The Hedgehog (Hh) signaling pathway plays an instructional role during development, and is frequently activated in cancer. Ligand-induced pathway activation requires signaling by the transmembrane protein Smoothened (Smo), a member of the G-protein-coupled receptor (GPCR) superfamily. The extracellular (EC) loops of canonical GPCRs harbor cysteine residues that engage in disulfide bonds, affecting active and inactive signaling states through regulating receptor conformation, dimerization and/or ligand binding. Although a functional importance for cysteines localized to the N-terminal extracellular cysteine-rich domain has been described, a functional role for a set of conserved cysteines in the EC loops of Smo has not yet been established. In this study, we mutated each of the conserved EC cysteines, and tested for effects on Hh signal transduction. Cysteine mutagenesis reveals that previously uncharacterized functional roles exist for Smo EC1 and EC2. We provide in vitro and in vivo evidence that EC1 cysteine mutation induces significant Hh-independent Smo signaling, triggering a level of pathway activation similar to that of a maximal Hh response in Drosophila and mammalian systems. Furthermore, we show that a single amino acid change in EC2 attenuates Hh-induced Smo signaling, whereas deletion of the central region of EC2 renders Smo fully active, suggesting that the conformation of EC2 is crucial for regulated Smo activity. Taken together, these findings are consistent with loop cysteines engaging in disulfide bonds that facilitate a Smo conformation that is silent in the absence of Hh, but can transition to a fully active state in response to ligand. 相似文献
Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins whose role as a drug target against pathogenic microbes has been explored because of their stereo- and regio-specific oxidation activity. We aimed to assess the CYP53 family''s role as a common alternative drug target against animal (including human) and plant pathogenic fungi and its role in fungal-mediated wood degradation. Genome-wide analysis of fungal species revealed the presence of CYP53 members in ascomycetes and basidiomycetes. Basidiomycetes had a higher number of CYP53 members in their genomes than ascomycetes. Only two CYP53 subfamilies were found in ascomycetes and six subfamilies in basidiomycetes, suggesting that during the divergence of phyla ascomycetes lost CYP53 P450s. According to phylogenetic and gene-structure analysis, enrichment of CYP53 P450s in basidiomycetes occurred due to the extensive duplication of CYP53 P450s in their genomes. Numerous amino acids (103) were found to be conserved in the ascomycetes CYP53 P450s, against only seven in basidiomycetes CYP53 P450s. 3D-modelling and active-site cavity mapping data revealed that the ascomycetes CYP53 P450s have a highly conserved protein structure whereby 78% amino acids in the active-site cavity were found to be conserved. Because of this rigid nature of ascomycetes CYP53 P450s'' active site cavity, any inhibitor directed against this P450 family can serve as a common anti-fungal drug target, particularly toward pathogenic ascomycetes. The dynamic nature of basidiomycetes CYP53 P450s at a gene and protein level indicates that these P450s are destined to acquire novel functions. Functional analysis of CYP53 P450s strongly supported our hypothesis that the ascomycetes CYP53 P450s ability is limited for detoxification of toxic molecules, whereas basidiomycetes CYP53 P450s play an additional role, i.e. involvement in degradation of wood and its derived components. This study is the first report on genome-wide comparative structural (gene and protein structure-level) and evolutionary analysis of a fungal P450 family. 相似文献
Diet is now one of the well established means in the management of diabetes. Bitter gourd and spent turmeric at 10% level were tested for their efficacy on glycosaminoglycan metabolism in various tissues viz., liver, spleen, lungs, heart and testis in control, diabetic and treated rats. The glycosaminoglycans (GAGs) were isolated from defatted and dried tissues. The contents of sulfated GAGs decreased in all the tissues and the decrease was more prominent in heart and testis. In the isolated GAGs, contents of total sugar, amino sugar, uronic acid and sulfate were studied. Decrease in total sugar content was maximum in testis. Amino sugar content decreased considerably in testis (38%) and lungs (15%). The content of uronic acid also decreased in testis (33%) besides heart (29%) and liver (25%). Sulfate groups in GAGs perform pivotal functions in many biological events and decrease in sulfate content was significant in heart (40%), testis (37%) and liver (37%). GAGs profile on the cellulose acetate electrophoresis revealed that heparan sulfate (HS), hyaluronic acid (HA) and chondroitin sulfate/dermatan sulfate (CS/DS) were present in liver, spleen and lungs. HS, CS were present in heart, DS/CS was observed in testis. The observed beneficial effects in GAGs metabolism during diabetes may be due to the presence of high amounts of dietary fibres present in bitter gourd and spent turmeric, besides, possible presence of bioactive compounds in one or both of them. 相似文献
Mannose-6-phosphate receptors (MPRs) have been identified in a wide range of species from humans to invertebrates such as
molluscs. A characteristic of all MPRs is their common property to recognize mannose-6-phosphate residues that are labelling
lysosomal enzymes and to mediate their targeting to lysosomes in mammalian cells by the corresponding receptor proteins. We
present here the analysis of full-length sequences for MPR 46 from zebrafish (Danio rerio) and its functional analysis. This is the first non-mammalian MPR 46 to be characterised. The amino acid sequences of the
zebrafish MPR 46 displays 70% similarity to the human MPR 46 protein. In particular, all essential cysteine residues, the
transmembrane domain as well as the cytoplasmic tail residues harbouring the signals for endocytosis and Golgi-localizing,
γ-ear-containing, ARF-binding protein (GGA)-mediated sorting at the trans-Golgi network, are highly conserved. The zebrafish
MPR 46 has the arginine residue known to be essential for mannose-6-phosphate binding and other additional characteristic
residues of the mannose-6-phosphate ligand-binding pocket. Like the mammalian MPR 46, zebrafish MPR 46 binds to the multimeric
mannose-6-phosphate ligand phosphomannan and can rescue the missorting of lysosomal enzymes in mammalian MPR-deficient cells.
The conserved C-terminal acidic dileucine motif (DxxLL) in the cytoplasmic domain of zebrafish MPR 46 essential for the interaction
of the GGAs with the receptor domains interacts with the human GGA1-VHS domain. Interestingly, the serine residue suggested
to regulate the interaction between the tail and the GGAs in a phosphorylation-dependent manner is substituted by a proline
residue in fish.
Electronic Supplementary Material Supplementary material is available for this article at .
The zebrafish MPR 46 sequence data have been submitted to the GenBank database under accession no. DQ089037. 相似文献
Although a relationship between PDZK1 expression and estrogen receptor (ER)-α stimulation has been suggested, the nature of such a connection and the function of PDZK1 in breast cancer remain unknown. Human tissue microarrays (cancer tissue: 262 cores; normal tissue: 87 cores) and breast cancer cell lines were used to conduct the study. We show that PDZK1 protein expression is tightly correlated with human breast malignancy, is negatively correlated with age and had no significant correlation with ER-α expression levels. PDZK1 exhibited an exclusive epithelial expression with mostly cytosolic subcellular localization. Additionally, 17β-estradiol induced PDZK1 expression above its basal level more than 24 h after treatment in MCF-7 cells. PDZK1 expression was indirectly regulated by ER-α stimulation, requiring insulinlike growth factor 1 receptor (IGF-1R) expression and function. The molecular link between PDZK1 and IGF-1R was supported by a significant correlation between protein and mRNA levels (r = 0.591, p < 0.001, and r = 0.537, p < 0.001, respectively) of the two factors in two different cohorts of human breast cancer tissues. Interestingly, PDZK1 knockdown in MCF-7 cells blocked ER-dependent growth and reduced c-Myc expression, whereas ectopic expression of PDZK1 enhanced cell proliferation in the presence or absence of 17β-estradiol potentially through an increase in c-Myc expression, suggesting that PDZK1 has oncogenic activity. PDKZ1 also appeared to interact with the Src/ER-α/epidermal growth factor receptor (EGFR) complex, but not with IGF-1R and enhanced EGFR-stimulated MEK/ERK1/2 signaling. Collectively, our results clarify the relationship between ER-α and PDZK1, propose a direct relationship between PDZK1 and IGF-1R, and identify a novel oncogenic activity for PDZK1 in breast cancer. 相似文献
Pancreatic cancer is a highly aggressive form of cancer with a poor prognosis, partly due to ‘austerity’, a phenomenon of tolerance to nutrient deprivation and survival in its hypovascular tumor microenvironment. Anti-austerity agents which preferentially diminish the survival of cancer cells under nutrition starvation is regarded as new generation anti-cancer agents. This study investigated the potential of Piper longum constituents as anti-austerity agents. The ethanolic extract of Piper longum was found to have preferential cytotoxicity towards PANC-1 human pancreatic cancer cells in a nutrient-deprived medium (NDM). Further investigation led to the identification of pipernonaline ( 3 ) as the lead compound with the strongest anti-austerity activity, inducing cell death and inhibiting migration in a normal nutrient medium, as well as strongly inhibiting the Akt/mTOR/autophagy pathway. Therefore, pipernonaline ( 3 ) holds promise as a novel antiausterity agent for the treatment of pancreatic cancer. 相似文献
This study evaluated the effect of inorganic mercury (Hg) on bacterial community and diversity in different soils. Three soils—neutral, alkaline and acidic—were spiked with six different concentrations of Hg ranging from 0 to 200 mg kg−1 and aged for 90 days. At the end of the ageing period, 18 samples from three different soils were investigated for bacterial community structure and soil physicochemical properties. Illumina MiSeq-based 16s ribosomal RNA (rRNA) amplicon sequencing revealed the alteration in the bacterial community between un-spiked control soils and Hg-spiked soils. Among the bacterial groups, Actinobacteria (22.65%) were the most abundant phyla in all samples followed by Proteobacteria (21.95%), Bacteroidetes (4.15%), Firmicutes (2.9%) and Acidobacteria (2.04%). However, the largest group showing increased abundance with higher Hg doses was the unclassified group (45.86%), followed by Proteobacteria. Mercury had a considerable negative impact on key soil functional bacteria such as ammonium oxidizers and nitrifiers. Canonical correspondence analysis (CCA) indicated that among the measured soil properties, Hg had a major influence on bacterial community structure. Furthermore, nonlinear regression analysis confirmed that Hg significantly decreased soil bacterial alpha diversity in lower organic carbon containing neutral and alkaline soils, whereas in acidic soil with higher organic carbon there was no significant correlation. EC20 values obtained by a nonlinear regression analysis indicated that Hg significantly decreased soil bacterial diversity in concentrations lower than several guideline values.
Conjugated linoleic acid (CLA), a dietary lipid, has been proposed as an antidiabetic agent. However, studies specifically addressing the molecular dynamics of CLA on skeletal muscle glucose transport and differences between the key isomers are limited. We demonstrate that acute exposure of L6 myotubes to cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12) CLA isomers mimics insulin action by stimulating glucose uptake and glucose transporter-4 (GLUT4) trafficking. Both c9,t10-CLA and t10,c12-CLA stimulate the phosphorylation of phosphatidylinositol 3-kinase (PI3-kinase) p85 subunit and Akt substrate-160 kDa (AS160), while showing isomer-specific effects on AMP-activated protein kinase (AMPK). CLA isomers showed synergistic effects with the AMPK activator, 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleoside (AICAR). Blocking PI3-kinase and AMPK prevented the stimulatory effects of t10,c12-CLA on AS160 phosphorylation and glucose uptake, indicating that this isomer acts via a PI3-kinase and AMPK-dependent mechanism, whereas the mechanism of c9,t11-CLA remains unclear. Intriguingly, CLA isomers sensitized insulin-Akt-responsive glucose uptake and prevented high insulin-induced Akt desensitisation. Together, these results establish that CLA exhibits isomer-specific effects on GLUT4 trafficking and the increase in glucose uptake induced by CLA treatment of L6 myotubes occurs via pathways that are distinctive from those utilised by insulin. 相似文献
Interest is increasing in developing fluorescent ligands for characterization of adenosine receptors (ARs), which hold a promise of usefulness in the drug discovery process. The size of a strategically labeled AR ligand can be greatly increased after the attachment of a fluorophore. The choice of dye moiety (e.g. Alexa Fluor 488), attachment point and linker length can alter the selectivity and potency of the parent molecule. Fluorescent derivatives of adenosine agonists and antagonists (e.g. XAC and other heterocyclic antagonist scaffolds) have been synthesized and characterized pharmacologically. Some are useful AR probes for flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer, and scanning confocal microscopy. Thus, the approach of fluorescent labeled GPCR ligands, including those for ARs, is a growing dynamic research field. 相似文献