N-acetyltransferase 10 facilitates tumorigenesis of diffuse large B-cell lymphoma by regulating AMPK/mTOR signalling through N4-acetylcytidine modification of SLC30A9.

BACKGROUND: Accumulating studies suggested that posttranscriptional modifications exert a vital role in the tumorigenesis of diffuse large B-cell lymphoma (DLBCL). N4-acetylcytidine (ac4C) modification, catalyzed by the N-acetyltransferase 10 (NAT10), was a novel type of chemical modification that improves translation efficiency and mRNA stability. METHODS: GEO databases and clinical samples were used to explore the expression and clinical value of NAT10 in DLBCL. CRISPER/Cas9-mediated knockout of NAT10 was performed to determine the biological functions of NAT10 in DLBCL. RNA sequencing, acetylated RNA immunoprecipitation sequencing (acRIP-seq), LC-MS/MS, RNA immunoprecipitation (RIP)-qPCR and RNA stability assays were performed to explore the mechanism by which NAT10 contributed to DLBCL progression. RESULTS: Here, we demonstrated that NAT10-mediated ac4C modification regulated the occurrence and progression of DLBCL. Dysregulated N-acetyltransferases expression was found in DLBCL samples. High expression of NAT10 was associated with poor prognosis of DLBCL patients. Deletion of NAT10 expression inhibited cell proliferation and induced G0/G1 phase arrest. Furthermore, knockout of NAT10 increased the sensitivity of DLBCL cells to ibrutinib. AcRIP-seq identified solute carrier family 30 member 9 (SLC30A9) as a downstream target of NAT10 in DLBCL. NAT10 regulated the mRNA stability of SLC30A9 in an ac4C-dependent manner. Genetic silencing of SLC30A9 suppressed DLBCL cell growth via regulating the activation of AMP-activated protein kinase (AMPK) pathway. CONCLUSION: Collectively, these findings highlighted the essential role of ac4C RNA modification mediated by NAT10 in DLBCL, and provided insights into novel epigenetic-based therapeutic strategies.

Inhibition of hippocampal melatonin synthesis by siRNA induced learning and memory deficits in male rats.

Melatonin, the multi-functional neurohormone, is synthesized in the extra-pineal tissues such as the hippocampus. The key enzyme in hippocampal melatonin synthesis is arylalkylamine-N-acetyltransferase (AANAT). The importance of melatonin synthesis in the hippocampus has not yet been determined. We investigated hippocampal AANAT role in cognitive function using gene silencing small interference RNA (siRNA) technology. The hippocampal local melatonin synthesis was inhibited by AANAT-siRNA injection. The time-gene silencing profile of AANAT-siRNA was obtained by RT-PCR technique. The cytotoxicity of siRNA dose was determined by MTT assay on the B65 neural cells. Animals received the selected dosage of AANAT-siRNA. Then, the spatial working memory (Y maze), object recognition memory and spatial reference memory (Morris's water maze, MWM) were evaluated. The anxiety-like behaviors were evaluated by the elevated plus maze. After one week, following the probe test of MWM, the rats were sacrificed for histological analysis. The hippocampal melatonin levels were measured using the liquid chromatography-mass spectrometry technique. The hippocampal melatonin levels in the AANAT-siRNA group decreased. Animals receiving the AANAT-siRNA showed deficits in spatial learning and working memory which were verified by increased escape latency and reduced spontaneous alternations, respectively. There was an increase in anxiety-like behaviors as well as a deficit in recognition memory in the AANAT-siRNA group. The Nissl staining and immunohistochemistry of activated caspase-3 showed the neuronal loss and cell apoptosis in hippocampal tissue of the AANAT-siRNA group. The 18F-FDG-PET imaging displayed lower glucose metabolism following the reduction in AANAT mRNA. Data suggest that the AANAT mRNA and hippocampal melatonin synthesis might be an essential factor for learning, memory and some aspects of cognition, as well as homeostasis of hippocampal cells.

Targeting N4-acetylcytidine suppresses hepatocellular carcinoma progression by repressing eEF2-mediated HMGB2 mRNA translation.

BACKGROUND: N4-acetylcytidine (ac4C) represents a novel messenger RNA (mRNA) modification, and its associated acetyltransferase N-acetyltransferase 10 (NAT10) plays a crucial role in the initiation and progression of tumors by regulating mRNA functionality. However, its role in hepatocellular carcinoma (HCC) development and prognosis is largely unknown. This study aimed to elucidate the role of NAT10-mediated ac4C in HCC progression and provide a promising therapeutic approach. METHODS: The ac4C levels were evaluated by dot blot and ultra-performance liquid chromatography-tandem mass spectrometry with harvested HCC tissues. The expression of NAT10 was investigated using quantitative real-time polymerase chain reaction, western blotting, and immunohistochemical staining across 91 cohorts of HCC patients. To explore the underlying mechanisms of NAT10-ac4C in HCC, we employed a comprehensive approach integrating acetylated RNA immunoprecipitation and sequencing, RNA sequencing and ribosome profiling analyses, along with RNA immunoprecipitation, RNA pull-down, mass spectrometry, and site-specific mutation analyses. The drug affinity responsive targets stability, cellular thermal shift assay, and surface plasmon resonance assays were performed to assess the specific binding of NAT10 and Panobinostat. Furthermore, the efficacy of targeting NAT10-ac4C for HCC treatment was elucidated through in vitro experiments using HCC cells and in vivo HCC mouse models. RESULTS: Our investigation revealed a significant increase in both the ac4C RNA level and NAT10 expression in HCC. Notably, elevated NAT10 expression was associated with poor outcomes in HCC patients. Functionally, silencing NAT10 suppressed HCC proliferation and metastasis in vitro and in vivo. Mechanistically, NAT10 stimulates the ac4C modification within the coding sequence (CDS) of high mobility group protein B2 (HMGB2), which subsequently enhances HMGB2 translation by facilitating eukaryotic elongation factor 2 (eEF2) binding to the ac4C sites on HMGB2 mRNA's CDS. Additionally, high-throughput compound library screening revealed Panobinostat as a potent inhibitor of NAT10-mediated ac4C modification. This inhibition significantly attenuated HCC growth and metastasis in both in vitro experiments using HCC cells and in vivo HCC mouse models. CONCLUSIONS: Our study identified a novel oncogenic epi-transcriptome axis involving NAT10-ac4C/eEF2-HMGB2, which plays a pivotal role in regulating HCC growth and metastasis. The drug Panobinostat validates the therapeutic potential of targeting this axis for HCC treatment.

Acetylcytidine modification of Amotl1 by N-acetyltransferase 10 contributes to cardiac fibrotic expansion in mice after myocardial infarction.

Cardiac fibrosis is a pathological scarring process that impairs cardiac function. N-acetyltransferase 10 (Nat10) is recently identified as the key enzyme for the N4-acetylcytidine (ac4C) modification of mRNAs. In this study, we investigated the role of Nat10 in cardiac fibrosis following myocardial infarction (MI) and the related mechanisms. MI was induced in mice by ligation of the left anterior descending coronary artery; cardiac function was assessed with echocardiography. We showed that both the mRNA and protein expression levels of Nat10 were significantly increased in the infarct zone and border zone 4 weeks post-MI, and the expression of Nat10 in cardiac fibroblasts was significantly higher compared with that in cardiomyocytes after MI. Fibroblast-specific overexpression of Nat10 promoted collagen deposition and induced cardiac systolic dysfunction post-MI in mice. Conversely, fibroblast-specific knockout of Nat10 markedly relieved cardiac function impairment and extracellular matrix remodeling following MI. We then conducted ac4C-RNA binding protein immunoprecipitation-sequencing (RIP-seq) in cardiac fibroblasts transfected with Nat10 siRNA, and revealed that angiomotin-like 1 (Amotl1), an upstream regulator of the Hippo signaling pathway, was the target gene of Nat10. We demonstrated that Nat10-mediated ac4C modification of Amotl1 increased its mRNA stability and translation in neonatal cardiac fibroblasts, thereby increasing the interaction of Amotl1 with yes-associated protein 1 (Yap) and facilitating Yap translocation into the nucleus. Intriguingly, silencing of Amotl1 or Yap, as well as treatment with verteporfin, a selective and potent Yap inhibitor, attenuated the Nat10 overexpression-induced proliferation of cardiac fibroblasts and prevented their differentiation into myofibroblasts in vitro. In conclusion, this study highlights Nat10 as a crucial regulator of myocardial fibrosis following MI injury through ac4C modification of upstream activators within the Hippo/Yap signaling pathway.

Integration of molecular docking and molecular dynamics simulations with subtractive proteomics approach to identify the novel drug targets and their inhibitors in Streptococcus gallolyticus.

Streptococcus gallolyticus (Sg) is a non-motile, gram-positive bacterium that causes infective endocarditis (inflammation of the heart lining). Because Sg has gained resistance to existing antibiotics and there is currently no drug available, developing effective anti-Sg drugs is critical. This study combined core proteomics with a subtractive proteomics technique to identify potential therapeutic targets for Sg. Several bioinformatics approaches were used to eliminate non-essential and human-specific homologous sequences from the bacterial proteome. Then, virulence, druggability, subcellular localization, and functional analyses were carried out to specify the participation of significant bacterial proteins in various cellular processes. The pathogen's genome contained three druggable proteins, glucosamine-1phosphate N-acetyltransferase (GlmU), RNA polymerase sigma factor (RpoD), and pantetheine-phosphate adenylyltransferase (PPAT) which could serve as effective targets for developing novel drugs. 3D structures of target protein were modeled through Swiss Model. A natural product library containing 10,000 molecules from the LOTUS database was docked against therapeutic target proteins. Following an evaluation of the docking results using the glide gscore, the top 10 compounds docked against each protein receptor were chosen. LTS001632, LTS0243441, and LTS0236112 were the compounds that exhibited the highest binding affinities against GlmU, PPAT, and RpoD, respectively, among the compounds that were chosen. To augment the docking data, molecular dynamics simulations and MM-GBSA binding free energy were also utilized. More in-vitro research is necessary to transform these possible inhibitors into therapeutic drugs, though computer validations were employed in this study. This combination of computational techniques paves the way for targeted antibiotic development, which addresses the critical need for new therapeutic strategies against S. gallolyticus infections.

Melatonin mediated tolerance to benzalkonium chloride phytotoxicity through improved growth, photochemical reactions, and antioxidant system in wild-type and snat2 mutant Arabidopsis lines.

Melatonin (Mel) is a phytohormone that plays a crucial role in various plant processes, including stress response. Despite numerous studies on the role of Mel in stress resistance, its significance in plants exposed to benzalkonium chloride (BAC) pollution remains unexplored. BAC, a common antiseptic, poses a threat to terrestrial plants due to its widespread use and inefficient removal, leading to elevated concentrations in the environment. This study investigated the impact of BAC (0.5 mg L-1) pollution on wild-type Col-0 and snat2 knockout mutant Arabidopsis lines, revealing reduced growth, altered water relations, and gas exchange parameters. On the other hand, exogenous Mel (100 µM) treatments mitigated BAC-induced phytotoxicity and increased the growth rate by 1.8-fold in Col-0 and 2-fold in snat2 plants. snat2 mutant seedlings had a suppressed carbon assimilation rate (A) under normal conditions, but BAC contamination led to further A repression by 71% and 48% in Col-0 and snat2 leaves, respectively. However, Mel treatment on stressed plants was successful in improving Fv/Fm and increased the total photosynthesis efficiency by regulating photochemical reactions. Excessive H2O2 accumulation in the guard cells of plants exposed to BAC pollution was detected by confocal microscopy. Mel treatments triggered almost all antioxidant enzyme activities (except POX) in both Arabidopsis lines under stress. This enhanced antioxidant activity, facilitated by foliar Mel application, contributed to the alleviation of oxidative damage, regulation of photosynthesis reactions, and promotion of plant growth in Arabidopsis. In addition to corroborating results observed in many agricultural plants regarding the development of tolerance to environmental stresses, this study provides novel insights into the action mechanisms of Mel under the emerging pollutant benzalkonium chloride.

Melatonin-Regulated Chaperone Binding Protein Plays a Key Role in Cadmium Stress Tolerance in Rice, Revealed by the Functional Characterization of a Novel Serotonin N-Acetyltransferase 3 (SNAT3) in Rice.

The study of the mechanisms by which melatonin protects against cadmium (Cd) toxicity in plants is still in its infancy, particularly at the molecular level. In this study, the gene encoding a novel serotonin N-acetyltransferase 3 (SNAT3) in rice, a pivotal enzyme in the melatonin biosynthetic pathway, was cloned. Rice (Oryza sativa) OsSNAT3 is the first identified plant ortholog of archaeon Thermoplasma volcanium SNAT. The purified recombinant OsSNAT3 catalyzed the conversion of serotonin and 5-methoxytryptamine to N-acetylserotonin and melatonin, respectively. The suppression of OsSNAT3 by RNAi led to a decline in endogenous melatonin levels followed by a reduction in Cd tolerance in transgenic RNAi rice lines. In addition, the expression levels of genes encoding the endoplasmic reticulum (ER) chaperones BiP3, BiP4, and BiP5 were much lower in RNAi lines than in the wild type. In transgenic rice plants overexpressing OsSNAT3 (SNAT3-OE), however, melatonin levels were higher than in wild-type plants. SNAT3-OE plants also tolerated Cd stress, as indicated by seedling growth, malondialdehyde, and chlorophyll levels. BiP4 expression was much higher in the SNAT3-OE lines than in the wild type. These results indicate that melatonin engineering could help crops withstand Cd stress, resulting in high yields in Cd-contaminated fields.

N4-acetylcytidine acetylation of neurexin 2 in the spinal dorsal horn regulates hypersensitivity in a rat model of cancer-induced bone pain.

Cancer-induced bone pain (CIBP) significantly impacts the quality of life and survival of patients with advanced cancer. Despite the established role of neurexins in synaptic structure and function, their involvement in sensory processing during injury has not been extensively studied. In this study using a rat model of CIBP, we observed increased neurexin 2 expression in spinal cord neurons. Knockdown of neurexin 2 in the spinal cord reversed CIBP-related behaviors, sensitization of spinal c-Fos neurons, and pain-related negative emotional behaviors. Additionally, increased acetylation of neurexin 2 mRNA was identified in the spinal dorsal horn of CIBP rats. Decreasing the expression of N-acetyltransferase 10 (NAT10) reduced neurexin 2 mRNA acetylation and neurexin 2 expression. In PC12 cells, we confirmed that neurexin 2 mRNA acetylation enhanced its stability, and neurexin 2 expression was regulated by NAT10. Finally, we discovered that the NAT10/ac4C-neurexin 2 axis modulated neuronal synaptogenesis. This study demonstrated that the NAT10/ac4C-mediated posttranscriptional modulation of neurexin 2 expression led to the remodeling of spinal synapses and the development of conscious hypersensitivity in CIBP rats. Therefore, targeting the epigenetic modification of neurexin 2 mRNA ac4C may offer a new therapeutic approach for the treatment of nociceptive hypersensitivity in CIBP.

Remodelin delays non-small cell lung cancer progression by inhibiting NAT10 via the EMT pathway.

BACKGROUND: Lung cancer remains the foremost reason of cancer-related mortality, with invasion and metastasis profoundly influencing patient prognosis. N-acetyltransferase 10 (NAT10) catalyzes the exclusive N (4)-acetylcytidine (ac4C) modification in eukaryotic RNA. NAT10 dysregulation is linked to various diseases, yet its role in non-small cell lung cancer (NSCLC) invasion and metastasis remains unclear. Our study delves into the clinical significance and functional aspects of NAT10 in NSCLC. METHODS: We investigated NAT10's clinical relevance using The Cancer Genome Atlas (TCGA) and a group of 98 NSCLC patients. Employing WB, qRT-PCR, and IHC analyses, we assessed NAT10 expression in NSCLC tissues, bronchial epithelial cells (BECs), NSCLC cell lines, and mouse xenografts. Further, knockdown and overexpression techniques (siRNA, shRNA, and plasmid) were employed to evaluate NAT10's effects. A series of assays were carried out, including CCK-8, colony formation, wound healing, and transwell assays, to elucidate NAT10's role in proliferation, invasion, and metastasis. Additionally, we utilized lung cancer patient-derived 3D organoids, mouse xenograft models, and Remodelin (NAT10 inhibitor) to corroborate these findings. RESULTS: Our investigations revealed high NAT10 expression in NSCLC tissues, cell lines and mouse xenograft models. High NAT10 level correlated with advanced T stage, lymph node metastasis and poor overall survive. NAT10 knockdown curtailed proliferation, invasion, and migration, whereas NAT10 overexpression yielded contrary effects. Furthermore, diminished NAT10 levels correlated with increased E-cadherin level whereas decreased N-cadherin and vimentin expressions, while heightened NAT10 expression displayed contrasting results. Notably, Remodelin efficiently attenuated NSCLC proliferation, invasion, and migration by inhibiting NAT10 through the epithelial-mesenchymal transition (EMT) pathway. CONCLUSIONS: Our data underscore NAT10 as a potential therapeutic target for NSCLC, presenting avenues for targeted intervention against lung cancer through NAT10 inhibition.

Structural analysis of neomycin B and kanamycin A binding Aminoglycosides Modifying Enzymes (AME) and bacterial ribosomal RNA.

Aminoglycosides are crucial antibiotics facing challenges from bacterial resistance. This study addresses the importance of aminoglycoside modifying enzymes in the context of escalating resistance. Drawing upon over two decades of structural data in the Protein Data Bank, we focused on two key antibiotics, neomycin B and kanamycin A, to explore how the aminoglycoside structure is exploited by this family of enzymes. A systematic comparison across diverse enzymes and the RNA A-site target identified common characteristics in the recognition mode, while assessing the adaptability of neomycin B and kanamycin A in various environments.

Effects of compound Anoectochilus roxburghii (Wall.) Lindl. oral liquid on relative metabolic enzymes and various biochemical indices in Wistar rats with isoniazid-induced liver injury.

Isoniazid (INH) is the first-line anti-tuberculosis drug in clinical practice, and its main adverse effect is drug-induced liver injury (DILI). This study aimed to investigate the hepatoprotective effect of Compound Anoectochilus roxburghii (Wall.) Lindl. Oral Liquid (CAROL) and to provide a new strategy for the search of potential drugs against INH-induced liver injury in Wistar rats. Animal experiment was based on INH (100 mg/kg) induced liver injury to explore the intervention effects of CAROL at doses of 1.35, 2.70, and 5.40 mL/kg. LC-QTOF-MS/MS was used to identify hepatoprotective components in CAROL and its' exposed components in rat serum. The hepatoprotective effect of CAROL was evaluated by pathological observation of rat liver tissue and changes in levels of biochemical indices and cytokines in serum or liver tissue. Of the 58 hepatoprotective components identified, 15 were detected in the serum of rats with liver-injured treated by high-dose CAROL. Results of animal experiments showed that the levels of various biochemical indexes and cytokines were significantly reversed with CAROL intervention. In particular, the expression level of cytokeratin-18 and high-mobility group box 1, as specific and sensitive indicators of DILI, was significantly reduced in the serum of rats with CAROL intervention compared with the INH model group. The same reversal was observed in the levels of TBIL, ALP, ALT, and AST in serum, as well as in the levels of TNF-α, IL-6, SOD, and MDA in liver tissue. For INH-metabolizing enzymes, an evident expression inhibition was observed in N-acetyltransferase 2 and glutathione S-transferases with CAROL intervention, which may be the key to controlling INH hepatotoxicity. CAROL has a favorable hepatoprotective effect on INH-induced liver injury. This study takes the first step in studying the hepatoprotective mechanism of CAROL against INH hepatotoxicity and provides reference for wider clinical applications.

Role of arylalkylamine N-acetyltransferase 7 in reproduction and limb pigmentation of Aedes aegypti.

Arylalkylamine N-acetyltransferase (aaNAT) is a crucial enzyme that catalyses the transfer of acetyl groups from acetyl coenzyme A to arylalkylamines and arylamines. Evolutionary studies have identified a distinct class of aaNATs specific to mosquitoes, yet their functions remain elusive. This study focuses on Ae-aaNAT7, a mosquito-unique gene in Aedes aegypti (Diptera:Culicidae), to explore its functionality. Temporal and spatial expression analysis of Ae-aaNAT7 mRNA revealed high expression during embryonic development and in first-instar larvae, with notable expression in the limbs of adult mosquitoes based on tissue expression profiling. By further employing CRISPR/Cas9 technology for loss-of-function studies, our investigation revealed a reduction in the area of white spotting in the limbs of Ae-aaNAT7 mutant adult mosquitoes. Further investigation revealed a significant decrease in the fecundity and hatchability of the mutants. Dissection of the ovaries from Ae-aaNAT7 heterozygous mutants showed a noticeable reduction in the oocyte area compared with wild type. Dissection of the exochorion of the eggs from Ae-aaNAT7 homozygous mutants consistently revealed a striking absence of mature embryos. In addition, RNA interference experiments targeting Ae-aaNAT7 in males resulted in a reduction in fecundity, but no effect on hatchability was observed. These collective insights underscore the substantial impact of Ae-aaNAT7 on reproduction and its pivotal contribution to adult limb pigmentation in Ae. aegypti. These revelations offer insights pivotal for the strategic design of future insecticide targets.

Characterization of an agmatine N-acetyltransferase from Bactrocera dorsalis that modulates ovary development.

Agmatine N-acetyltransferase (AgmNAT), which catalyzes the formation of N-acetylagmatine from acetyl-CoA and agmatine, is a member of the GCN5-related N-acetyltransferase family. So far, knowledge of the physiological roles of AgmNAT in insects is limited. Here, we identified one gene encoding protein homologous to that of Drosophila AgmNAT using sequence information from an activity-verified Drosophila AgmNAT in a BLAST search of the Bactrocera dorsalis genome. We expressed and purified B. dorsalis AgmNAT in Escherichia coli and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Our application of the screening strategy to BdorAgmNAT led to the identification of agmatine as the best amine substrate for this enzyme, with the highest kcat/Km value. We successfully obtained a BdorAgmNAT knockout strain based on a wild-type strain (WT) using the CRISPR/Cas9 technique. The ovary development of the BdorAgmNAT knockout mutants was delayed for 10 days compared with the WT specimens. Moreover, mutants had a much smaller mature ovary size and laid far fewer eggs than WT. Loss of function of BdorAgmNAT caused by RNAi with mature WT females did not affect their fecundity. These findings indicate that BdorAgmNAT is critical for oogenesis. Our data provide the first evidence for AgmNAT in regulating ovary development.

tRNA-derived small RNA (tsr007330) regulates myocardial fibrosis after myocardial infarction through NAT10-mediated ac4C acetylation of EGR3 mRNA.

Small non-coding ribonucleic acids (sncRNAs) play an important role in cell regulation and are closely related to the pathogenesis of heart diseases. However, the role and molecular mechanism of transfer RNA-derived small RNAs (tsRNAs) in myocardial fibrosis after myocardial infarction (MI) remain unknown. In this study, we identified and validated sncRNAs (mainly miRNA and tsRNA) associated with myocardial fibrosis after MI through PANDORA sequencing of rat myocardial tissue. As a key enzyme of N4-acetylcytidine (ac4C) acetylation modification, N-acetyltransferase 10 (NAT10) plays an important role in regulating messenger RNA (mRNA) stability and translation efficiency. We found that NAT10 is highly expressed in infarcted myocardial tissue, and the results of acetylated RNA immunoprecipitation sequencing (acRIP-seq) analysis suggest that early growth response 3 (EGR3) may be an important molecule in the pathogenesis of NAT10-mediated myocardial fibrosis. Both in vivo and in vitro experiments have shown that inhibition of NAT10 can reduce the expression of EGR3 and alleviate myocardial fibrosis after MI. tsRNA can participate in gene regulation by inhibiting target genes. The expression of tsr007330 was decreased in myocardial infarction tissue. We found that overexpression of tsr007330 in rat myocardial tissue could antagonize NAT10, improve myocardial function in MI and alleviate myocardial fibrosis. In conclusion, tsRNAs (rno-tsr007330) may regulate the occurrence of myocardial fibrosis by regulating NAT10-mediated EGR3 mRNA acetylation. This study provides new insights into the improvement of myocardial fibrosis after MI by targeting tsRNA therapy.

Engineering the Activity of a Newly Identified Arylalkylamine N-Acetyltransferase in the Acetylation of 5-Hydroxytryptamine.

Arylalkylamine N-acetyltransferase (AANAT) serves as a key enzyme in the biosynthesis of melatonin by transforming 5-hydroxytryptamine (5-HT) to N-acetyl-5-hydroxytryptamine (NAS), while its low activity may hinder melatonin yield. In this study, a novel AANAT derived from Sus scrofa (SsAANAT) was identified through data mining using 5-HT as a model substrate, and a rational design of SsAANAT was conducted in the quest to improving its activity. After four rounds of mutagenesis procedures, a triple combinatorial dominant mutant M3 was successfully obtained. Compared to the parent enzyme, the conversion of the whole-cell reaction bearing the best variant M3 exhibted an increase from 50 % to 99 % in the transformation of 5-HT into NAS. Additionally, its catalytic efficiency (kcat/Km) was enhanced by 2-fold while retaining the thermostability (Tm>45 °C). In the up-scaled reaction with a substrate loading of 50 mM, the whole-cell system incorporating variant M3 achieved a 99 % conversion of 5-HT in 30 h with an 80 % yield. Molecular dynamics simulations were ultilized to shed light on the origin of improved activity. This study broadens the repertoire of AANAT for the efficient biosynthesis of melatonin.

Comparative genomics of N-acetyl-5-methoxytryptamine members in four Prunus species with insights into bud dormancy and abiotic stress responses in Prunus avium.

KEY MESSAGE: This study provides novel insights into the evolution, diversification, and functions of melatonin biosynthesis genes in Prunus species, highlighting their potential role in regulating bud dormancy and abiotic stresses. The biosynthesis of melatonin (MEL) in plants is primarily governed by enzymatic reactions involving key enzymes such as serotonin N-acetyltransferase (SNAT), tryptamine 5-hydroxylase (T5H), N-acetylserotonin methyltransferase (ASMT) and tryptophan decarboxylase (TDC). In this study, we analyzed Melatonin genes in four Prunus species such as Prunus avium (Pavi), Prunus pusilliflora (Ppus), Prunus serulata (Pser), and Prunus persica (Pper) based on comparative genomics approach. Among the four Prunus species, a total of 29 TDCs, 998 T5Hs, 16 SNATs, and 115 ASMTs within the genome of four Prunus genomes. A thorough investigation of melatonin-related genes was carried out using systematic biological methods and comparative genomics. Through phylogenetic analysis, orthologous clusters, Go enrichment, syntenic relationship, and gene duplication analysis, we discovered both similarities and variations in Melatonin genes among these Prunus species. Additionally, our study revealed the existence of unique subgroup members in the Melatonin genes of these species, which were distinct from those found in Arabidopsis genes. Furthermore, the transcriptomic expression analysis revealed the potential significance of melatonin genes in bud dormancy regulation and abiotic stresses. Our extensive results offer valuable perspectives on the evolutionary patterns, intricate expansion, and functions of PavMEL genes. Given their promising attributes, PavTDCs, PavT5H, PavNAT, and three PavASMT genes warrant in-depth exploration as prime candidates for manipulating dormancy in sweet cherry. This was done to lay the foundation for future explorations into the structural and functional aspects of these factors in Prunus species. This study offers significant insights into the functions of ASMT, SNAT, T5H, and TDC genes and sheds light on their roles in Prunus avium. Moreover, it established a robust foundation for further exploration functional characterization of melatonin genes in fruit species.

NAT10-mediated ac4C-modified ANKZF1 promotes tumor progression and lymphangiogenesis in clear-cell renal cell carcinoma by attenuating YWHAE-driven cytoplasmic retention of YAP1.

BACKGROUND: Lymphatic metastasis is one of the most common metastatic routes and indicates a poor prognosis in clear-cell renal cell carcinoma (ccRCC). N-acetyltransferase 10 (NAT10) is known to catalyze N4-acetylcytidine (ac4C) modification of mRNA and participate in many cellular processes. However, its role in the lymphangiogenic process of ccRCC has not been reported. This study aimed to elucidate the role of NAT10 in ccRCC lymphangiogenesis, providing valuable insights into potential therapeutic targets for intervention. METHODS: ac4C modification and NAT10 expression levels in ccRCC were assessed using public databases and clinical samples. Functional investigations involved manipulating NAT10 expression in cellular and mouse models to study its role in ccRCC. Mechanistic insights were gained through a combination of RNA sequencing, mass spectrometry, co-immunoprecipitation, RNA immunoprecipitation, immunofluorescence, and site-specific mutation analyses. RESULTS: We found that ac4C modification and NAT10 expression levels increased in ccRCC. NAT10 promoted tumor progression and lymphangiogenesis of ccRCC by enhancing the nuclear import of Yes1-associated transcriptional regulator (YAP1). Subsequently, we identified ankyrin repeat and zinc finger peptidyl tRNA hydrolase 1 (ANKZF1) as the functional target of NAT10, and its upregulation in ccRCC was caused by NAT10-mediated ac4C modification. Mechanistic analyses demonstrated that ANKZF1 interacted with tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (YWHAE) to competitively inhibit cytoplasmic retention of YAP1, leading to transcriptional activation of pro-lymphangiogenic factors. CONCLUSIONS: These results suggested a pro-cancer role of NAT10-mediated acetylation in ccRCC and identified the NAT10/ANKZF1/YAP1 axis as an under-reported pathway involving tumor progression and lymphangiogenesis in ccRCC.

Recent advances in the potential role of RNA N4-acetylcytidine in cancer progression.

N4-acetylcytidine (ac4C) is a highly conserved chemical modification widely found in eukaryotic and prokaryotic RNA, such as tRNA, rRNA, and mRNA. This modification is significantly associated with various human diseases, especially cancer, and its formation depends on the catalytic activity of N-acetyltransferase 10 (NAT10), the only known protein that produces ac4C. This review discusses the detection techniques and regulatory mechanisms of ac4C and summarizes ac4C correlation with tumor occurrence, development, prognosis, and drug therapy. It also comments on a new biomarker for early tumor diagnosis and prognosis prediction and a new target for tumor therapy. Video Abstract.

Towards pharmacogenomics-guided tuberculosis (TB) therapy: N-acetyltransferase-2 genotypes among TB-infected Kenyans of mixed ethnicity.

BACKGROUND: Though persons of African descent have one of the widest genetic variability, genetic polymorphisms of drug-metabolising enzymes such as N-Acetyltransferase-2 (NAT2) are understudied. This study aimed to identify prevalent NAT2 single nucleotide polymorphisms (SNPs) and infer their potential effects on enzyme function among Kenyan volunteers with tuberculosis (TB) infection. Genotypic distribution at each SNP and non-random association of alleles were evaluated by testing for Hardy-Weinberg Equilibrium (HWE) and Linkage Disequilibrium (LD). METHODS: We isolated genomic DNA from cryopreserved Peripheral Blood Mononuclear Cells of 79 volunteers. We amplified the protein-coding region of the NAT2 gene by polymerase chain reaction (PCR) and sequenced PCR products using the Sanger sequencing method. Sequencing reads were mapped and aligned to the NAT2 reference using the Geneious software (Auckland, New Zealand). Statistical analyses were performed using RStudio version 4.3.2 (2023.09.1 + 494). RESULTS: The most frequent haplotype was the wild type NAT2*4 (37%). Five genetic variants: 282C > T (NAT2*13), 341 T > C (NAT2*5), 803A > G (NAT2*12), 590G > A (NAT2*6) and 481C > T (NAT2*11) were observed with allele frequencies of 29%, 18%, 6%, 6%, and 4% respectively. According to the bimodal distribution of acetylation activity, the predicted phenotype was 76% rapid (mainly consisting of the wildtype NAT2*4 and the NAT2*13A variant). A higher proportion of rapid acetylators were female, 72% vs 28% male (p = 0.022, odds ratio [OR] 3.48, 95% confidence interval [CI] 1.21 to 10.48). All variants were in HWE. NAT2 341 T > C was in strong complete LD with the 590G > A variant (D' = 1.0, r2 = - 0.39) but not complete LD with the 282C > T variant (D' = 0.94, r2 = - 0.54). CONCLUSION: The rapid acetylation haplotypes predominated. Despite the LD observed, none of the SNPs could be termed tag SNP. This study adds to the genetic characterisation data of African populations at NAT2, which may be useful for developing relevant pharmacogenomic tools for TB therapy. To support optimised, pharmacogenomics-guided TB therapy, we recommend genotype-phenotype studies, including studies designed to explore gender-associated differences.

Role of Transporters and Enzymes in Metabolism and Distribution of 4-Chlorokynurenine (AV-101).

4-Chlorokynurenine (4-Cl-KYN, AV-101) is a prodrug of a NMDA receptor antagonist and is in clinical development for potential CNS indications. We sought to further understand the distribution and metabolism of 4-Cl-KYN, as this information might provide a strategy to enhance the clinical development of this drug. We used excretion studies in rats, in vitro transporter assays, and pharmacogenetic analysis of clinical trial data to determine how 4-Cl-KYN and metabolites are distributed. Our data indicated that a novel acetylated metabolite (N-acetyl-4-Cl-KYN) did not affect the uptake of 4-Cl-KYN across the blood-brain barrier via LAT1. 4-Cl-KYN and its metabolites were found to be renally excreted in rodents. In addition, we found that N-acetyl-4-Cl-KYN inhibited renal and hepatic transporters involved in excretion. Thus, this metabolite has the potential to limit the excretion of a range of compounds. Our pharmacogenetic analysis found that a SNP in N-acetyltransferase 8 (NAT8, rs13538) was linked to levels of N-acetyl-4-Cl-KYN relative to 4-Cl-KYN found in the plasma and that a SNP in SLC7A5 (rs28582913) was associated with the plasma levels of the active metabolite, 7-Cl-KYNA. Thus, we have a pharmacogenetics-based association for plasma drug level that could aid in the drug development of 4-Cl-KYN and have investigated the interaction of a novel metabolite with drug transporters.