ABSTRACT Peroxy natural products, including endoperoxides, acyclic peroxides, and hydroperoxides, are widely distributed across all domains of life, with many, such as artemisinin and prostaglandins, serving as clinically important agents. However, the enzymatic mechanisms by which nature installs O─O bonds have largely remained elusive. To date, only a limited number of endoperoxide‐forming enzymes have been identified, while the enzymatic basis for acyclic peroxide assembly remains unknown. Here, we identify two homologous flavin‐dependent enzymes, OxaJ and OtnJ, that catalyze enantioselective acyclic peroxide formation in the biosynthesis of oxanthromicin natural products. A conserved structural motif acts as a redox gate by blocking NADPH access to the active site, thereby promoting peroxide installation. Removal of this motif permits NADPH binding and redirects the enzyme's activity toward hydroxylation. This work establishes the first example of peroxide formation by a flavin‐dependent enzyme and introduces redox gating as a previously unrecognized strategy for controlling oxidative divergence in enzymatic catalysis.

Expansion of a hexanucleotide repeat in a noncoding region of the C9ORF72 gene is responsible for a significant fraction of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) cases, but mechanisms linking mutant gene products to neuronal toxicity remain debatable. Pathogenesis was proposed to involve the production of toxic RNA species and/or accumulation of toxic dipeptide repeats (DPRs) but distinguishing between these mechanisms has been challenging. In this study, we first use complementary model systems for analyzing pathogenesis in adult-onset neurodegenerative diseases to characterize the pathogenicity of DPRs produced by Repeat Associated Non-ATG translation of C9ORF72 in specific cellular compartments: isolated axoplasm and giant synapse from the squid. Results showed selective axonal and presynaptic toxicity of GP-DPRs, independent of associated RNA. These effects involved a MAPK signaling pathway that affects fast axonal transport and synaptic function, a pathogenic mechanism shared with other mutant proteins associated with familial ALS, like SOD1 and FUS. In primary cultured neurons, GP but not other DPRs promote the "dying-back" axonopathy seen in ALS. Interestingly, GR- and PR-DPRs, which had no effect on axonal transport or synaptic transmission, were found to disrupt the nuclear membrane, promoting "dying-forward" neuropathy. All C9-DPR-mediated toxic effects observed in these studies are independent of whether the corresponding mRNAs contained hexanucleotide repeats or alternative codons. Finally, C9ORF72 human tissues confirmed a close association between GP and active P38 in degenerating motor neurons as well as GR-associated nuclear damage in the cortex. Collectively, our studies establish compartment-specific toxic effects of C9-DPRs associated with degeneration, suggesting that two independent pathogenic mechanisms may contribute to disease heterogeneity and/or synergize on disease progression in C9ORF72 patients with ALS and/or FTD symptoms.

Biosynthesis of atypical angucyclines involves unique oxidative B-ring cleavage and rearrangement reactions, which are catalyzed by AlpJ-family oxygenases, including AlpJ, JadG, and GilOII. Prior investigations established the essential requirement for FADH2/FMNH2 as cofactors when utilizing the quinone intermediate dehydrorabelomycin as a substrate. In this study, we unveil a previously unrecognized facet of these enzymes as cofactor-independent oxygenases when employing the hydroquinone intermediate CR1 as a substrate. The enzymes autonomously drive oxidative ring cleavage and rearrangement reactions of CR1, yielding products identical to those observed in cofactor-dependent reactions of AlpJ-family oxygenases. Furthermore, the AlpJ- and JadG-catalyzed reactions of CR1 could be quenched by superoxide dismutase, supporting a catalytic mechanism wherein the substrate CR1 reductively activates molecular oxygen, generating a substrate radical and the superoxide anion O2•−. Our findings illuminate a substrate-controlled catalytic mechanism of AlpJ-family oxygenases, expanding the realm of cofactor-independent oxygenases. Notably, AlpJ-family oxygenases stand as a pioneering example of enzymes capable of catalyzing oxidative reactions in either an FADH2/FMNH2-dependent or cofactor-independent manner.

Input analysis of corresponding Co-IP.

The synergistic effect between metformin and ABT-263 is independent of the cell cycle changes and metformin sensitizes ABT-263 not via changing the levels of BCL-2, BCL-XL, cIAP1 and cIAP2.

The influence of the cotreatment with metformin and ABT-263 on different translation-relevant kinase signaling pathways.

Combination of metformin with ABT-263 had no significant growth inhibition effect on HEK-293 cells.

Metformin attenuates ABT-263-induced MCL-1, XIAP and SURVIVIN through neither transcriptional nor posttranslational regulation.

Metformin and ABT-263 synergistically inhibit the growth of p53-defective (but not p53-WT) cancer cells and the metformin/ABT-263 combination more effectively elicits apoptosis in p53-defective xenograft tumors compared to p53-WT xenograft tumors.

Excessive consumption of fructose in the Western diet contributes to cancer development. However, it is still unclear how cancer cells coordinate glucose and fructose metabolism during tumor malignant progression. We demonstrate here that glioblastoma multiforme (GBM) cells switch their energy supply from glycolysis to fructolysis in response to glucose deprivation. Mechanistically, glucose deprivation induces expression of two essential fructolytic proteins GLUT5 and ALDOB through selectively activating translation of activating transcription factor 4 (ATF4). Functionally, genetic or pharmacological disruption of ATF4-dependent fructolysis significantly inhibits growth and colony formation of GBM cells in vitro and GBM growth in vivo. In addition, ATF4, GLUT5, and ALDOB levels positively correlate with each other in GBM specimens and are poor prognostic indicators in GBM patients. This work highlights ATF4-dependent fructolysis as a metabolic feature and a potential therapeutic target for GBM.

Itaconate is a newly discovered endogenous metabolite promoting an anti-inflammatory program during innate immune response, but the precise mechanisms underlying its effect remains poorly understood owing primarily to the limitations of available itaconate-monitoring techniques. Here, we develop and validate a genetically encoded fluorescent itaconate biosensor, BioITA, for directly monitoring itaconate dynamics in subcellular compartments of living macrophages. Utilizing BioITA, we monitor the itaconate dynamics in response to lipopolysaccharide (LPS) stimulation in the context of modulating itaconate transportation and metabolism. Moreover, we show that STING activation induces itaconate production, and injection of AAVs expressing cytosolic BioITA into mice allows directly reporting elevation of itaconate level in activated macrophages derived from LPS-injected mice. Thus, BioITA enables subcellular resolution imaging of itaconate in living macrophages.

Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR interference and programmable base editing have transformed the manipulation of eukaryotic genomes for potential therapeutic applications 1 – 4 . Here, we exploited CRISPR interference and programmable base editing to determine their potential in editing a TERT gene promoter-activating mutation, which occurs in many diverse cancer types, particularly glioblastoma 5 – 8 . Correction of the −124C>T TERT promoter mutation to −124C was achieved using a single guide RNA (sgRNA)-guided and catalytically impaired Campylobacter jejuni CRISPR-associated protein 9-fused adenine base editor (CjABE). This modification blocked the binding of members of the E26 transcription factor family to the TERT promoter, reduced TERT transcription and TERT protein expression, and induced cancer-cell senescence and proliferative arrest. Local injection of adeno-associated viruses expressing sgRNA-guided CjABE inhibited the growth of gliomas harbouring TERT -promoter mutations. These preclinical proof-of-concept studies establish the feasibility of gene editing as a therapeutic approach for cancer and validate activated TERT -promoter mutations as a cancer-specific therapeutic target.

Expressed in macrophages and endothelial cells, the receptor for angiopoietin, tyrosine kinase with immunoglobulin and epidermal growth factor homology-2 (Tie2), is required for the reconstruction of blood vessels in tumor recurrence after chemotherapy. Thus, small therapeutic peptides that target and block Tie2 activity are promising as a therapeutic for the prevention of tumor relapse after chemotherapy. However, such small peptides often have low bioavailability, undergo rapid enzymatic degradation, and exhibit a short circulation half-life, making them ineffective in cancer therapy. Herein, we designed a dual-responsive amphiphilic peptide (mPEG1000-K(DEAP)-AAN-NLLMAAS) to modify the small peptide T4 (NLLMAAS) as a Tie2 inhibitor, endowing it with the ability to endure in circulation and specifically target tumor tissue. The ultimate nanoformulation (P-T4) releases T4 in response to the combination of the acidic tumor microenvironment and the presence of legumain, which is commonly overexpressed in tumor tissue. Compared with free T4, P-T4 decreases vessel density significantly (free T4: 2.44 ± 1.20%, P-T4: 0.90 ± 0.75%), delays tumor regrowth after chemotherapy (free T4: 43.2 ± 11.8%, P-T4: 63.6 ± 13.9%), and reduces distant metastasis formation (free T4: 4.50 ± 2.40%, P-T4: 0.67 ± 0.32%). These effects of P-T4 are produced by the local blockage of Tie2 signals in Tie2-positive macrophages and endothelial cells. In addition to describing a potential strategy to enhance circulation half-life and the accumulation of an active peptide at tumor sites, our approach exemplifies the successful targeting of multiple cell types that overexpress a key molecule in conditions associated with tumors.

Nogo-B receptor (NgBR) plays fundamental roles in regulating angiogenesis, vascular development, and the epithelial-mesenchymal transition (EMT) of cancer cells. However, the therapeutic effect of NgBR blockade on tumor vasculature and malignancy is unknown, investigations on which requires an adequate delivery system for small interfering RNA against NgBR (NgBR siRNA). Here a surface charge switchable polymeric nanoparticle that was sensitive to the slightly acidic tumor microenvironment was developed for steady delivery of NgBR siRNA to tumor tissues. The nanoformulation was constructed by conjugating 2, 3-dimethylmaleic anhydride (DMMA) molecules to the surface amines of micelles formed by cationic co-polymer poly(lactic-co-glycolic acid)2-poly(ethylenimine) and subsequent absorption of NgBR siRNAs. The nanoparticles remained negatively charged in physiological condition and smartly converted to positive surface charge due to tumor-acidity-activated shedding of DMMA. The charge conversion facilitated cellular uptake of siRNAs and in turn efficiently depleted the expression of NgBR in tumor-bearing tissues. Silencing of NgBR suppressed endothelial cell migration and tubule formation, and reverted the EMT process of breast cancer cells. Delivery of the nanoformulation to mice bearing orthotopic breast carcinoma showed no effect on tumor growth, but led to remarkable decrease of distant metastasis by normalizing tumor vessels and suppressing the EMT of breast cancer cells. This study demonstrated that NgBR is a promising therapeutic target in abnormal tumor vasculature and aggressive cancer cells, and the tumor-responsive nanoparticle with the feature of charge transformation offers great potential for tumor-specific delivery of gene therapeutics.

Photothermal-based combination therapy using functional nanomaterials shows great promise in eradication of aggressive tumors and improvement of drug sensitivity. The therapeutic efficacy and adverse effects of drug combinations depend on the precise control of timely tumor-localized drug release. Here a polymer-dopamine nanocomposite is designed for combination therapy, thermo-responsive drug release and prevention of uncontrolled drug leakage. The thermo-sensitive co-polymer poly (2-(2-methoxyethoxy) ethyl methacrylate-co-oligo (ethylene glycol) methacrylate)-co-2-(dimethylamino) ethyl methacrylate-b-poly (D, l-lactide-co-glycolide) is constructed into core-shell structured nanoparticles for co-encapsulation of two cytotoxic drugs and absorption of small interfering RNAs against survivin. The drug-loaded nanoparticles are surface-coated with polydopamine which confers the nanoformulation with photothermal activity and protects drugs from burst release. Under tumor-localized laser irradiation, polydopamine generates sufficient heat, resulting in nanoparticle collapse and instant drug release within the tumor. The combination strategy of photothermal, chemo-, and gene therapy leads to triple-negative breast cancer regression, with a decrease in the chemotherapeutic drug dosage to about 1/20 of conventional dose. This study establishes a powerful nanoplatform for precisely controlled combination therapy, with dramatic improvement of therapeutic efficacy and negligible side effects.

C-C bond ring cleaving oxygenases represent a unique family of enzymes involved in the B ring cleavage reaction only observed in atypical angucycline biosynthesis. B ring cleavage is the key reaction leading to dramatic divergence in the final structures of atypical angucyclines. Here, we present the crystal structure of AlpJ, the first structure of this family of enzymes. AlpJ has been verified as the enzyme catalyzing C-C bond cleavage in kinamycin biosynthesis. The crystal structure of the AlpJ monomer resembles the dimeric structure of ferredoxin-like proteins. The N- and C-terminal halves of AlpJ are homologous, and both contain a putative hydrophobic substrate binding pocket in the "closed" and "open" conformations, respectively. Structural comparison of AlpJ with ActVA-Orf6 and protein-ligand docking analysis suggest that the residues including Asn60, Trp64, and Trp181 are possibly involved in substrate recognition. Site-directed mutagenesis results supported our hypothesis, as mutation of these residues led to nearly a complete loss of the activity of AlpJ. Structural analysis also revealed that AlpJ possesses an intramolecular domain-domain interface, where the residues His50 and Tyr178 form a hydrogen bond that probably stabilizes the three-dimensional structure of AlpJ. Site-directed mutagenesis showed that the two residues, His50 and Tyr178, were vital for the activity of AlpJ. Our findings shed light on the structure and catalytic mechanism of the AlpJ family of oxygenases, which presumably involves two active sites that might function in a cooperative manner.

The antifungal activities of eleven C21-steroidal compounds isolated from Cynanchum wilfordii, together with thirty-six derivatives of caudatin and qingyangshengenin were evaluated on Sclerotinia sclerotiorum and other five fungal strains by the mycelium growth rate method. Four derivatives 1k, 1y, 10d, and 10j exhibited much stronger inhibitions on growth of S. sclerotiorum with IC50 values of 0.0084, 0.0049, 0.0053, and 0.0034 μM, respectively.

The naturally occurring diazobenzofluorenes, kinamycins, fluostatins and lomaiviticins, possess highly oxygenated A-rings, via which the last forms a dimeric pharmacophore. However, neither the A-ring transformation nor the dimerization mechanisms have been explored thus far. Here we propose a unified biosynthetic logic for the three types of antibiotics and verify one key reaction via detailed genetic and enzymatic experiments. Alp1U and Lom6 from the kinamycin and lomaiviticin biosynthesis, respectively, are shown to catalyse epoxy hydrolysis on a substrate that is obtained by chemical deacetylation of a kinamycin-pathway-derived intermediate. Thus, our study provides the first evidence for the existence of an epoxy intermediate in lomaiviticin biosynthesis. Furthermore, our results suggest that the dimerization in the lomaiviticin biosynthesis proceeds after dehydration of a product generated by Lom6.

The biosynthesis of diazobenzofluorene kinamycins requires a hitherto uncharacterized B-ring contraction. Via detailed genetic and enzymatic analyses we unambiguously characterized the conserved pairs of oxidases, AlpJ and AlpK homologs, as nature's machinery for benzofluorenone formation, which paves the way for the investigation of the following diazo assembly.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.