Structural and Functional Analysis of the Natural JNK1 Inhibitor Quercetagetin

c-Jun NH2-terminal kinases (JNKs) and phosphatidylinositol 3-kinase (PI3-K) play critical roles in chronic diseases such as cancer, type II diabetes, and obesity. We describe here the binding of quercetagetin (3,3′,4′,5,6,7-hydroxyflavone), related flavonoids, and SP600125 to JNK1 and PI3-K by ATP-competitive and immobilized metal ion affinity-based fluorescence polarization assays and measure the effect of quercetagetin on JNK1 and PI3-K activities. Quercetagetin attenuated the phosphorylation of c-Jun and AKT, suppressed AP-1 and NF-κB promoter activities, and also reduced cell transformation. It attenuated tumor incidence and reduced tumor volumes in a two-stage skin carcinogenesis mouse model.Our crystallographic structure determination data show that quercetagetin binds to the ATP-binding site of JNK1. Notably, the interaction between Lys55, Asp169, and Glu73 of JNK1 and the catechol moiety of quercetagetin reorients the N-terminal lobe of JNK1, thereby improving compatibility of the ligand with its binding site. The results of a theoretical docking study suggest a binding mode of PI3-K with the hydroxyl groups of the catechol moiety forming hydrogen bonds with the side chains of Asp964 and Asp841 in the p110γ catalytic subunit. These interactions could contribute to the high inhibitory activity of quercetagetin against PI3-K. Our study suggests the potential use of quercetagetin in the prevention or therapy of cancer and other chronic diseases.     

Wnt/β-Catenin Signaling Protects Mouse Liver against Oxidative Stress-induced Apoptosis through the Inhibition of Forkhead Transcription Factor FoxO3

Numerous liver diseases are associated with extensive oxidative tissue damage. It is well established that Wnt/β-catenin signaling directs multiple hepatocellular processes, including development, proliferation, regeneration, nutrient homeostasis, and carcinogenesis. It remains unexplored whether Wnt/β-catenin signaling provides hepatocyte protection against hepatotoxin-induced apoptosis. Conditional, liver-specific β-catenin knockdown (KD) mice and their wild-type littermates were challenged by feeding with a hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet to induce chronic oxidative liver injury. Following the DDC diet, mice with β-catenin-deficient hepatocytes demonstrate increased liver injury, indicating an important role of β-catenin signaling for liver protection against oxidative stress. This finding was further confirmed in AML12 hepatocytes with β-catenin signaling manipulation in vitro using paraquat, a known oxidative stress inducer. Immunofluorescence staining revealed an intense nuclear FoxO3 staining in β-catenin-deficient livers, suggesting active FoxO3 signaling in response to DDC-induced liver injury when compared with wild-type controls. Consistently, FoxO3 target genes p27 and Bim were significantly induced in β-catenin KD livers. Conversely, SGK1, a β-catenin target gene, was significantly impaired in β-catenin KD hepatocytes that failed to inactivate FoxO3. Furthermore, shRNA-mediated deletion of FoxO3 increased hepatocyte resistance to oxidative stress-induced apoptosis, confirming a proapoptotic role of FoxO3 in the stressed liver. Our findings suggest that Wnt/β-catenin signaling is required for hepatocyte protection against oxidative stress-induced apoptosis. The inhibition of FoxO through its phosphorylation by β-catenin-induced SGK1 expression reduces the apoptotic function of FoxO3, resulting in increased hepatocyte survival. These findings have relevance for future therapies directed at hepatocyte protection, regeneration, and anti-cancer treatment.     

Translocation and Stability of Replicative DNA Helicases upon Encountering DNA-Protein Cross-links

DNA-protein cross-links (DPCs) are formed when cells are exposed to various DNA-damaging agents. Because DPCs are extremely large, steric hindrance conferred by DPCs is likely to affect many aspects of DNA transactions. In DNA replication, DPCs are first encountered by the replicative helicase that moves at the head of the replisome. However, little is known about how replicative helicases respond to covalently immobilized protein roadblocks. In the present study we elucidated the effect of DPCs on the DNA unwinding reaction of hexameric replicative helicases in vitro using defined DPC substrates. DPCs on the translocating strand but not on the nontranslocating strand impeded the progression of the helicases including the phage T7 gene 4 protein, simian virus 40 large T antigen, Escherichia coli DnaB protein, and human minichromosome maintenance Mcm467 subcomplex. The impediment varied with the size of the cross-linked proteins, with a threshold size for clearance of 5.0–14.1 kDa. These results indicate that the central channel of the dynamically translocating hexameric ring helicases can accommodate only small proteins and that all of the helicases tested use the steric exclusion mechanism to unwind duplex DNA. These results further suggest that DPCs on the translocating and nontranslocating strands constitute helicase and polymerase blocks, respectively. The helicases stalled by DPC had limited stability and dissociated from DNA with a half-life of 15–36 min. The implications of the results are discussed in relation to the distinct stabilities of replisomes that encounter tight but reversible DNA-protein complexes and irreversible DPC roadblocks.     

The Sulfonylurea Receptor 1 (Sur1)-Transient Receptor Potential Melastatin 4 (Trpm4) Channel

The sulfonylurea receptor 1 (Sur1)-NC_Ca-ATP channel plays a central role in necrotic cell death in central nervous system (CNS) injury, including ischemic stroke, and traumatic brain and spinal cord injury. Here, we show that Sur1-NC_Ca-ATP channels are formed by co-assembly of Sur1 and transient receptor potential melastatin 4 (Trpm4). Co-expression of Sur1 and Trpm4 yielded Sur1-Trpm4 heteromers, as shown in experiments with Förster resonance energy transfer (FRET) and co-immunoprecipitation. Co-expression of Sur1 and Trpm4 also yielded functional Sur1-Trpm4 channels with biophysical properties of Trpm4 and pharmacological properties of Sur1. Co-assembly with Sur1 doubled the affinity of Trpm4 for calmodulin and doubled its sensitivity to intracellular calcium. Experiments with FRET and co-immunoprecipitation showed de novo appearance of Sur1-Trpm4 heteromers after spinal cord injury in rats. Our findings depart from the long-held view of an exclusive association between Sur1 and K_ATP channels and reveal an unexpected molecular partnership with far-ranging implications for CNS injury.     

Modulation of Lipid Kinase PI4KIIα Activity and Lipid Raft Association of Presenilin 1 Underlies γ-Secretase Inhibition by Ginsenoside (20S)-Rg3

Amyloid β-peptide (Aβ) pathology is an invariant feature of Alzheimer disease, preceding any detectable clinical symptoms by more than a decade. To this end, we seek to identify agents that can reduce Aβ levels in the brain via novel mechanisms. We found that (20S)-Rg3, a triterpene natural compound known as ginsenoside, reduced Aβ levels in cultured primary neurons and in the brains of a mouse model of Alzheimer disease. The (20S)-Rg3 treatment induced a decrease in the association of presenilin 1 (PS1) fragments with lipid rafts where catalytic components of the γ-secretase complex are enriched. The Aβ-lowering activity of (20S)-Rg3 directly correlated with increased activity of phosphatidylinositol 4-kinase IIα (PI4KIIα), a lipid kinase that mediates the rate-limiting step in phosphatidylinositol 4,5-bisphosphate synthesis. PI4KIIα overexpression recapitulated the effects of (20S)-Rg3, whereas reduced expression of PI4KIIα abolished the Aβ-reducing activity of (20S)-Rg3 in neurons. Our results substantiate an important role for PI4KIIα and phosphoinositide modulation in γ-secretase activity and Aβ biogenesis.     

Latency Locus Complements MicroRNA 155 Deficiency In Vivo

MicroRNA-155 (miR-155) is expressed in many cancers. It also executes evolutionary conserved functions in normal B cell development. We show that the Kaposi''s sarcoma-associated herpesvirus (KSHV) latency locus, which contains an ortholog of miR-155, miR-K12-11, complements B cell deficiencies in miR-155 knockout mice. Germinal center (GC) formation was rescued in spleen, lymph node, and Peyer''s patches. Immunoglobulin levels were restored. This demonstrates that KSHV can complement the normal, physiological function of miR-155.     

Sequence polymorphisms in Pvs48/45 and Pvs47 gametocyte and gamete surface proteins in Plasmodium vivax isolated in Korea

Nucleotide sequence analyses of the Pvs48/45 and Pvs47 genes were conducted in 46 malaria patients from the Republic of Korea (ROK) (n = 40) and returning travellers from India (n = 3) and Indonesia (n = 3). The domain structures, which were based on cysteine residue position and secondary protein structure, were similar between Plasmodium vivax (Pvs48/45 and Pvs47) and Plasmodium falciparum (Pfs48/45 and Pfs47). In comparison to the Sal-1 reference strain (Pvs48/45, PVX_083235 and Pvs47, PVX_083240), Korean isolates revealed seven polymorphisms (E35K, H211N, K250N, D335Y, A376T, I380T and K418R) in Pvs48/45. These isolates could be divided into five haplotypes with the two major types having frequencies of 47.5% and 20%, respectivelfy. In Pvs47, 10 polymorphisms (F22L, F24L, K27E, D31N, V230I, M233I, E240D, I262T, I273M and A373V) were found and they could be divided into four haplotypes with one major type having a frequency of 75%. The Pvs48/45 isolates from India showed a unique amino acid substitution site (K26R). Compared to the Sal-1 and ROK isolates, the Pvs47 isolates from travellers returning from India and Indonesia had amino acid substitutions (S57T and I262K). The current data may contribute to the development of the malaria transmission-blocking vaccine in future clinical trials.     

Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration

Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO₂). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO₂, a major greenhouse gas, making this a promising alternative for chemical CO₂ mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO₂ sequestration by mineral carbonation, a process with the potential to store large quantities of CO₂. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO₂ compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO₃) formation and exerted a striking impact on the maximal amount of CaCO₃ produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO₂ sequestration.     

Stimulation of Rg3 ginsenoside biosynthesis in ginseng hairy roots elicited by methyl jasmonate

It has been recognized that ginsenoside Rg3 is not naturally produced in ginseng although this ginsenoside can accumulate in red ginseng as the result of a thermal process. In order to determine whether or not Rg3 is synthesized in ginseng, hairy roots were treated with methyl jasmonate (MJ). From HPLC analysis, no peak for Rg3 was observed in the controls. However, Rg3 did accumulate in hairy roots that were MJ-treated for 7?days. Rg3 content was 0.42?mg/g (dry weight). To gain more insight into the effects of MJ on UDP-glucosyltransferase (UGT) activity, we attempted to evaluate ginsenoside Rg3 biosynthesis by UGT. A new peak for putative Rg3 was observed, which was confirmed by LC-MS/MS analysis. Our findings indicate that the proteins extracted from our hairy root lines can catalyze Rg3 from Rh2. This suggests that our ginseng hairy root lines possess Rg3 biosynthesis capacity.     

Combinatorial biosynthesis and antibacterial evaluation of glycosylated derivatives of 12-membered macrolide antibiotic YC-17

Expression plasmids carrying different deoxysugar biosynthetic gene cassettes and the gene encoding a substrate-flexible glycosyltransferase DesVII were constructed and introduced into Streptomyces venezuelae YJ003 mutant strain bearing a deletion of a desosamine biosynthetic (des) gene cluster. The resulting recombinants produced macrolide antibiotic YC-17 analogs possessing unnatural sugars replacing native d-desosamine. These metabolites were isolated and further purified using chromatographic techniques and their structures were determined as d-quinovosyl-10-deoxymethynolide, l-rhamnosyl-10-deoxymethynolide, l-olivosyl-10-deoxymethynolide, and d-boivinosyl-10-deoxymethynolide on the basis of 1D and 2D NMR and MS analyses and the stereochemistry of sugars was confirmed using coupling constant values and NOE correlations. Their antibacterial activities were evaluated in vitro against erythromycin-susceptible and -resistant Enterococcus faecium and Staphylococcus aureus. Substitution with l-rhamnose displayed better antibacterial activity than parent compound YC-17 containing native sugar d-desosamine. The present study on relationships between chemical structures and antibacterial activities could be useful in generation of novel advanced antibiotics utilizing combinatorial biosynthesis approach.