The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/β-catenin signaling, ...however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced β-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced β-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated β-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4-/- crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.
Reoccurring/high-risk neuroblastoma (NB) tumors have the enrichment of non-RAS/RAF mutations along the mitogen-activated protein kinase (MAPK) signaling pathway, suggesting that activation of MEK/ERK ...is critical for their survival. However, based on preclinical data, MEK inhibitors are unlikely to be active in NB and have demonstrated dose-limiting toxicities that limit their use. Here, we explore an alternative way to target the MAPK pathway in high-risk NB. We find that NB models are among the most sensitive among over 900 tumor-derived cell lines to the allosteric SHP2 inhibitor SHP099. Sensitivity to SHP099 in NB is greater in models with loss or low expression of the RAS GTPase activation protein (GAP) neurofibromin 1 (NF1). Furthermore, NF1 is lower in advanced and relapsed NB and NF1 loss is enriched in high-risk NB tumors regardless of MYCN status. SHP2 inhibition consistently blocks tumor growth in high-risk NB mouse models, revealing a new drug target in relapsed NB.
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•We identify neuroblastoma models as sensitive to SHP2 inhibition•SHP099 sensitivity is correlated with low expression of neurofibromin (NF1)•SHP2 inhibitors inhibit pERK in neuroblastoma cells better than in normal cells•NF1 mutation/expression may predict SHP2 inhibitor response in neuroblastoma
In this paper, Cai et al. demonstrate that high-risk neuroblastomas with low NF1 expression are sensitive to SHP2 inhibitors, which may have treatment advantages over MEK inhibitors. Targeting SHP2 blocks neuroblastoma tumor growth. As several SHP2 inhibitors are in clinical trials, SHP2 inhibitors may benefit high-risk NB patients.
Protein tyrosine phosphatase SHP2 is an oncoprotein associated with cancer as well as a potential immune modulator because of its role in the programmed cell death PD-L1/PD-1 pathway. In the ...preceding manuscript, we described the optimization of a fused, bicyclic screening hit for potency, selectivity, and physicochemical properties in order to further expand the chemical diversity of allosteric SHP2 inhibitors. In this manuscript, we describe the further expansion of our approach, morphing the fused, bicyclic system into a novel monocyclic pyrimidinone scaffold through our understanding of SAR and use of structure-based design. These studies led to the identification of SHP394 (1), an orally efficacious inhibitor of SHP2, with high lipophilic efficiency, improved potency, and enhanced pharmacokinetic properties. We also report other pyrimidinone analogues with favorable pharmacokinetic and potency profiles. Overall, this work improves upon our previously described allosteric inhibitors and exemplifies and extends the range of permissible chemical templates that inhibit SHP2 via the allosteric mechanism.
The Wnt/beta-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/beta-catenin signaling, ...however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced beta-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced beta-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated beta-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4-/- crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.
Obesity, type 2 diabetes and cancer are serious threats to human health and are increasing at an alarming rate. Dysregulation of energy homeostasis, both at the whole body and cellular level, has ...been implicated in the pathogenesis of those disorders. Cellular energy homeostasis is a delicate balance between ATP production and consumption. ATP production, mainly from oxidative phosphorylation in mitochondria, is responsive to nutrient availability and cellular energy demand. Therefore, nutrient sensing is critical to maintain cellular energy homeostasis. There are two well-characterized nutrient sensing kinases, AMPK and mTOR. We studied another nutrient-responsive kinase named PASK. Using PASK-/- mice, the role of PASK in cellular energy homeostasis was revealed. PASK deletion caused nearly complete protection from the deleterious effects of a high-fat diet, including obesity, insulin resistance and hepatic steatosis. This protection is likely due to the increased metabolic rate of PASK -/- mice, which can be recapitulated in cultured cells upon PASK knockdown. Therefore, we conclude that PASK acts as a cell-autonomous metabolic sensor to maintain cellular energy homeostasis. In addition to PASK, we studied a previously uncharacterized but highly conserved mitochondrial protein that we later named Sdh5. Using yeast as primary model system, we showed that Sdh5 is necessary and probably sufficient for insertion of the requisite FAD cofactor into the catalytic subunit of the succinate dehydrogenase (SDH) complex. SDH deficiency has been associated with several types of cancer, predominantly paraganglioma (PGL). Indeed, three out of four familial PGL genes have been mapped to SDH subunits. We discovered a point mutation in the human SDH5 gene that perfectly cosegregates with disease in a Dutch PGL2 lineage. This mutation completely abolishes hSDH5 function and, as expected, PGL2 tumors exhibit a loss of FAD insertion in succinate dehydrogenase. Our studies of PASK and SDH5 not only contribute significant conceptual advances in cellular nutrient sensing and mitochondrial metabolism, but also provides new screening biomarkers and therapeutic targets for diagnosing and treating the metabolic syndrome and cancer.
Considerable knowledge has been accumulated on the lactic acid bacteria (LAB) that affect the aroma and flavour of yogurt. This review focuses on the role of LAB in the production of flavour ...compounds during yogurt fermentation. The biochemical processes of flavour compound formation by LAB including glycolysis, proteolysis, and lipolysis are summarised, with some key compounds described in detail. The flavour-related activities of LAB mostly depend on the species used for yogurt fermentation, and some strategies have been developed to obtain more control of the flavour-forming process. Metabolic engineering can be a powerful tool to reroute the metabolic flux towards the efficient accumulation of the desired flavour compounds with the knowledge of the complex network of flavour-forming pathways and the availability of genetic tools. Further progress made in the omics-based techniques and the use of systems biology approaches are needed to fully understand, control, and steer flavour formation in yogurt fermentation processes.
A novel photoelectrochemical hydrogen peroxide (H2O2) sensor was constructed with platinum (Pt) and nickel hydroxyl-oxide (NiOOH) double layers modified n-silicon electrode (NiOOH/Pt/n–n+-Si). About ...40nm Pt layer and about 100nm Ni layer were successively coated on the front surface of n–n+-Si (111) wafers by vacuum evaporating. A stable layer of NiOOH was formed through oxidation of the Ni layer on the coated silicon wafer by the electrochemical method. The surface of modified electrode was characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The NiOOH/Pt/n–n+-Si electrode has been used for determination of H2O2 with a two-electrode cell in the absence of reference electrode by photocurrent measurement at a zero bias. The photoelectrochemical sensor showed a good linear response to H2O2 concentrations in a range from 1.0×10−5 to 6×10−5M with a determination limit (S/N=3) of 2.2μM. The NiOOH/Pt/n–n+-Si electrode exhibited excellent reproducibility and stability. Particularly, the facile measurement requirements made this novel modified electrode promising for the development of outdoor H2O2 sensors.
•We introduce a novel photoelectrochemical sensor for rapid detection of hydrogen peroxide.•Nickel hydroxyl-oxide double layer coated n-silicon electrode was used as the photoelectrode.•The flexible sensor lies in its two-electrode system with no reference electrode and no bias voltage.•The new platform opens new applications in portable sensor of hydrogen peroxide.
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The aroma types of fermented milk produced by
L. bulgaricus
were divided into milky-type, cheesy-type, fermented-type and miscellaneous-type.
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The flavor fingerprints of different aroma types were ...established by GC-IMS.
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Acetaldehyde, 2,3-butanedione, acetic acid, butanoic acid, hexanoic acid and δ-decalactone of different aroma types were determined by Flavoromics.
The aroma of the fermented milk produced by twenty-eight
Lactobacillus delbrueckii
subsp.
bulgaricus
strains was evaluated via quantitative descriptive analysis. According to the sensory analysis results, the fermented milks were grouped into milky-type, cheesy-type, fermented-type and miscellaneous-type. The representative samples of cheese-type and fermented-type were analyzed by headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) and flavoromics. A total of 95 volatile compounds were identified and particularly, 12 aroma-active compounds were detected by using gas chromatography-olfactometry-mass spectrometry (GC-O-MS). Among the different aroma types, 2,3-butanedione, δ-decalactone, acetaldehyde, butanoic acid, acetic acid and hexanoic acid were finally screened out as the key aroma-active compounds by quantitative and odor activity value (OAV) analysis combined with aroma recombination, omission and addition experiments. These findings were valuable in developing specific fermented milk products with different aroma profiles.
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