Effects of mitochondrial dysfunction on cellular function: Role in atherosclerosis.

Atherosclerosis, an immunoinflammatory disease of medium and large arteries, is associated with life-threatening clinical events, such as acute coronary syndromes and stroke. Chronic inflammation and impaired lipoprotein metabolism are considered to be among the leading causes of atherosclerosis, while numerous risk factors, including arterial hypertension, diabetes mellitus, obesity, and aging, can contribute to the development of the disease. In recent years, emerging evidence has underlined the key role of mitochondrial dysfunction in the pathogenesis of atherosclerosis. Mitochondrial dysfunction is believed to result in an increase in reactive oxygen species, leading to oxidative stress, chronic inflammation, and intracellular lipid deposition, all of which can contribute to the pathogenesis of atherosclerosis. Critical cells, including endothelial cells, vascular smooth muscle cells, and macrophages, play an important role in atherosclerosis. Mitochondrial function is also involved in maintaining the normal function of these cells. To better understand the relationship between mitochondrial dysfunction and atherosclerosis, this review summarizes the findings of recent studies and discusses the role of mitochondrial dysfunction in the risk factors and critical cells of atherosclerosis. FACTS: OPEN QUESTIONS.

Structural and mechanistic investigations on CC bond forming α-oxoamine synthase allowing L-glutamate as substrate.

Carbon­carbon (C-C) bonds serve as the fundamental structural backbone of organic molecules. As a critical CC bond forming enzyme, α-oxoamine synthase is responsible for the synthesis of α-amino ketones by performing the condensation reaction between amino acids and acyl-CoAs. We previously identified an α-oxoamine synthase (AOS), named as Alb29, involved in albogrisin biosynthesis in Streptomyces albogriseolus MGR072. This enzyme belongs to the α-oxoamine synthase family, a subfamily under the pyridoxal 5'-phosphate (PLP) dependent enzyme superfamily. In this study, we report the crystal structures of Alb29 bound to PLP and L-Glu, which provide the atomic-level structural insights into the substrate recognition by Alb29. We discover that Alb29 can catalyze the amino transformation from L-Gln to L-Glu, besides the condensation of L-Glu with ß-methylcrotonyl coenzyme A. Subsequent structural analysis has revealed that one flexible loop in Alb29 plays an important role in both amino transformation and condensation. Based on the crystal structure of the S87G mutant in the loop region, we capture two distinct conformations of the flexible loop in the active site, compared with the wild-type Alb29. Our study offers valuable insights into the catalytic mechanism underlying substrate recognition of Alb29.

Combined inhibition of HER2 and VEGFR synergistically improves therapeutic efficacy via PI3K-AKT pathway in advanced ovarian cancer.

BACKGROUND: Ovarian cancer (OC) is a prevalent malignancy in the female reproductive system, and developing effective targeted therapies for this disease remains challenging. The aim of this study was to use clinically-relevant OC models to evaluate the therapeutic effectiveness of RC48, an antibody-drug conjugate (ADC) targeting HER2, either alone or in combination with the VEGFR inhibitor Cediranib Maleate (CM), for the treatment of advanced OC. METHODS: OC tumor specimens and cell lines were analyzed to determine HER2 and VEGFR expression by Western blot, immunocytochemistry and immunofluorescence. Moreover, the OC cell lines, cell-derived xenograft (CDX) and patient-derived xenograft (PDX) models were treated with RC48 and/or CM and then subjected to cell proliferation, viability, apoptosis, and tumor growth analyses to evaluate the feasibility of combination therapy for OC both in vitro and in vivo. Additionally, RNA-Seq was performed to investigate the critical mechanism underlying the combination therapy of RC48 and CM. RESULTS: Our results demonstrated that RC48 alone effectively targeted and inhibited the growth of HER2-positive OC tumors in both cell lines and PDX models. Furthermore, the combination of RC48 and CM synergistically induced tumor regression in human OC cell lines, as well as CDX and PDX models. Mechanistically, we observed that the combination treatment inhibited the growth of OC cells involved inducing apoptosis and suppressing cell motility. RNA-seq analysis provided further mechanistic insights and revealed that co-administration of RC48 and CM downregulated multiple cancer-related pathways, including the AKT/mTOR pathway, cell cycle, and cell proliferation. Notably, our data further confirmed that the PI3K-AKT pathway played a key role in the inhibition of proliferation triggered by combinational treatment of RC48 and CM in OC cells. CONCLUSIONS: These findings provide a preclinical framework supporting the potential of dual targeting HER2 and VEGFR as a promising therapeutic strategy to improve outcomes in patients with OC.

The emerging role of ectodermal neural cortex 1 in cancer.

Ectodermal neural cortex 1 (ENC1) is a protein that plays a crucial role in the regulation of various cellular processes such as cell proliferation, differentiation, and apoptosis. Numerous studies have shown that ENC1 is overexpressed in various types of cancers, including breast, lung, pancreatic, and colorectal cancer, and its upregulation is correlated with a poorer prognosis. In addition to its role in cancer growth and spreading, ENC1 has also been linked to neuronal process development and neural crest cell differentiation. In this review, we provide an overview of the current knowledge on the relationship between ENC1 and cancer. We discuss the molecular mechanisms by which ENC1 contributes to tumorigenesis, including its involvement in multiple oncogenic signaling pathways. We also summarize the potential of targeting ENC1 for cancer therapy, as its inhibition has been shown to significantly reduce cancer cell invasion, growth, and metastasis. Finally, we highlight the remaining gaps in our understanding of ENC1's role in cancer and propose potential directions for future research.

Upregulated ENC1 predicts unfavorable prognosis and correlates with immune infiltration in endometrial cancer.

A better knowledge of the molecular process behind uterine corpus endometrial carcinoma (UCEC) is important for prognosis prediction and the development of innovative targeted gene therapies. The purpose of this research is to discover critical genes associated with UCEC. We analyzed the gene expression profiles of TCGA-UCEC and GSE17025, respectively, using Weighted Gene Co-expression Network Analysis (WGCNA) and differential gene expression analysis. From four sets of findings, a total of 95 overlapping genes were retrieved. On the 95 overlapping genes, KEGG pathway and GO enrichment analysis were conducted. Then, we mapped the PPI network of 95 overlapping genes using the STRING database. Twenty hub genes were evaluated using the Cytohubba plugin, including NR3C1, ATF3, KLF15, THRA, NR4A1, FOSB, PER3, HLF, NTRK3, EGR3, MAPK13, ARNTL2, PKM2, SCD, EIF5A, ADHFE1, RERGL, TUB, and ENC1. The expression levels of NR3C1, PKM2, and ENC1 were shown to be adversely linked with the survival time of UCEC patients using univariate Cox regression analysis and Kaplan-Meier survival calculation. ENC1 were also overexpressed in UCEC tumor tissues or cell lines, as shown by quantitative real-time PCR and Western blotting. Then we looked into it further and discovered that ENC1 expression was linked to tumor microenvironment and predicted various immunological checkpoints. In conclusion, our data indicate that ENC1 may be required for the development of UCEC and may serve as a future biomarker for diagnosis and therapy.

PAD4 silencing inhibits inflammation whilst promoting trophoblast cell invasion and migration by inactivating the NEMO/NF-κB pathway.

Preeclampsia (PE), presenting with onset hypertension and proteinuria, is a pregnancy-specific disorder that can result in maternal and fetal morbidity and mortality. Insufficient trophoblast invasion and migration has been considered to be an important cause of this disease. The present study aimed to investigate the role of peptidyl arginine deiminase 4 (PAD4), whose knockdown has been previously indicated to reduce inflammation and susceptibility to pregnancy loss in mice, in the development of PE in vitro. Lipopolysaccharide (LPS) was used to treat a human trophoblast cell line (HTR8/SVneo). After PAD4 silencing via transfection with short hairpin RNA against PAD4, the concentrations of inflammatory factors IL-6, IL-12 and monocyte chemoattractant protein (MCP)-1 were measured using ELISA. Cell viability was also measured using Cell Counting Kit-8 assay. HTR8/SVneo cell invasion and migration were detected using Transwell and wound healing assays, respectively. Western blotting was used to measure the expression of citrullinated NF-κB essential modulator (NEMO) and nuclear NF-κB p65 protein levels. TNF-α was applied for evaluating the potential regulatory effects of PAD4 on NF-κB in LPS-stimulated HTR8/SVneo cells. LPS increased the levels of IL-6, IL-12 and MCP-1 and reduced the migration and invasion of HTR8/SVneo cells. PAD4-knockdown was found to markedly reduce the levels of IL-6, IL-12 and MCP-1 secretion. HTR8/SVneo cell invasion and migration was also significantly elevated after PAD4 silencing following LPS exposure. In addition, LPS stimulation notably upregulated the protein levels of citrullinated NEMO and nuclear NF-κB p65, which was restored by PAD4 knockdown. Furthermore, TNF-α treatment partially counteracted the effects of PAD4 knockdown on the secretion of IL-6, MCP-1 and IL-12, which are markers of inflammation, and invasion and migration in LPS-induced HTR8/SVneo cells. To conclude, these results suggest that PAD4 silencing can suppress inflammation whilst promoting invasion and migration by trophoblast cells through inhibiting the NEMO/NF-κB pathway. These findings furthered the understanding in the complex molecular mechanism that can trigger PE and provide a promising target for the treatment of this disease.

Effects of betaine on non-alcoholic liver disease.

The increasing prevalence of non-alcoholic fatty liver disease (NAFLD) poses a growing challenge in terms of its prevention and treatment. The 'multiple hits' hypothesis of multiple insults, such as dietary fat intake, de novo lipogenesis, insulin resistance, oxidative stress, mitochondrial dysfunction, gut dysbiosis and hepatic inflammation, can provide a more accurate explanation of the pathogenesis of NAFLD. Betaine plays important roles in regulating the genes associated with NAFLD through anti-inflammatory effects, increased free fatty oxidation, anti-lipogenic effects and improved insulin resistance and mitochondrial function; however, the mechanism of betaine remains elusive.

Identification of an α-Oxoamine Synthase and a One-Pot Two-Step Enzymatic Synthesis of α-Amino Ketones.

Alb29, an α-oxoamine synthase involved in albogrisin biosynthesis in Streptomyces albogriseolus MGR072, was characterized and responsible for the incorporation of l-glutamate to acyl-coenzyme A substrates. Combined with Alb29 and Mgr36 (an acyl-coenzyme A ligase), a one-pot enzymatic system was established to synthesize seven α-amino ketones. When these α-amino ketones were fed into the alb29 knockout strain Δalb29, respectively, the albogrisin analogs with different side chains were observed.

Constructing Microbial Hosts for the Production of Benzoheterocyclic Derivatives.

Natural products containing benzoheterocyclic skeletons are widely found in plants and exhibit various pharmacological activities. To address the current limited availability of these compounds, we herein demonstrate the production of benzopyran, furanocoumarins, and pyranocoumarins in Streptomyces xiamenensis by employing prenyltransferases and two substrate-promiscuous enzymes, XimD and XimE. To avoid the degradation in S. xiamenensis, furanocoumarins and pyranocoumarins were also successfully produced in Escherichia coli. The production of linear furanocoumarins (marmesin) and angular pyranocoumarins (decursinol) reached 3.6 and 3.7 mg/L in shake flasks, respectively. To the best of our knowledge, this is the first report of the microbial production of the plant metabolites furanocoumarins and pyranocoumarins. Our study complements the missing link in the biosynthesis of pyranocoumarins by leveraging the catalytic promiscuity of microbial enzymes.

Ikarugamycin inhibits pancreatic cancer cell glycolysis by targeting hexokinase 2.

Mangrove-derived actinobacteria strains are well-known for producing novel secondary metabolites. The polycyclic tetramate macrolactam (PTM), ikarugamycin (IKA) isolated from Streptomyces xiamenensis 318, exhibits antiproliferative activities against pancreatic ductal adenocarcinoma (PDAC) in vitro. However, the protein target for bioactive IKA is unclear. In this study, whole transcriptome-based profiling revealed that the glycolysis pathway is significantly affected by IKA. Metabolomic studies demonstrated that IKA treatment induces a significant drop in glucose-6-phosphate and a slight increase in intracellular glucose level. Analysis of glucose consumption, lactate production, and the extracellular acidification rate confirmed the inhibitory role of IKA on the glycolytic flux in PDAC cells. Surface plasmon resonance (SPR) experiments and docking studies identified the key enzyme of glycolysis, hexokinase 2 (HK2), as a molecular target of IKA. Moreover, IKA reduced tumor size without overt cytotoxicity in mice with PDAC xenografts and increased chemotherapy response to gemcitabine in PDAC cells in vitro. Taken together, IKA can block glycolysis in pancreatic cancer by targeting HK2, which may be a potential drug candidate for PDAC treatment.

New alkylated benzoquinones from mangrove plant Aegiceras corniculatum with anticancer activity.

Three new alkylated benzoquinones, 2-hydroxy-5-ethoxy-3-nonyl-1,4-benzoquinone (1), 5-O-butyl-embelin (2), and 2,5-dihydroxy-6-methyl-3-pentadecyl-1,4-benzoquinone (3), together with seven known analogues (4-10), were isolated from the stems and twigs of mangrove plant, Aegiceras corniculatum. Their structural elucidation was achieved by spectroscopic methods, chemical exchanging experiments, and semisynthesis method. The cytotoxic activities of all the isolated compounds were evaluated by MTT assay. Compounds 1, 2, 8, 9, and 10 possess varying degrees of selective cytotoxicity against HL-60, HepG2, BGC-823, and A2780 cell lines.[Formula: see text].

Three transcriptional regulators positively regulate the biosynthesis of polycyclic tetramate macrolactams in Streptomyces xiamenensis 318.

Polycyclic tetramate macrolactams (PTMs) are a widely distributed class of structurally complex natural products, and most of them exhibit multiple biological activities. However, the transcriptional regulators (TRs) involved in the regulation of PTM production have seldom been reported. Here, we identified three TRs, i.e., Sxim_22880, CvnABCSx, and WblASx, and revealed their positive roles in the regulation of PTM biosynthesis in mangrove-derived Streptomyces xiamenensis 318. This strain produces a considerable amount of PTMs at 30 °C, but the production of PTMs is mostly blocked at 37 °C. Quantitative real-time PCR analysis confirmed that the transcriptions of PTM biosynthetic genes were downregulated. We determined that the transcriptions of several putative TRs, i.e., WblASx, Sxim_22880, and CvnCSx, were significantly downregulated under such heat-shock conditions. We showed that the transcription of PTM biosynthetic genes and the production of PTMs could be restored at 37 °C if the impaired transcriptions of wblASx, sxim_22880, and cvnABCSx were restored. Electrophoretic mobility shift assays showed that none of these TRs could bind to the promoter region of the PTM gene cluster, suggesting their indirect but positive involvement in the regulation on PTM production. Moreover, concurrent overexpression of the three TRs in S. xiamenensis 318 resulted in a 242.5% increase in PTM production when the strain was cultured at 30 °C. Furthermore, overexpression of these three TRs in Streptomyces sp. FR-008 and S. albus J1074 stimulated the production of new secondary metabolites, indicating that these conserved TRs could be used to activate cryptic secondary metabolite gene clusters in Streptomyces.

Total synthesis and anti-inflammatory evaluation of violacin A and its analogues.

A concise total synthesis of an exceedingly potent anti-inflammatory agent violacin A as well as the preparation of thirty analogues of this lead from commercially available orcinol are described. Highlights of our synthetic efforts involve Friedel-Crafts acylation, the regioselective etherification and esterification of phenolic hydroxyl groups, and Baker-Venkatamaran rearrangement to form basic skeleton of violacin A. The deprotection reaction with Pd-catalytic was involved to avoid the elimination of the hemiacetal hydroxyl at C2. In addition, all synthetic compounds were screened for anti-inflammatory activity against nitric oxide (NO) production using lipopolysaccharide (LPS)-induced Raw264.7 cells. A range of violacin A derivatives 11b, 11d, 11f, 12e, 12g, 13g, 17d-g exhibited stronger anti-inflammatory effect than that of violacin A. Notably, halogeno-benzyloxy substituent at C-7 were favourable for anti-inflammatory activities of violacin A derivatives. Additionally, Western blot results indicated halogeno-benzyloxy derivatives inhibited pro-inflammatory cytokines releases correlated with the suppression of NF-κB signaling pathway.

Rational engineering of amide synthetase enables bioconversion to diverse xiamenmycin derivatives.

XimA is a unique amide synthetase that belongs to the ANL superfamily of adenylating enzymes, but with a special structural fold. In order to improve the enzyme promiscuity, we engineered XimA by site-directed mutagenesis at a specific position based on our theoretical model of XimA. Thus, we were able to produce diverse benzopyran derivatives with up to 15 different l-form and d-form amino acid substitutions, catalyzed by several XimA variants. Molecular docking and molecular dynamics simulations conducted for various XimA systems provide further structural insights into the substitution effects of the phenylalanine-201 as an active site residue on protein dynamics and enzyme catalysis.

Characterization and Nonenzymatic Transformation of Three Types of Alkaloids from Streptomyces albogriseolus MGR072 and Discovery of Inhibitors of Indoleamine 2,3-Dioxygenase.

The known benzonaphthyridine alkaloid, albogrisin A (1), and six new compounds, including two pyrazinone stereoisomers, albogrisin B (2)/B' (2'), together with four 4H-pyrroloquinolinones, two diastereoisomers, albogrisin C (3)/C' (3'), and their methyl esters, albogrisin D (4)/D' (4'), were isolated from mangrove-derived Streptomyces albogriseolus MGR072. 2 and 2' are converted into 1 in acidic aqueous solution but into 3/3' and 4/4' in 0.05% trifluoroacetic acid acetonitrile. 4 and 4' are new indoleamine 2,3-dioxygenase 1 inhibitors.

Correction to: 'Dynamical modelling of secondary metabolism and metabolic switches in Streptomyces xiamenensis 318'.

[This corrects the article DOI: 10.1098/rsos.190418.].

Dynamical modelling of secondary metabolism and metabolic switches in Streptomyces xiamenensis 318.

The production of secondary metabolites, while important for bioengineering purposes, presents a paradox in itself. Though widely existing in plants and bacteria, they have no definite physiological roles. Yet in both native habitats and laboratories, their production appears robust and follows apparent metabolic switches. We show in this work that the enzyme-catalysed process may improve the metabolic stability of the cells. The latter can be responsible for the overall metabolic behaviours such as dynamic metabolic landscape, metabolic switches and robustness, which can in turn affect the genetic formation of the organism in question. Mangrove-derived Streptomyces xiamenensis 318, with a relatively compact genome for secondary metabolism, is used as a model organism in our investigation. Integrated studies via kinetic metabolic modelling, transcriptase measurements and metabolic profiling were performed on this strain. Our results demonstrate that the secondary metabolites increase the metabolic fitness of the organism via stabilizing the underlying metabolic network. And the fluxes directing to NADH, NADPH, acetyl-CoA and glutamate provide the key switches for the overall and secondary metabolism. The information may be helpful for improving the xiamenmycin production on the strain.

A Novel AdpA Homologue Negatively Regulates Morphological Differentiation in Streptomyces xiamenensis 318.

The pleiotropic transcriptional regulator AdpA positively controls morphological differentiation and regulates secondary metabolism in most Streptomyces species. Streptomyces xiamenensis 318 has a linear chromosome 5.96 Mb in size. How AdpA affects secondary metabolism and morphological differentiation in such a naturally minimized genomic background is unknown. Here, we demonstrated that AdpA Sx , an AdpA orthologue in S. xiamenensis, negatively regulates cell growth and sporulation and bidirectionally regulates the biosynthesis of xiamenmycin and polycyclic tetramate macrolactams (PTMs) in S. xiamenensis 318. Overexpression of the adpASx gene in S. xiamenensis 318 had negative effects on morphological differentiation and resulted in reduced transcription of putative ssgA, ftsZ, ftsH, amfC, whiB, wblA1, wblA2, wblE, and a gene encoding sporulation-associated protein (sxim_29740), whereas the transcription of putative bldD and bldA genes was upregulated. Overexpression of adpASx led to significantly enhanced production of xiamenmycin but had detrimental effects on the production of PTMs. As expected, the transcriptional level of the xim gene cluster was upregulated, whereas the PTM gene cluster was downregulated. Moreover, AdpA Sx negatively regulated the transcription of its own gene. Electrophoretic mobility shift assays revealed that AdpA Sx can bind the promoter regions of structural genes of both the xim and PTM gene clusters as well as to the promoter regions of genes potentially involved in the cell growth and differentiation of S. xiamenensis 318. We report that an AdpA homologue has negative effects on morphological differentiation in S. xiamenensis 318, a finding confirmed when AdpA Sx was introduced into the heterologous host Streptomyces lividans TK24.IMPORTANCE AdpA is a key regulator of secondary metabolism and morphological differentiation in Streptomyces species. However, AdpA had not been reported to negatively regulate morphological differentiation. Here, we characterized the regulatory role of AdpA Sx in Streptomyces xiamenensis 318, which has a naturally streamlined genome. In this strain, AdpA Sx negatively regulated cell growth and morphological differentiation by directly controlling genes associated with these functions. AdpA Sx also bidirectionally controlled the biosynthesis of xiamenmycin and PTMs by directly regulating their gene clusters rather than through other regulators. Our findings provide additional evidence for the versatility of AdpA in regulating morphological differentiation and secondary metabolism in Streptomyces.

Combinatory Biosynthesis of Prenylated 4-Hydroxybenzoate Derivatives by Overexpression of the Substrate-Promiscuous Prenyltransferase XimB in Engineered E. coli.

Prenylated aromatic compounds are important intermediates in the biosynthesis of bioactive molecules such as 3-chromanols from plants, ubiquinones from prokaryotes and meroterpenoids from sponges. Biosynthesis of prenylated aromatic compounds using prokaryotic microorganisms has attracted increasing attention in the field of synthetic biology. In this study, we demonstrated that the production of 3-geranyl-4-hydroxybenzoic acid (GBA) and a variety of GBA analogues was feasible in a metabolically engineered E. coli by using XimB, a special prenyltransferase involved in the biosynthesis of xiamenmycin A in Streptomyces xiamenensis 318. XimB exhibits broad substrate specificity and can catalyze the transfer reaction of prenyl moieties with different carbon chain lengths to both the natural substrate 4-hydroxybenzoate (4-HBA) and to different substituted 4-HBA derivatives at C-2 and C-3. Feeding 4-HBA to an engineered E. coli equipped with a hybrid mevalonate pathway increased the production of GBA up to 94.30 mg/L. Considerable amounts of other GBA derivatives, compounds 4, 5, 6, 7, and 9, can be achieved by feeding precursors. The plug-and-play design for inserting C5, C15, and C20 prenyl diphosphate synthetases under the control of the T7 promoter resulted in targeted production of 3-dimethylallyl, 3-farnesyl-, and 3-geranylgeranyl-4-hydroxybenzoic acid, respectively. Furthermore, the valuable benzopyran xiamenmycin B was successfully produced in E. coli R7-MVA by coexpression of a complete biosynthetic gene cluster, which contains ximBDE.

Ancistrocyclinones A and B, unprecedented pentacyclic N,C-coupled naphthylisoquinoline alkaloids, from the Chinese liana Ancistrocladus tectorius.

Two unique pentacyclic N,C-coupled naphthylisoquinolines, the ancistrocyclinones A (5) and B (6), were discovered in the Chinese liana Ancistrocladus tectorius. Furthermore, six known, likewise N,C-coupled alkaloids, viz., ancistrocladinium A (7a) and its mono- and bisphenolic analogs 8a and 9a were isolated, along with their atropo-diastereomers 7b, 8b, and 9b. The stereostructures of 5 and 6 were determined by HRESIMS, 1D and 2D NMR, oxidative degradation, and ECD calculations. The pentacyclic ancistrocyclinones A (5) and B (6) are structurally similar to berberine alkaloids - yet arising from a most different biosynthetic pathway: they are apparently formed by N,C-coupling of their polyketide-derived molecular halves, followed by oxidative cyclo-condensation. Biomimetic conversion of the co-occurring 4'-O-demethylancistrocladinium A (8a) to ancistrocyclinone A (5) via a quinoid intermediate supported the postulated pathway. Ancistrocyclinone A (5) was found to significantly inhibit the viability of drug-sensitive human leukemia (CCRF-CEM) and multidrug-resistant tumor cells (CEM/ADR5000) with comparable efficacies.