Cyclic immonium ion of lactyllysine reveals widespread lactylation in the human proteome.

Lactylation was initially discovered on human histones. Given its nascence, its occurrence on nonhistone proteins and downstream functional consequences remain elusive. Here we report a cyclic immonium ion of lactyllysine formed during tandem mass spectrometry that enables confident protein lactylation assignment. We validated the sensitivity and specificity of this ion for lactylation through affinity-enriched lactylproteome analysis and large-scale informatic assessment of nonlactylated spectral libraries. With this diagnostic ion-based strategy, we confidently determined new lactylation, unveiling a wide landscape beyond histones from not only the enriched lactylproteome but also existing unenriched human proteome resources. Specifically, by mining the public human Meltome Atlas, we found that lactylation is common on glycolytic enzymes and conserved on ALDOA. We also discovered prevalent lactylation on DHRS7 in the draft of the human tissue proteome. We partially demonstrated the functional importance of lactylation: site-specific engineering of lactylation into ALDOA caused enzyme inhibition, suggesting a lactylation-dependent feedback loop in glycolysis.

The marine-derived compound TAG alleviates Parkinson's disease by restoring RUBCN-mediated lipid metabolism homeostasis.

Parkinson's disease (PD) is the second most common neurodegenerative disease, and its prevalence is increasing. Currently, no effective therapies for PD exist. Marine-derived natural compounds are considered important resources for the discovery of new drugs due to their distinctive structures and diverse activities. In this study, tetrahydroauroglaucin (TAG), a polyketide isolated from a marine sponge, was found to have notable neuroprotective effects on MPTP/MPP+-induced neurotoxicity. RNA sequencing analysis and metabolomics revealed that TAG significantly improved lipid metabolism disorder in PD models. Further investigation indicated that TAG markedly decreased the accumulation of lipid droplets (LDs), downregulated the expression of RUBCN, and promoted autophagic flux. Moreover, conditional knockdown of Rubcn notably attenuated PD-like symptoms and the accumulation of LDs, accompanied by blockade of the neuroprotective effect of TAG. Collectively, our results first indicated that TAG, a promising PD therapeutic candidate, could suppress the accumulation of LDs through the RUBCN-autophagy pathway, which highlighted a novel and effective strategy for PD treatment.

Spirodalesol analog 8A inhibits NLRP3 inflammasome activation and attenuates inflammatory disease by directly targeting adaptor protein ASC.

Pharmacological inhibition of the Nod-like receptor family protein 3 (NLRP3) inflammasome contributes to the treatment of numerous inflammation-related diseases, making it a desirable drug target. Spirodalesol, derived from the ascomycete fungus Daldinia eschscholzii, has been reported to inhibit NLRP3 inflammasome activation. Based on the structure of spirodalesol, we synthesized and screened a series of analogs to find a more potent inhibitor. Analog compound 8A was identified as the most potent selective inhibitor for NLRP3 inflammasome assembly, but 8A did not inhibit the priming phase of the inflammasome. Specifically, while 8A did not reduce NLRP3 oligomerization, we found that it inhibited the oligomerization of adaptor protein apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), as ASC speck formation was significantly reduced. Also, 8A interrupted the assembly of the NLRP3 inflammasome complex and inhibited the activation of caspase-1. Subsequently, we used a cellular thermal shift assay and microscale thermophoresis assay to demonstrate that 8A interacts directly with ASC, both in vitro and ex vivo. Further, 8A alleviated lipopolysaccharide-induced endotoxemia, as well as monosodium urate-induced peritonitis and gouty arthritis in mice by suppressing NLRP3 inflammasome activation. Thus, 8A was identified as a promising ASC inhibitor to treat inflammasome-driven diseases.

Characterization of LTr1 derived from cruciferous vegetables as a novel anti-glioma agent via inhibiting TrkA/PI3K/AKT pathway.

Malignant glioma is the most fatal, invasive brain cancer with limited treatment options. Our previous studies show that 2-(indol-3-ylmethyl)-3,3'-diindolylmethane (LTr1), a major metabolite of indole-3-carbinol (I3C) derived from cruciferous vegetables, produces anti-tumour effect against various tumour cell lines. In this study we characterized LTr1 as a novel anti-glioma agent. Based on screening 134 natural compounds and comparing the candidates' efficacy and toxicity, LTr1 was selected as the lead compound. We showed that LTr1 potently inhibited the viability of human glioma cell lines (SHG-44, U87, and U251) with IC50 values of 1.97, 1.84, and 2.03 µM, respectively. Furthermore, administration of LTr1 (100,300 mg· kg-1 ·d-1, i.g. for 18 days) dose-dependently suppressed the tumour growth in a U87 xenograft nude mouse model. We demonstrated that LTr1 directly bound with TrkA to inhibit its kinase activity and the downstream PI3K/AKT pathway thus inducing significant S-phase cell cycle arrest and apoptosis in SHG-44 and U87 cells by activating the mitochondrial pathway and inducing the production of reactive oxygen species (ROS). Importantly, LTr1 could cross the blood-brain barrier to achieve the therapeutic concentration in the brain. Taken together, LTr1 is a safe and promising therapeutic agent against glioma through inhibiting TrkA/PI3K/AKT pathway.

Three new polyketide derivatives of the endophytic fungus Aspergillus cristatus 2H1.

Three new polyketide derivatives, 2-ethoxycarbonyl-endocrocin (1), 6-methoxy-2-ethoxycarbonyl-endocrocin (2) and pannorin C (3), along with sixteen known compounds (4-19) were isolated from a plant endophytic fungus Aspergillus cristatus 2H1. Their structures were elucidated by 1D/2D NMR and HR-ESI-MS data analysis. Compound 3 showed weak antibacterial activity against Staphylococcus aureus (MIC 20 µg/ml). Compounds 14 and 15 showed effective cytotoxicity on human melanoma A375 cells (IC50 4.13 µM for 14, 3.39 µM for 15).

Two new azaphilone pigments from Talaromyces albobiverticillius and their anti-inflammatory activity.

Two new azaphilone pigments, talaralbols A and B (3 and 7), along with five known azaphilone metabolites (1, 2, and 4-6), were isolated from the culture of Talaromyces albobiverticillius associated with the isopod Armadillidium vulgare. Their structures were elucidated by a combination of 1 D and 2 D NMR data, ECD calculations, chemical transformations, and NMR data analogy with model compounds. Talaralbol A (3) showed a moderate inhibition on the lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW264.7 cells with the inhibitory rate being 31.0% at the concentration of 10 µM.[Formula: see text].

G1 phase cell cycle arrest in NSCLC in response to LZ-106, an analog of enoxacin, is orchestrated through ROS overproduction in a P53-dependent manner.

LZ-106, a newly synthetized analog of quinolone, has been shown to be highly effective in non-small cell lung cancer (NSCLC) in both cultured cells and xenograft mouse model with low toxicity, yet the molecular mechanisms still require exploration. Here, we substantiated the involvement of P53 activation in intracellular reactive oxygen species (ROS) generation upon LZ-106 treatment and related P53 to the ROS-induced viability inhibition and apoptosis, which was exhibited in the previous research. P53 was shown to play an indispensable role in the elevated levels of intracellular ROS in LZ-106-treated NSCLC cells through ROS detection. We further identified the anti-proliferation effect of LZ-106 in NSCLC cells through G1 phase cell cycle arrest by cell cycle analysis, with the expression analysis of the key proteins, and discovered that the cell cycle arrest effect is also mediated by induction of ROS in a P53-dependent manner. In addition, the tumor suppression effect exhibited in vivo was demonstrated to be similar to that in vitro, which requires the participation of P53. Thus, LZ-106 is a potent antitumor drug possessing potent proliferation inhibition and apoptosis induction ability through the P53-dependent ROS modulation both in vitro and in vivo.

CARM1-mediated methylation of protein arginine methyltransferase 5 represses human γ-globin gene expression in erythroleukemia cells.

Protein arginine methyltransferase 5 (PRMT5) is a member of the arginine methyltransferase protein family that critically mediates the symmetric dimethylation of Arg-3 at histone H4 (H4R3me2s) and is involved in many key cellular processes, including hematopoiesis. However, the post-translational modifications (PTMs) of PRMT5 that may affect its biological functions remain less well-understood. In this study, using MS analyses, we found that PRMT5 itself is methylated in human erythroleukemia Lys-562 cells. Biochemical assays revealed that coactivator-associated arginine methyltransferase 1 (CARM1) interacts directly with and methylates PRMT5 at Arg-505 both in vivo and in vitro. Substitutions at Arg-505 significantly reduced PRMT5's methyltransferase activity, decreased H4R3me2s enrichment at the γ-globin gene promoter, and increased the expression of the γ-globin gene in Lys-562 cells. Moreover, CARM1 knockdown consistently reduced PRMT5 activity and activated γ-globin gene expression. Importantly, we show that CARM1-mediated methylation of PRMT5 is essential for the intracellular homodimerization of PRMT5 to its active form. These results thus reveal a critical PTM of PRMT5 that represses human γ-globin gene expression. We conclude that CARM1-mediated asymmetric methylation of PRMT5 is critical for its dimerization and methyltransferase activity leading to the repression of γ-globin expression. Given PRMT5's crucial role in diverse cellular processes, these findings may inform strategies for manipulating its methyltransferase activity for managing hemoglobinopathy or cancer.

Oroxylin A increases the sensitivity of temozolomide on glioma cells by hypoxia-inducible factor 1α/hedgehog pathway under hypoxia.

Microenvironmental hypoxia-mediated drug resistance is responsible for the failure of cancer therapy. To date, the role of the hedgehog pathway in resistance to temozolomide (TMZ) under hypoxia has not been investigated. In this study, we discovered that the increasing hypoxia-inducible factor 1α (HIF-1α) activated the hedgehog pathway in hypoxic microenvironment by promoting autocrine secretion of sonic hedgehog protein (Shh), and then upregulating transfer of Gli1 to the nucleus, finally contributed to TMZ resistance in glioma cells. Oroxylin A (C16H12O5), a bioactive flavonoid, could induce HIF-1α degradation via prolyl-hydroxylases-VHL signaling pathway, resulting in the inactivation of the hedgehog. Besides, oroxylin A increased the expression of Sufu, which is a negative regulator of Gli1. By this mechanism, oroxylin A sensitized TMZ on glioma cells. U251 intracranial transplantation model and GL261 xenograft model were used to confirm the reversal effects of oroxylin A in vivo. In conclusion, our results demonstrated that HIF-1α/hedgehog pathway conferred TMZ resistance under hypoxia, and oroxylin A was capable of increasing the sensitivity of TMZ on glioma cells in vitro and in vivo by inhibiting HIF-1α/hedgehog pathway and depressing the activation of Gli1 directly.

Divergent Total Synthesis of Chaetoglines C to F.

The first total syntheses of chaetoglines C-F via a bioinspired and divergent synthetic strategy are reported. Chaetolines C and D were obtained from the condensation of hemiacetal and tryptophan methyl ester building blocks followed by functional group transformations. The synthesis of chaetogline E employed the diastereoselective Pictet-Spengler reaction, and the tetrahydro-carboline skeleton was further utilized as a precursor for an oxidative aromatization reaction to introduce the ß-carboline moiety of chaetogline F.

Cytotoxic Trichothecene Macrolides Produced by the Endophytic Myrothecium roridum.

Six new macrolides named myrothecines D-G (1-4), 16-hydroxymytoxin B (5), and 14'-dehydrovertisporin (6), including four 10,13-cyclotrichothecane derivatives, in addition to 12 known compounds (7-18), were isolated from three endophytic Myrothecium roridum, IFB-E008, IFB-E009, and IFB-E012. The isolated compounds were characterized by MS, NMR, CD, and single-crystal X-ray crystallography. The isolated macrolides exhibited an antiproliferation effect against chronic myeloid leukemia K562 and colorectal carcinoma SW1116 cell lines. Compounds 1-6 were cytotoxic, with IC50 values ranging between 56 nM and 16 µM. Since slight structural changes led to obvious activity differences, the CoMFA (comparative molecular field analysis) and CoMSIA (comparative molecular similarity indices analysis) methods were then used to explore the 3D QSAR (three-dimensional quantitative structure-activity relationship) of these macrolides. The result showed that the steric, electrostatic, hydrophobic, and H-bond acceptor factors were involved in their cytotoxicity and provided an in-depth understanding of the structure-activity relationships of these metabolites.

Pharmacodynamic Effect of Ellagic Acid on Ameliorating Cerebral Ischemia/Reperfusion Injury.

Cerebral ischemia/reperfusion (I/R) injury causes a larger population of disable patients and deaths annually. Three Tibetan prescriptions have been applied in alleviating the I/R injury for a 1,000 years. Interestingly, ellagic acid (EA) is one of the commonly dominated phytochemicals in these 3 prescriptions. Therefore, it is noteworthy to evaluate the association between the pharmacodynamics effects of EA and I/R injury alleviation. In this study, we reveal that the EA can effectively reduce the infarction area, and prevent the neuron from apoptosis and damage in permanent middle cerebral artery occlusion rat model. The results of the histopathological study indicate that alleviation of brain damage is positively correlated with the EA dose. Further by biochemical analysis, it indicates that the EA can alleviate the brain damage by the anti-inflammatory and anti-oxidative response mediated by EA. The upregulation of zonula occludens-1 and down-regulation of Aquaporin 4 and matrix metalloprotein 9 (MMP-9) in injured brain tissues after being treated with EA suggested that the reconstruction of brain-blood-barrier (BBB), which can further prevent the brain from further injury by the other xenobiotics. In addition, EA will not activate the coagulation factors XII to induce coagulation formation during the treatment process. Therefore, EA is a promising candidate oral drug for I/R injury therapy.

Heterochromatin Protein 1γ Is a Novel Epigenetic Repressor of Human Embryonic ϵ-Globin Gene Expression.

Production of hemoglobin during development is tightly regulated. For example, expression from the human ß-globin gene locus, comprising ß-, δ-, ϵ-, and γ-globin genes, switches from ϵ-globin to γ-globin during embryonic development and then from γ-globin to ß-globin after birth. Expression of human ϵ-globin in mice has been shown to ameliorate anemia caused by ß-globin mutations, including those causing ß-thalassemia and sickle cell disease, raising the prospect that reactivation of ϵ-globin expression could be used in managing these conditions in humans. Although the human globin genes are known to be regulated by a variety of multiprotein complexes containing enzymes that catalyze epigenetic modifications, the exact mechanisms controlling ϵ-globin gene silencing remain elusive. Here we found that the heterochromatin protein HP1γ, a multifunctional chromatin- and DNA-binding protein with roles in transcriptional activation and elongation, represses ϵ-globin expression by interacting with a histone-modifying enzyme, lysine methyltransferase SUV4-20h2. Silencing of HP1γ expression markedly decreased repressive histone marks and the multimethylation of histone H3 lysine 9 and H4 lysine 20, leading to a significant decrease in DNA methylation at the proximal promoter of the ϵ-globin gene and greatly increased ϵ-globin expression. In addition, using chromatin immunoprecipitation, we showed that SUV4-20h2 facilitates the deposition of HP1γ on the ϵ-globin-proximal promoter. Thus, these data indicate that HP1γ is a novel epigenetic repressor of ϵ-globin gene expression and provide a potential strategy for targeted therapies for ß-thalassemia and sickle cell disease.

Modulating calcium-mediated NFATc1 and mitogen-activated protein kinase deactivation underlies the inhibitory effects of kavain on osteoclastogenesis and bone resorption.

Osteoclasts are responsible for bone resorption during the process of bone remodeling. Increased osteoclast numbers and bone resorption activity are the main factors contributing to bone loss-related diseases such as osteoporosis. Therefore, modulating the formation and function of osteoclasts is critical for the effective treatment of osteolysis and osteoporosis. Kavain is the active ingredient extracted from the root of the kava plant, which possesses known anti-inflammatory properties. However, the effects of kavain on osteoclastogenesis and bone resorption remain unclear. In this study, we found that kavain inhibits receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and fusion using tartrate-resistant acid phosphatase staining and immunofluorescence. Furthermore, kavain inhibited bone resorption performed by osteoclasts. Using reverse transcription-polymerase chain reaction and western blot analysis, we found that kavain downregulates the expression of osteoclast marker genes, such as nuclear factor of activated T cells, cytoplasmic 1 (Nfatc1), v-atpase d2 (Atp6v0d2), dendrocyte expressed seven transmembrane protein (Dcstamp), matrix metallopeptidase 9 (Mmp9), cathepsin K (Ctsk), and Acp5. Additionally, kavain repressed RANKL-induced calcium oscillations, nuclear factor of activated T cells activation, and mitogen-activated protein kinase phosphorylation, while leaving NF-κB unaffected. We found no effects of kavain on either osteoblast proliferation or differentiation. Besides, kavain inhibited bone loss in ovariectomized mice by suppressing osteoclastogenesis. Collectively, these data suggest a potential use for kavain as a candidate drug for the treatment of osteolytic diseases.

Lumichrome inhibits osteoclastogenesis and bone resorption through suppressing RANKL-induced NFAT activation and calcium signaling.

The dynamic balance between bone resorption and bone formation is crucial to maintain bone mass. Osteoclasts are key cells that perform bone resorption while osteoblasts and osteocytes function in bone formation. Osteoporosis, a bone metabolism disease characterized by bone loss and degradation of bone microstructure, occurs when osteoclastic bone resorption outstrips osteoblastic bone synthesis. The interaction between receptor activator of nuclear factor κB ligand (RANKL) and RANK on the surface of bone marrow macrophages promotes osteoclast differentiation and activation. In this study, we found that lumichrome, a photodegradation product of riboflavin, inhibits RANKL-induced osteoclastogenesis and bone resorption as determined by tartrate-resistant acid phosphatase staining, immunofluorescence, reverse transcription-polymerase chain reaction, and western blot. Our results showed that lumichrome represses the expression of osteoclast marker genes, including cathepsin K (Ctsk) and Nfatc1. In addition, lumichrome suppressed RANKL-induced calcium oscillations, NFATc1, NF-κB, and MAPK signaling activation. Moreover, lumichrome promoted osteoblast differentiation at an early stage, as demonstrated by upregulated expression of osteoblast marker genes Alp, Runx2, and Col1a1. We also found that lumichrome reduces bone loss in ovariectomized mice by inhibiting osteoclastogenesis. In summary, our data suggest the potential of lumichrome as a therapeutic drug for osteolytic diseases.

Synthesis and bioevaluation of new tacrine-cinnamic acid hybrids as cholinesterase inhibitors against Alzheimer's disease.

Small molecule cholinesterases inhibitor (ChEI) provides an effective therapeutic strategy to treat Alzheimer's disease (AD). Currently, the discovery of new ChEI with multi-target effect is still of great importance. Herein, we report the synthesis, structure-activity relationship study and biological evaluation of a series of tacrine-cinnamic acid hybrids as new ChEIs. All target compounds are evaluated for their in vitro cholinesterase inhibitory activities. The representatives which show potent activity on cholinesterase, are evaluated for the amyloid ß-protein self-aggregation inhibition and in vivo assays. The optimal compound 19, 27, and 30 (human AChE IC50 = 10.2 ± 1.2, 16.5 ± 1.7, and 15.3 ± 1.8 nM, respectively) show good performance in ameliorating the scopolamine-induced cognition impairment and preliminary safety in hepatotoxicity evaluation. These compounds deserve further evaluation for the development of new therapeutic agents against AD.

Novel chaetospirolactone and orsellide F from an endophytic fungus Chaetomium sp.

Chaetospirolactone (1), a novel spiro-lactone bearing a rare 1-oxaspiro [4.4] non-7-ene-2,6-dione skeleton, and orsellide F (2), together with six known compounds (3-8), were isolated from an endophytic fungus Chaetomium sp. NF00754. Their structures were determined by interpretation of spectroscopic data. The absolute configurations of 1 and 2 were established by analysis of single X-ray crystallographic data and CD spectra. Compounds 3, 4, and 6 showed moderate acetylcholinesterase inhibitory activity with IC50 values of 7.34, 5.19, and 7.67 µM, respectively.

New p-terphenyls from the endophytic fungus Aspergillus sp. YXf3.

Five new p-terphenyls named prenylterphenyllin D (1), prenylterphenyllin E (2), 2'-O-methylprenylterphenyllin (3), 4-O-methylprenylterphenyllin (4) and 3'-O-methylterphenyllin (5) together with seven known compounds (6-12), were isolated from cultures of Aspergillus sp. YXf3. The structures of the new compounds were elucidated by extensive MS and NMR analyses. The NMR and MS data of 5 is reported for the first time, as its structure was listed in SciFinder Scholar with no associated reference. Compounds 6 and 7 were distinguished from each other on the basis of 2D NMR experiments. Compounds 1, 2, 3 and 8 showed antibacterial activities against X. oryzae pv. oryzicola Swings and E. amylovora with the same MIC values of 20µg/mL while 10 exhibited activities against E. amylovora with an MIC value of 10µg/mL.

Strepchazolins A and B: Two New Alkaloids from a Marine Streptomyces chartreusis NA02069.

Two new alkaloids, strepchazolins A (1) and B (2), together with a previously reported compound, streptazolin (3), were isolated from a marine actinomycete, Streptomyces chartreusis NA02069, collected in the Coast of Hainan Island, China. The structures of new compounds were determined by extensive NMR, mass spectroscopic and X-ray crystallographic analysis, as well as modified Mosher's method. Compound 1 showed weak anti-Bacillus subtilis activity with the MIC value of 64.0 µM, and weak inhibitory activity against acetylcholinesterase (AChE) in vitro with IC50 value of 50.6 µM, while its diastereoisomer, Compound 2, is almost inactive.

Eriodictyol Inhibits RANKL-Induced Osteoclast Formation and Function Via Inhibition of NFATc1 Activity.

Receptor activator of nuclear factor kappa-B ligand (RANKL) induces differentiation and function of osteoclasts through triggering multiple signaling cascades, including NF-κB, MAPK, and Ca(2+) -dependent signals, which induce and activate critical transcription factor NFATc1. Targeting these signaling cascades may serve as an effective therapy against osteoclast-related diseases. Here, by screening a panel of natural plant extracts with known anti-inflammatory, anti-tumor, or anti-oxidant properties for possible anti-osteoclastogenic activities we identified Eriodictyol. This flavanone potently suppressed RANKL-induced osteoclastogenesis and bone resorption in a dose-dependent manner without detectable cytotoxicity, suppressing RANKL-induced NF-κB, MAPK, and Ca(2+) signaling pathways. Eriodictyol also strongly inhibited RANKL-induction of c-Fos levels (a critical component of AP-1 transcription factor required by osteoclasts) and subsequent activation of NFATc1, concomitant with reduced expression of osteoclast specific genes including cathepsin K (Ctsk), V-ATPase-d2 subunit, and tartrate resistant acid phosphatase (TRAcP/Acp5). Taken together, these data provide evidence that Eriodictyol could be useful for the prevention and treatment of osteolytic disorders associated with abnormally increased osteoclast formation and function. J. Cell. Physiol. 231: 1983-1993, 2016. © 2016 Wiley Periodicals, Inc.