Oxidative stress contributes to hypermethylation of Histone H3 lysine 9 in placental trophoblasts from preeclamptic pregnancies.

Background and objective: Aberrant epigenetic regulation and increased oxidative stress in the placenta play a significant role in placental pathophysiology and fetal programming in preeclampsia, a hypertensive disorder in human pregnancy. The purpose of the study is to investigate if hypermethylation of histone H3K9 occurs in placental trophoblasts from preeclampsia. Methods: Trophoblasts were isolated and cultured from 14 placentas, 7 from normotensive pregnant women and 7 from preeclamptic pregnancies. Methylated H3K9 expression and antioxidant superoxide dismutase expression were determined by Western blot. We also examined consequences of oxidative stress and the downstream effects of histone methyltransferase inhibition on H3K9 expression associated with antioxidant CuZn-SOD and Mn-SOD expression in placental trophoblasts. Results: We found that expression of mono-, di-, and tri-methylation of histone H3 lysine 9 (H3K9me1, H3K9me2 and H3K9me3) was significantly increased, p<0.01, which correlated with downregulation of antioxidant superoxide dismutase CuZn-SOD and Mn-SOD expression, in trophoblasts from preeclamptic placentas compared to those from uncomplicated control placentas. We further demonstrated hypoxia could promote histone H3K9 methylation in placental trophoblasts, and hypoxia-induced upregulation of H3K9me1, H3K9me2 and H3K9me3 expression was reversible when hypoxic condition was removed. In addition, we also uncovered that inhibition of methyltransferase not only prevented hypoxia-induced upregulation of H3K9me1, H3K9me2 and H3K9me3 expression, but also abolished hypoxia-induced downregulation of CuZn-SOD and Mn-SOD expression in placental trophoblasts. Conclusions: These findings are noteworthy and provide further evidence that increased oxidative stress in the intrauterine environment is likely a mechanism to induce aberrant histone modification in placental trophoblasts in preeclampsia. Moreover, CuZn-SOD and Mn-SOD expression/activity are possibly H3K9 methylation-dependent in placental trophoblasts, which further suggest that oxidative stress and aberrant histone modification have significant impact on placental trophoblasts/fetal programming in preeclampsia.

Impact of Combinatorial Histone Modifications on Acetyllysine Recognition by the ATAD2 and ATAD2B Bromodomains.

The ATPase family AAA+ domain containing 2 (ATAD2) protein and its paralog ATAD2B have a C-terminal bromodomain (BRD) that functions as a reader of acetylated lysine residues on histone proteins. Using a structure-function approach, we investigated the ability of the ATAD2/B BRDs to select acetylated lysine among multiple histone post-translational modifications. The ATAD2B BRD can bind acetylated histone ligands that also contain adjacent methylation or phosphorylation marks, while the presence of these modifications significantly weakened the acetyllysine binding activity of the ATAD2 BRD. Our structural studies provide mechanistic insights into how ATAD2/B BRD-binding pocket residues coordinate the acetyllysine group in the context of adjacent post-translational modifications. Furthermore, we investigated how sequence changes in amino acids of the histone ligands impact the recognition of an adjacent acetyllysine residue. Our study highlights how the interplay between multiple combinations of histone modifications influences the reader activity of the ATAD2/B BRDs, resulting in distinct binding modes.

Methylation of ESCRT-III components regulates the timing of cytokinetic abscission.

Abscission is the final stage of cytokinesis, which cleaves the intercellular bridge (ICB) connecting two daughter cells. Abscission requires tight control of the recruitment and polymerization of the Endosomal Protein Complex Required for Transport-III (ESCRT-III) components. We explore the role of post-translational modifications in regulating ESCRT dynamics. We discover that SMYD2 methylates the lysine 6 residue of human CHMP2B, a key ESCRT-III component, at the ICB, impacting the dynamic relocation of CHMP2B to sites of abscission. SMYD2 loss-of-function (genetically or pharmacologically) causes CHMP2B hypomethylation, delayed CHMP2B polymerization and delayed abscission. This is phenocopied by CHMP2B lysine 6 mutants that cannot be methylated. Conversely, SMYD2 gain-of-function causes CHMP2B hypermethylation and accelerated abscission, specifically in cells undergoing cytokinetic challenges, thereby bypassing the abscission checkpoint. Additional experiments highlight the importance of CHMP2B methylation beyond cytokinesis, namely during ESCRT-III-mediated HIV-1 budding. We propose that lysine methylation signaling fine-tunes the ESCRT-III machinery to regulate the timing of cytokinetic abscission and other ESCRT-III dependent functions.

Lactylproteome analysis indicates histone H4K12 lactylation as a novel biomarker in triple-negative breast cancer.

Objective: The established link between posttranslational modifications of histone and non-histone lysine (K) residues in cell metabolism, and their role in cancer progression, is well-documented. However, the lactylation expression signature in triple-negative breast cancer (TNBC) remains underexplored. Methods: We conducted a comprehensive lactylproteome profiling of eight pairs of TNBC samples and their matched adjacent tissues. This was achieved through 4-Dimensional label-free quantitative proteomics combined with lactylation analysis (4D-LFQP-LA). The expression of identified lactylated proteins in TNBC was detected using immunoblotting and immunohistochemistry (IHC) with specific primary antibodies, and their clinicopathological and prognostic significance was evaluated. Results: Our analysis identified 58 lactylation sites on 48 proteins, delineating the protein lactylation alteration signature in TNBC. Bioinformatic and functional analyses indicated that these lactylated proteins play crucial roles in regulating key biological processes in TNBC. Notably, lactylation of lysine at position 12 (H4K12lac) in the histone H4 domain was found to be upregulated in TNBC. Further investigations showed a high prevalence of H4K12lac upregulation in TNBC, with positive rates of 93.19% (137/147) and 92.93% (92/99) in TNBC tissue chip and validation cohorts, respectively. H4K12lac expression correlated positively with Ki-67 and inversely with overall survival (OS) in TNBC (HR [hazard ratio] =2.813, 95%CI [credibility interval]: 1.242-6.371, P=0.0164), suggesting its potential as an independent prognostic marker (HR=3.477, 95%CI: 1.324-9.130, P=0.011). Conclusions: Lactylation is a significant post-translational modification in TNBC proteins. H4K12lac emerges as a promising biomarker for TNBC, offering insights into the lactylation profiles of TNBC proteins and linking histone modifications to clinical implications in TNBC.

YOD1 protects against MRSA sepsis-induced DIC through Lys33-linked deubiquitination of NLRP3.

Disseminated intravascular coagulation (DIC) is considered to be the most common and lethal complication of sepsis. NLR-family pyrin domain-containing-3 (NLRP3) inflammasome plays an important role in host defense against microbial pathogens, and its deregulation may cause coagulation cascade and should be strictly managed. Here, we identified the deubiquitinase YOD1, which played a vital role in regulating coagulation in a NLRP3 inflammasome-dependent manner in sepsis induced by methicillin-resistant Staphylococcus aureus (MRSA). YOD1 interacted with NLRP3 to remove K33-linked ubiquitination of NLRP3 based on its deubiquitinating enzyme activity and specifically inhibited expression of NLRP3 as well as activation of NLRP3 inflammasome. Deficiency of YOD1 expression enhanced NLRP3 inflammasome activation and coagulation both in vitro and in vivo. In addition, pharmacological inhibition of the NLRP3 effectively improved coagulation and alleviated organ injury in Yod1-/- mice infected with MRSA. Thus, our study reported that YOD1 is a key regulator of coagulation during MRSA infection, and provided YOD1 as a potential therapeutic target for the treatment of NLRP3 inflammasome-related diseases, especially MRSA sepsis-induced DIC.

High histone crotonylation modification in bovine fibroblasts promotes cell proliferation and the developmental efficiency of preimplantation nuclear transfer embryos.

Lysine crotonylation (Kcr) is a recently discovered histone acylation modification that is closely associated with gene expression, cell proliferation, and the maintenance of stem cell pluripotency and indicates the transcriptional activity of genes and the regulation of various biological processes. During cell culture, the introduction of exogenous croconic acid disodium salt (Nacr) has been shown to modulate intracellular Kcr levels. Although research on Kcr has increased, its role in cell growth and proliferation and its potential regulatory mechanisms remain unclear compared to those of histone methylation and acetylation. Our investigation demonstrated that the addition of 5 mM Nacr to cultured bovine fibroblasts increased the expression of genes associated with Kcr modification, ultimately promoting cell growth and stimulating cell proliferation. Somatic cell nuclear transfer of donor cells cultured in 5 mM Nacr resulted in 38.1% blastocyst development, which was significantly greater than that in the control group (25.2%). This research is important for elucidating the crotonylation modification mechanism in fibroblast proliferation to promote the efficacy of somatic cell nuclear transfer.

Influence of microorganisms on flavor substances and functional components of sojae semen praeparatum during fermentation: A study integrating comparative metabolomics and high-throughput sequencing.

Sojae semen praeparatum (SSP), a fermented product known for its distinctive flavor and medicinal properties, undergoes a complex fermentation process due to the action of various microorganisms. Despite its widespread use, the effect of these microorganisms on the flavor compounds and functional components of SSP remains poorly understood. This study aimed to shed light on this aspect by identifying 20 metabolites as potential key flavor substances in SSP. Moreover, glycine and lysine were identified as crucial flavor substances. Additionally, 24 metabolites were identified as key functional components. The dominant microorganisms involved in the fermentation process were examined, revealing six genera of fungi and 12 genera of bacteria. At the species level, 16 microorganisms were identified as dominant through metagenome sequencing. Spearman correlation analysis demonstrated a strong association between dominant microorganisms and both flavor substances and functional components. Furthermore, the study validated the significance of four core functional microorganisms in improving the flavor and quality of SSP. This comprehensive exploration of functional microorganisms of SSP on key flavor substances/functional components during SSP fermentation. The study findings serve as a valuable reference for enhancing the overall flavor and quality of SSP.

The crotonylated and succinylated proteins of jujube involved in phytoplasma-stress responses.

BACKGROUND: Protein posttranslational modifications (PTMs) are fast and early responses to environmental changes, including pathogen infection. Jujube witches' broom (JWB) is a phytoplasma disease causing great economic loss in jujube production. After phytoplasma infection, the transcriptional, translational, and metabolic levels in jujube were activated, enabling it to survive during phytoplasma invasion. However, no study has yet reported on PTMs in jujube. Lysine crotonylation (Kcr) and lysine succinylation (Ksu) have been popular studies in recent years and their function in plant phytoplasma-stress responses remains unclear. RESULTS: Here, 1656 crotonylated and 282 succinylated jujube proteins were first identified under phytoplasma-stress, of which 198 were simultaneously crotonylated and succinylated. Comparative analysis revealed that 656 proteins, 137 crotonylated and 43 succinylated proteins in jujube were regulated by phytoplasma infection, suggesting that Kcr was more universal than Ksu. Kcr differentially expressed proteins (DEPs) were related to ribosomes, photosynthetic and carbon metabolism, while Ksu DEPs were mainly involved in carbon metabolism, the TCA cycle and secondary metabolite biosynthesis. The crosstalk network among proteome, crotonylome and succinylome showed that DEPs related to ribosomal, peroxidases and glutathione redox were enriched. Among them, ZjPOD51 and ZjPHGPX2 significantly increased at the protein and Kcr level under phytoplasma-stress. Notably, 7 Kcr sites were identified in ZjPHGPX2, a unique antioxidant enzyme. After inhibitor nicotinamide (NAM) treatment, GPX enzyme activity in jujube seedlings was reduced. Further, site-directed mutagenesis of key Kcr modification sites K130 and/or K135 in ZjPHGPX2 significantly reduced its activity. CONCLUSIONS: This study firstly provided large-scale datasets of Kcr and Ksu in phytoplasma-infected jujube and revealed that Kcr modification in ZjPHGPX2 positively regulates its activity.

K48- and K63-linked ubiquitin chain interactome reveals branch- and length-specific ubiquitin interactors.

The ubiquitin (Ub) code denotes the complex Ub architectures, including Ub chains of different lengths, linkage types, and linkage combinations, which enable ubiquitination to control a wide range of protein fates. Although many linkage-specific interactors have been described, how interactors are able to decode more complex architectures is not fully understood. We conducted a Ub interactor screen, in humans and yeast, using Ub chains of varying lengths, as well as homotypic and heterotypic branched chains of the two most abundant linkage types-lysine 48-linked (K48) and lysine 63-linked (K63) Ub. We identified some of the first K48/K63-linked branch-specific Ub interactors, including histone ADP-ribosyltransferase PARP10/ARTD10, E3 ligase UBR4, and huntingtin-interacting protein HIP1. Furthermore, we revealed the importance of chain length by identifying interactors with a preference for Ub3 over Ub2 chains, including Ub-directed endoprotease DDI2, autophagy receptor CCDC50, and p97 adaptor FAF1. Crucially, we compared datasets collected using two common deubiquitinase inhibitors-chloroacetamide and N-ethylmaleimide. This revealed inhibitor-dependent interactors, highlighting the importance of inhibitor consideration during pulldown studies. This dataset is a key resource for understanding how the Ub code is read.

An integrated cofactor and co-substrate recycling pathway for the biosynthesis of 1,5-pentanediol.

BACKGROUND: 1,5-pentanediol (1,5-PDO) is a linear diol with an odd number of methylene groups, which is an important raw material for polyurethane production. In recent years, the chemical methods have been predominantly employed for synthesizing 1,5-PDO. However, with the increasing emphasis on environmentally friendly production, it has been a growing interest in the biosynthesis of 1,5-PDO. Due to the limited availability of only three reported feasible biosynthesis pathways, we developed a new biosynthetic pathway to form a cell factory in Escherichia coli to produce 1,5-PDO. RESULTS: In this study, we reported an artificial pathway for the synthesis of 1,5-PDO from lysine with an integrated cofactor and co-substrate recycling and also evaluated its feasibility in E.coli. To get through the pathway, we first screened aminotransferases originated from different organisms to identify the enzyme that could successfully transfer two amines from cadaverine, and thus GabT from E. coli was characterized. It was then cascaded with lysine decarboxylase and alcohol dehydrogenase from E. coli to achieve the whole-cell production of 1,5-PDO from lysine. To improve the whole-cell activity for 1,5-PDO production, we employed a protein scaffold of EutM for GabT assembly and glutamate dehydrogenase was also validated for the recycling of NADPH and α-ketoglutaric acid (α-KG). After optimizing the cultivation and bioconversion conditions, the titer of 1,5-PDO reached 4.03 mM. CONCLUSION: We established a novel pathway for 1,5-PDO production through two consecutive transamination reaction from cadaverine, and also integrated cofactor and co-substrate recycling system, which provided an alternative option for the biosynthesis of 1,5-PDO.

Histone Lactylation Is Involved in Mouse Oocyte Maturation and Embryo Development.

Numerous post-translational modifications are involved in oocyte maturation and embryo development. Recently, lactylation has emerged as a novel epigenetic modification implicated in the regulation of diverse cellular processes. However, it remains unclear whether lactylation occurs during oocyte maturation and embryo development processes. Herein, the lysine lactylation (Kla) modifications were determined during mouse oocyte maturation and early embryo development by immunofluorescence staining. Exogenous lactate was supplemented to explore the consequences of modulating histone lactylation levels on oocyte maturation and embryo development processes by transcriptomics. Results demonstrated that lactylated proteins are widely present in mice with tissue- and cell-specific distribution. During mouse oocyte maturation, immunofluorescence for H3K9la, H3K14la, H4K8la, and H4K12la was most intense at the germinal vesicle (GV) stage and subsequently weakened or disappeared. Further, supplementing the culture medium with 10 mM sodium lactate elevated both the oocyte maturation rate and the histone Kla levels in GV oocytes, and there were substantial increases in Kla levels in metaphase II (MII) oocytes. It altered the transcription of molecules involved in oxidative phosphorylation. Moreover, histone lactylation levels changed dynamically during mouse early embryogenesis. Sodium lactate at 10 mM enhanced early embryo development and significantly increased lactylation, while impacting glycolytic gene transcription. This study reveals the roles of lactylation during oocyte maturation and embryo development, providing new insights to improving oocyte maturation and embryo quality.

Macrophage MCT4 inhibition activates reparative genes and protects from atherosclerosis by histone H3 lysine 18 lactylation.

Macrophage activation is a hallmark of atherosclerosis, accompanied by a switch in core metabolism from oxidative phosphorylation to glycolysis. The crosstalk between metabolic rewiring and histone modifications in macrophages is worthy of further investigation. Here, we find that lactate efflux-associated monocarboxylate transporter 4 (MCT4)-mediated histone lactylation is closely related to atherosclerosis. Histone H3 lysine 18 lactylation dependent on MCT4 deficiency activated the transcription of anti-inflammatory genes and tricarboxylic acid cycle genes, resulting in the initiation of local repair and homeostasis. Strikingly, histone lactylation is characteristically involved in the stage-specific local repair process during M1 to M2 transformation, whereas histone methylation and acetylation are not. Gene manipulation and protein hydrolysis-targeted chimerism technology are used to confirm that MCT4 deficiency favors ameliorating atherosclerosis. Therefore, our study shows that macrophage MCT4 deficiency, which links metabolic rewiring and histone modifications, plays a key role in training macrophages to become repair and homeostasis phenotypes.

Polyphosphate attachment to lysine repeats is a non-covalent protein modification.

Polyphosphate (polyP) is a chain of inorganic phosphate that is present in all domains of life and affects diverse cellular phenomena, ranging from blood clotting to cancer. A study by Azevedo et al. described a protein modification whereby polyP is attached to lysine residues within polyacidic serine and lysine (PASK) motifs via what the authors claimed to be covalent phosphoramidate bonding. This was based largely on the remarkable ability of the modification to survive extreme denaturing conditions. Our study demonstrates that lysine polyphosphorylation is non-covalent, based on its sensitivity to ionic strength and lysine protonation and absence of phosphoramidate bond formation, as analyzed via 31P NMR. Ionic interaction with lysine residues alone is sufficient for polyP modification, and we present a new list of non-PASK lysine repeat proteins that undergo polyP modification. This work clarifies the biochemistry of polyP-lysine modification, with important implications for both studying and modulating this phenomenon. This Matters Arising paper is in response to Azevedo et al. (2015), published in Molecular Cell. See also the Matters Arising Response by Azevedo et al. (2024), published in this issue.

On the covalent nature of lysine polyphosphorylation.

Post-translational modifications of proteins (PTMs) introduce an extra layer of complexity to cellular regulation. Although phosphorylation of serine, threonine, and tyrosine residues is well-known as PTMs, lysine is, in fact, the most heavily modified amino acid, with over 30 types of PTMs on lysine having been characterized. One of the most recently discovered PTMs on lysine residues is polyphosphorylation, which sees linear chains of inorganic polyphosphates (polyP) attached to lysine residues. The labile nature of phosphoramidate bonds raises the question of whether this modification is covalent in nature. Here, we used buffers with very high ionic strength, which would disrupt any non-covalent interactions, and confirmed that lysine polyphosphorylation occurs covalently on proteins containing PASK domains (polyacidic, serine-, and lysine-rich), such as the budding yeast protein nuclear signal recognition 1 (Nsr1) and the mammalian protein nucleolin. This Matters Arising Response paper addresses the Neville et al. (2024) Matters Arising paper, published concurrently in Molecular Cell.

MitoTempo protects against nε-carboxymethyl lysine-induced mitochondrial dyshomeostasis and neuronal cells injury.

Enhanced formation of advanced glycation end products (AGEs) is a pivotal factor in diabetes pathophysiology, increasing the risk of diabetic complications. Nε-carboxy-methyl-lysine (CML) is one of the most relevant AGEs found in several tissues including the peripheral blood of diabetic subjects. Despite recognizing diabetes as a risk factor for neurodegenerative diseases and the documented role of mitochondrial abnormalities in this connection, the impact of CML on neuronal mitochondria and its contribution to diabetes-related neurodegeneration remain uncertain. Here, we evaluated the effects of CML in differentiated SH-SY5Y human neuroblastoma cells. Due to the association between mitochondrial dysfunction and increased production of reactive oxygen species (ROS), the possible protective effects of MitoTempo, a mitochondria-targeted antioxidant, were also evaluated. Several parameters were assessed namely cells viability, mitochondrial respiration and membrane potential, ATP and ROS production, Ca2+ levels, mitochondrial biogenesis and dynamics, mito/autophagy, endoplasmic reticulum (ER) stress and amyloidogenic and synaptic integrity markers. CML caused pronounced mitochondrial defects characterized by a significant decrease in mitochondrial respiration, membrane potential, and ATP production and an increase in ROS production. An accumulation of individual mitochondria associated with disrupted mitochondrial networks was also observed. Furthermore, CML caused mitochondrial fusion and a decrease in mitochondrial mass and induced ER stress associated with altered unfolded protein response and Ca2+ dyshomeostasis. Moreover, CML increased the protein levels of ß-secretase-1 and amyloid precursor protein, key proteins involved in Alzheimer's Disease pathophysiology. All these effects contributed to the decline in neuronal cells viability. Notable, MitoTempo was able to counteract most of CML-mediated mitochondrial defects and neuronal cells injury and death. Overall, these findings suggest that CML induces pronounced defects in neuronal mitochondria and ER stress, predisposing to neurodegenerative events. More, our observations suggest that MitoTempo holds therapeutic promise in mitigating CML-induced mitochondrial imbalance and neuronal damage and death.

New Insights into the Genetic Basis of Lysine Accumulation in Rice Revealed by Multi-Model GWAS.

Lysine is an essential amino acid that cannot be synthesized in humans. Rice is a global staple food for humans but has a rather low lysine content. Identification of the quantitative trait nucleotides (QTNs) and genes underlying lysine content is crucial to increase lysine accumulation. In this study, five grain and three leaf lysine content datasets and 4,630,367 single nucleotide polymorphisms (SNPs) of 387 rice accessions were used to perform a genome-wide association study (GWAS) by ten statistical models. A total of 248 and 71 common QTNs associated with grain/leaf lysine content were identified. The accuracy of genomic selection/prediction RR-BLUP models was up to 0.85, and the significant correlation between the number of favorable alleles per accession and lysine content was up to 0.71, which validated the reliability and additive effects of these QTNs. Several key genes were uncovered for fine-tuning lysine accumulation. Additionally, 20 and 30 QTN-by-environment interactions (QEIs) were detected in grains/leaves. The QEI-sf0111954416 candidate gene LOC_Os01g21380 putatively accounted for gene-by-environment interaction was identified in grains. These findings suggested the application of multi-model GWAS facilitates a better understanding of lysine accumulation in rice. The identified QTNs and genes hold the potential for lysine-rich rice with a normal phenotype.

KbhbXG: A Machine learning architecture based on XGBoost for prediction of lysine ß-Hydroxybutyrylation (Kbhb) modification sites.

Lysine ß-hydroxybutyrylation is an important post-translational modification (PTM) involved in various physiological and biological processes. In this research, we introduce a novel predictor KbhbXG, which utilizes XGBoost to identify ß-hydroxybutyrylation modification sites based on protein sequence information. The traditional experimental methods employed for the identification of ß-hydroxybutyrylated sites using proteomic techniques are both costly and time-consuming. Thus, the development of computational methods and predictors can play a crucial role in facilitating the rapid identification of ß-hydroxybutyrylation sites. Our proposed KbhbXG model first utilizes machine learning algorithm XGBoost to predict ß-hydroxybutyrylation modification sites. On the independent test set, KbhbXG achieves an accuracy of 0.7457, specificity of 0.7771, and an impressive area under the curve (AUC) score of 0.8172. The high AUC score achieved by our method demonstrates its potential for effectively identifying novel ß-hydroxybutyrylation sites, thereby facilitating further research and exploration of the ß-hydroxybutyrylation process. Also, functional analyses have revealed that different organisms preferentially engage in distinct biological processes and pathways, which can provide valuable insights for understanding the mechanism of ß-hydroxybutyrylation and guide experimental verification. To promote transparency and reproducibility, we have made both the codes and dataset of KbhbXG publicly available. Researchers interested in utilizing our proposed model can access these resources at https://github.com/Lab-Xu/KbhbXG.

Predicting lysine methylation sites using a convolutional neural network.

Protein lysine methylation is a particular type of post translational modification that plays an important role in both histone and non-histone function regulation in proteins. Deregulation caused by lysine methyltransferases has been identified as the cause of several diseases including cancer as well as both mental and developmental disorders. Identifying lysine methylation sites is a critical step in both early diagnosis and drug design. This study proposes a new Machine Learning method called CNN-Meth for predicting lysine methylation sites using a convolutional neural network (CNN). Our model is trained using evolutionary, structural, and physicochemical-based presentation along with binary encoding. Unlike previous studies, instead of extracting handcrafted features, we use CNN to automatically extract features from different presentations of amino acids to avoid information loss. Automated feature extraction from these representations of amino acids as well as CNN as a classifier have never been used for this problem. Our results demonstrate that CNN-Meth can significantly outperform previous methods for predicting methylation sites. It achieves 96.0%, 85.1%, 96.4%, and 0.65 in terms of Accuracy, Sensitivity, Specificity, and Matthew's Correlation Coefficient (MCC), respectively. CNN-Meth and its source code are publicly available at https://github.com/MLBC-lab/CNN-Meth.

An Active Site Tyr Residue Guides the Regioselectivity of Lysine Hydroxylation by Nonheme Iron Lysine-4-hydroxylase Enzymes through Proton-Coupled Electron Transfer.

Lysine dioxygenase (KDO) is an important enzyme in human physiology involved in bioprocesses that trigger collagen cross-linking and blood pressure control. There are several KDOs in nature; however, little is known about the factors that govern the regio- and stereoselectivity of these enzymes. To understand how KDOs can selectively hydroxylate their substrate, we did a comprehensive computational study into the mechanisms and features of 4-lysine dioxygenase. In particular, we selected a snapshot from the MD simulation on KDO5 and created large QM cluster models (A, B, and C) containing 297, 312, and 407 atoms, respectively. The largest model predicts regioselectivity that matches experimental observation with rate-determining hydrogen atom abstraction from the C4-H position, followed by fast OH rebound to form 4-hydroxylysine products. The calculations show that in model C, the dipole moment is positioned along the C4-H bond of the substrate and, therefore, the electrostatic and electric field perturbations of the protein assist the enzyme in creating C4-H hydroxylation selectivity. Furthermore, an active site Tyr233 residue is identified that reacts through proton-coupled electron transfer akin to the axial Trp residue in cytochrome c peroxidase. Thus, upon formation of the iron(IV)-oxo species in the catalytic cycle, the Tyr233 phenol loses a proton to the nearby Asp179 residue, while at the same time, an electron is transferred to the iron to create an iron(III)-oxo active species. This charged tyrosyl residue directs the dipole moment along the C4-H bond of the substrate and guides the selectivity to the C4-hydroxylation of the substrate.

The histone lysine acetyltransferase KAT2B inhibits cholangiocarcinoma growth: evidence for interaction with SP1 to regulate NF2-YAP signaling.

BACKGROUND: Cholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tract with poor prognosis. Further mechanistic insights into the molecular mechanisms of CCA are needed to develop more effective target therapy. METHODS: The expression of the histone lysine acetyltransferase KAT2B in human CCA was analyzed in human CCA tissues. CCA xenograft was developed by inoculation of human CCA cells with or without KAT2B overexpression into SCID mice. Western blotting, ChIP-qPCR, qRT-PCR, protein immunoprecipitation, GST pull-down and RNA-seq were performed to delineate KAT2B mechanisms of action in CCA. RESULTS: We identified KAT2B as a frequently downregulated histone acetyltransferase in human CCA. Downregulation of KAT2B was significantly associated with CCA disease progression and poor prognosis of CCA patients. The reduction of KAT2B expression in human CCA was attributed to gene copy number loss. In experimental systems, we demonstrated that overexpression of KAT2B suppressed CCA cell proliferation and colony formation in vitro and inhibits CCA growth in mice. Mechanistically, forced overexpression of KAT2B enhanced the expression of the tumor suppressor gene NF2, which is independent of its histone acetyltransferase activity. We showed that KAT2B was recruited to the promoter region of the NF2 gene via interaction with the transcription factor SP1, which led to enhanced transcription of the NF2 gene. KAT2B-induced NF2 resulted in subsequent inhibition of YAP activity, as reflected by reduced nuclear accumulation of oncogenic YAP and inhibition of YAP downstream genes. Depletion of NF2 was able to reverse KAT2B-induced reduction of nuclear YAP and subvert KAT2B-induced inhibition of CCA cell growth. CONCLUSIONS: This study provides the first evidence for an important tumor inhibitory effect of KAT2B in CCA through regulation of NF2-YAP signaling and suggests that this signaling cascade may be therapeutically targeted for CCA treatment.