S6K1 controls adiponectin expression by inducing a transcriptional switch: BMAL1-to-EZH2.

Adiponectin (encoded by Adipoq), a fat-derived hormone, alleviates risk factors associated with metabolic disorders. Although many transcription factors are known to control adiponectin expression, the mechanism underlying its fluctuation with regard to metabolic status remains unclear. Here, we show that ribosomal protein S6 kinase 1 (S6K1) controls adiponectin expression by inducing a transcriptional switch between two transcriptional machineries, BMAL1 and EZH2. Active S6K1 induced a suppressive histone code cascade, H2BS36p-EZH2-H3K27me3, leading to suppression of adiponectin expression. Moreover, active S6K1 phosphorylated BMAL1, an important transcription factor regulating the circadian clock system, at serine 42, which led to its dissociation from the Adipoq promoter region. This response resulted in EZH2 recruitment and subsequent H3K27me3 modification of the Adipoq promoter. Upon fasting, inactivation of S6K1 induced the opposite transcriptional switch, EZH2-to-BMAL1, promoting adiponectin expression. Consistently, S6K1-depleted mice exhibited lower H3K27me3 levels and elevated adiponectin expression. These findings identify a novel epigenetic switch system by which S6K1 controls the production of adiponectin, which displays beneficial effects on metabolism.

Nuclear S6K1 regulates cAMP-responsive element-dependent gene transcription through activation of mTOR signal pathway.

S6K1 serves as an important signaling regulator of cell proliferation and growth in the mTOR signaling pathway. Excessive activation of the mTOR/S6K1 signaling pathway promotes abnormal cell growth and survival, thereby resulting in tumorigenesis. The roles of S6K1 in protein synthesis and metabolism are well known, but an additional role of S6K1 as a gene transcription regulator has not been much understood. Here, we demonstrated that S6K1 is dynamically distributed in the cytoplasm and nuclei of human cervical cancer cells. S6K1 nuclear localization was serum dependent and serum deprivation or rapamycin treatment inhibited S6K1 Thr389 phosphorylation and, thereby, S6K1 was retained in the cytoplasm. Furthermore, we found that endogenous S6K1 interacted with CREB in the cervical cancer cells. Additionally, S6K1 upregulated the CRE-driven promoter luciferase activity. The proto-oncogene c-JUN, which has several CREs, was attenuated in the S6K1 knockdown cervical cancer cells. The binding of CREB/S6K1 to the c-JUN promoter, altered by serum restimulation, was associated with active epigenetic markers. In HeLa cell, 891 promoter regions, to which S6K1 directly binds, were detected. Our findings suggested that active S6K1, which is dynamically translocated into the nucleus, directly binds to chromatin and could play a role in epigenetic mechanisms or transcription factor recruitment.

Transcriptomics-Based Repositioning of Natural Compound, Eudesmin, as a PRC2 Modulator.

Extensive epigenetic remodeling occurs during the cell fate determination of stem cells. Previously, we discovered that eudesmin regulates lineage commitment of mesenchymal stem cells through the inhibition of signaling molecules. However, the epigenetic modulations upon eudesmin treatment in genomewide level have not been analyzed. Here, we present a transcriptome profiling data showing the enrichment in PRC2 target genes by eudesmin treatment. Furthermore, gene ontology analysis showed that PRC2 target genes downregulated by eudesmin are closely related to Wnt signaling and pluripotency. We selected DKK1 as an eudesmin-dependent potential top hub gene in the Wnt signaling and pluripotency. Through the ChIP-qPCR and RT-qPCR, we found that eudesmin treatment increased the occupancy of PRC2 components, EZH2 and SUZ12, and H3K27me3 level on the promoter region of DKK1, downregulating its transcription level. According to the analysis of GEO profiles, DEGs by depletion of Oct4 showed an opposite pattern to DEGs by eudesmin treatment. Indeed, the expression of pluripotency markers, Oct4, Sox2, and Nanog, was upregulated upon eudesmin treatment. This finding demonstrates that pharmacological modulation of PRC2 dynamics by eudesmin might control Wnt signaling and maintain pluripotency of stem cells.

Increased synthesis of hyaluronic acid by enhanced penetration of CTP-EGF recombinant in human keratinocytes.

BACKGROUND: Epidermal growth factor (EGF) plays an important role in regeneration and proliferation of skin cells. It synthesizes fibrous proteins, such as collagen, and induces the proliferation of keratinocytes and fibroblasts. It can also induce hyaluronic acid synthesis, which subsequently leads to improved skin elasticity, wrinkle improvement, and moisturizing effects. Thus, the EGF is an attractive cosmetic additive for skin care. OBJECTIVES: We tested the use of cytoplasmic transduction peptide (CTP) as a delivery peptide for EGF into skin cells. Additionally, we characterized the skin permeability of CTP-EGF for its potential use in skin antiaging and antiwrinkle cosmetics. METHODS: Skin penetration by recombinant CTP-EGF protein was confirmed using fluorescent imaging techniques. The ability to synthesize hyaluronic acid was confirmed by immunoblotting and ELISA. RESULTS: CTP-EGF displayed cell membrane permeability and could penetrate skin cells. Treatment with CTP-EGF increased collagen protein formation, which is a major regulator of skin elasticity. Further, CTP-EGF treatment led to increased expression of HAS3 enzyme and subsequently boosted hyaluronic acid synthesis. The CTP-EGF also performed better than natural EGF in wound healing assays. CONCLUSIONS: CTP-EGF has a superior ability, compared with natural EGF, to permeate skin and induce hyaluronic acid synthesis and collagen formation. Thus, it has great potential to be used in cosmetics and therapeutic agents to improve wrinkles and health of the skin.

Helicobacter pylori flagellin: TLR5 evasion and fusion-based conversion into a TLR5 agonist.

Helicobacter pylori is a flagellated bacterium of the Epsilonproteobacteria class that causes peptic ulcers. Flagellin is a primary structural protein that assembles into the flagellar filament. Flagellins from bacteria that belong to the Gammaproteobacteria and Firmicutes groups are detected by Toll-like receptor 5 (TLR5) in the host, triggering the innate immune response, and thus have been studied for the development of vaccines against diverse infections through fusion with protein antigens. However, H. pylori flagellin (hFlg) does not stimulate TLR5, allowing H. pylori to evade TLR5-mediated immune surveillance. The unresponsiveness of TLR5 to hFlg, along with the tendency of the hFlg protein to precipitate, limits the utility of hFlg for H. pylori vaccine development. Here, we report a soluble hFlg derivative protein that activates TLR5. We performed expression and purification screens with full-length and fragment hFlg proteins and identified the hypervariable domains as the soluble part of hFlg. The hypervariable domains of hFlg were engineered into a TLR5 agonist through fusion with the TLR5-activating Bacillus subtilis flagellin. Furthermore, based on comparative sequence and mutation analyses, we reveal that hFlg evolved to evade TLR5 detection by modifying residues that correspond to a TLR5-activation hot spot.

A conserved TLR5 binding and activation hot spot on flagellin.

Flagellin is a bacterial protein that polymerizes into the flagellar filament and is essential for bacterial motility. When flagellated bacteria invade the host, flagellin is recognized by Toll-like receptor 5 (TLR5) as a pathogen invasion signal and eventually evokes the innate immune response. Here, we provide a conserved structural mechanism by which flagellins from Gram-negative γ-proteobacteria and Gram-positive Firmicutes bacteria bind and activate TLR5. The comparative structural analysis using our crystal structure of a complex between Bacillus subtilis flagellin (bsflagellin) and TLR5 at 2.1 Šresolution, combined with the alanine scanning analysis of the binding interface, reveals a common hot spot in flagellin for TLR5 activation. An arginine residue (bsflagellin R89) of the flagellin D1 domain and its adjacent residues (bsflagellin E114 and L93) constitute a hot spot that provides shape and chemical complementarity to a cavity generated by the loop of leucine-rich repeat 9 in TLR5. In addition to the flagellin D1 domain, the D0 domain also contributes to TLR5 activity through structurally dispersed regions, but not a single focal area. These results establish the groundwork for the future design of flagellin-based therapeutics.

S6K1 Phosphorylation of H2B Mediates EZH2 Trimethylation of H3: A Determinant of Early Adipogenesis.

S6K1 has been implicated in a number of key metabolic responses, which contribute to obesity. Critical among these is the control of a transcriptional program required for the commitment of mesenchymal stem cells to the adipocytic lineage. However, in contrast to its role in the cytosol, the functions and targets of nuclear S6K1 are unknown. Here, we show that adipogenic stimuli trigger nuclear translocation of S6K1, leading to H2BS36 phosphorylation and recruitment of EZH2 to H3, which mediates H3K27 trimethylation. This blocks Wnt gene expression, inducing the upregulation of PPARγ and Cebpa and driving increased adipogenesis. Consistent with this finding, white adipose tissue from S6K1-deficient mice exhibits no detectable H2BS36 phosphorylation or H3K27 trimethylation, whereas both responses are highly elevated in obese humans or in mice fed a high-fat diet. These findings define an S6K1-dependent mechanism in early adipogenesis, contributing to the promotion of obesity.

Structural and biochemical characterization of bacterial YpgQ protein reveals a metal-dependent nucleotide pyrophosphohydrolase.

The optimal balance of cellular nucleotides and the efficient elimination of non-canonical nucleotides are critical to avoiding erroneous mutation during DNA replication. One such mechanism involves the degradation of excessive or abnormal nucleotides by nucleotide-hydrolyzing enzymes. YpgQ contains the histidine-aspartate (HD) domain that is involved in the hydrolysis of nucleotides or nucleic acids, but the enzymatic activity and substrate specificity of YpgQ have never been characterized. Here, we unravel the catalytic activity and structural features of YpgQ to report the first Mn(2+)-dependent pyrophosphohydrolase that hydrolyzes (deoxy)ribonucleoside triphosphate [(d)NTP] to (deoxy)ribonucleoside monophosphate and pyrophosphate using the HD domain. YpgQ from Bacillus subtilis (bsYpgQ) displays a helical structure and assembles into a unique dimeric architecture that has not been observed in other HD domain-containing proteins. Each bsYpgQ monomer accommodates a metal ion and a nucleotide substrate in a cavity located between the N- and C-terminal lobes. The metal cofactor is coordinated by the canonical residues of the HD domain, namely, two histidine residues and two aspartate residues, and is positioned in close proximity to the ß-phosphate group of the nucleotide, allowing us to propose a nucleophilic attack mechanism for the nucleotide hydrolysis reaction. YpgQ enzymes from other bacterial species also catalyze pyrophosphohydrolysis but exhibit different substrate specificity. Comparative structural and mutational studies demonstrated that residues outside the major substrate-binding site of bsYpgQ are responsible for the species-specific substrate preference. Taken together, our structural and biochemical analyses highlight the substrate-recognition mode and catalysis mechanism of YpgQ in pyrophosphohydrolysis.

Purification, crystallization and preliminary X-ray analysis of a putative nucleotide phosphohydrolase, YpgQ, from Bacillus subtilis.

The histidine-aspartate (HD) domain exerts phosphohydrolase activity on nucleotides and functions in nucleotide metabolism. Sequence analysis suggested that YpgQ from Bacillus subtilis contains the HD domain, but the structure and function of YpgQ remain to be revealed. The recombinant YpgQ protein was overexpressed in an Escherichia coli cell expression system and was purified to homogeneity by Ni-NTA affinity and anion-exchange chromatography. Crystals in space group P21 were obtained in PEG 600 solutions and diffracted X-rays to 2.3 Šresolution. Moreover, X-ray fluorescence scans on YpgQ crystals demonstrated the metal-binding ability of YpgQ.

Destabilization of TNF-α mRNA by Rapamycin.

Stimulation of mast cells through the high affinity IgE receptor (FcεRI) induces degranulation, lipid mediator release, and cytokine secretion leading to allergic reactions. Although various signaling pathways have been characterized to be involved in the FcεRI-mediated responses, little is known about the precious mechanism for the expression of tumor necrosis factor-α (TNF-α) in mast cells. Here, we report that rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR), reduces the expression of TNF-α in rat basophilic leukemia (RBL-2H3) cells. IgE or specific antigen stimulation of RBL-2H3 cells increases the expression of TNF-α and activates various signaling molecules including S6K1, Akt and p38 MAPK. Rapamycin specifically inhibits antigen-induced TNF-α mRNA level, while other kinase inhibitors have no effect on TNF-α mRNA level. These data indicate that mTOR signaling pathway is the main regulation mechanism for antigen-induced TNF-α expression. TNF-α mRNA stability analysis using reporter construct containing TNF-α adenylate/uridylate-rich elements (AREs) shows that rapamycin destabilizes TNF-α mRNA via regulating the AU-rich element of TNF-α mRNA. The antigen-induced activation of S6K1 is inhibited by specific kinase inhibitors including mTOR, PI3K, PKC and Ca(2+)chelator inhibitor, while TNF-α mRNA level is reduced only by rapamycin treatment. These data suggest that the effects of rapamycin on the expression of TNF-α mRNA are not mediated by S6K1 but regulated by mTOR. Taken together, our results reveal that mTOR signaling pathway is a novel regulation mechanism for antigen-induced TNF-α expression in RBL-2H3 cells.