhibition of the upstream mediator MAT2A using the HT22 hippocampal neuronal cell line suggest a mechanistic role for PRMT7 via MAT2A in vitro. Collectively, we have identified PRMT7 as a novel target in rmTBI pathology in vivo and a mechanistic link between PRMT7 and upstream mediator MAT2A in vitro.

Comparative S-adenosyl-L-methionine analogue generation for selective biocatalytic Friedel-Crafts alkylation.

Methyltransferases provide excellent specificity in late-stage alkylation of biomolecules. Their dependence on S-adenosyl-L-methionine (SAM) mandates efficient access to SAM analogues for biocatalytic applications. We directly compared halide methyltransferase (HMT) and methionine adenosyltransferase (MAT) to access SAM analogues and explored their utility in cascade reactions with NovO for regioselective, late-stage Friedel-Crafts alkylation of a coumarin. The HMT cascade efficiently provided SAM for methylation, while the MAT cascade also supplied high levels of SAM analogues for alkylation reactions.

Enhanced selenocysteine biosynthesis for seleno-methylselenocysteine production in Bacillus subtilis.

Seleno-methylselenocysteine (SeMCys) is an effective component for selenium supplementation with anti-carcinogenic potential and can ameliorate neuropathology and cognitive deficits. In this study, we aimed to engineer Bacillus subtilis 168 for the microbial production of SeMCys. First, the accumulation of intracellular selenocysteine (SeCys) as the precursor of SeMCys was enhanced through overexpression of serine O-acetyltransferase, which was desensitized against feedback inhibition by cysteine. Next, the S-adenosylmethionine (SAM) synthetic pathway was optimized to improve methyl donor availability through expression of S-adenosylmethionine synthetase. Further, SeMCys was successfully produced through expression of the selenocysteine methyltransferase in SeCys and SAM-producing strain. The increased expression level of selenocysteine methyltransferase benefited the SeMCys production. Finally, all the heterologous genes were integrated into the genome of B. subtilis, and the strain produced SeMCys at a titer of 18.4 µg/L in fed-batch culture. This is the first report on the metabolic engineering of B. subtilis for microbial production of SeMCys and provides a good starting point for future pathway engineering to achieve the industrial-grade production of SeMCys. KEY POINTS: • Expression of the feedback-insensitive serine O-acetyltransferase provided B. subtilis the ability of accumulating SeCys. • SAM production was enhanced through expressing S-adenosylmethionine synthetase in B. subtilis. • Expression of selenocysteine methyltransferase in SeCys and SAM-accumulating strain facilitated SeMCys production.