共查询到20条相似文献,搜索用时 15 毫秒
1.
Akira Minami Tadamune Otsubo Daisuke Ieno Kiyoshi Ikeda Hiroaki Kanazawa Kosuke Shimizu Ko Ohata Tsunehiro Yokochi Yuuki Horii Hokuto Fukumoto Risa Taguchi Tadanobu Takahashi Naoto Oku Takashi Suzuki 《PloS one》2014,9(1)
Sialidase removes sialic acid from sialoglycoconjugates and plays crucial roles in many physiological and pathological processes. Various human cancers express an abnormally high level of the plasma membrane-associated sialidase isoform.Visualization of sialidase activity in living mammalian tissues would be useful not only for understanding sialidase functions but also for cancer diagnosis. However, since enzyme activity of mammalian sialidase is remarkably weak compared with that of bacterial and viral sialidases, it has been difficult to detect sialidase activity in mammalian tissues. We synthesized a novel benzothiazolylphenol-based sialic acid derivative (BTP-Neu5Ac) as a fluorescent sialidase substrate. BTP-Neu5Ac can visualize sialidase activities sensitively and selectively in acute rat brain slices. Cancer cells implanted orthotopically in mouse colons and human colon cancers (stages T3-T4) were also clearly detected with BTP-Neu5Ac. The results suggest that BTP-Neu5Ac is useful for histochemical imaging of sialidase activities. 相似文献
2.
哺乳动物细胞内的某些蛋白质或脂类可以被糖基化修饰,而糖链末端往往存在唾液酸化的现象,催化添加唾液酸的酶为糖基转移酶(sialyltransferase,ST),而去除唾液酸的为唾液酸酶(sialidase,SA或称为neuraminidase,NEU).本实验检测了人膀胱正常上皮细胞HCV29、非浸润性膀胱癌细胞KK47和浸润性膀胱癌细胞YTS-1中唾液酸的表达,发现恶性肿瘤细胞中唾液酸的含量高于正常细胞;进一步分析唾液酸酶和唾液酸转移酶的表达,发现唾液酸酶Neu1在正常细胞中表达最高,在良性肿瘤细胞中次之,在恶性肿瘤细胞中表达最低,推测在膀胱癌中Neu1对唾液酸的异常表达起着主要作用.同时,膀胱癌细胞中Toll样受体1,2,3,4(toll-like receptors,TLRs)表达趋势也与Neu1一致.利用TGF-β处理HCV29,使之发生上皮间质转化(epithelial-mesenchymal transition,EMT),细胞中Neu1和TLR3表达明显减少;将Neu1基因沉默后,TLR3表达也明显减少.此外,在YTS-1细胞中过表达Neu1,TLR3表达增高且激活了下游NF-κB通路.这一结果说明膀胱癌中Neu1与TLR3的表达有着密切的关系,这为膀胱癌的分子机理研究提供了工作基础. 相似文献
3.
Modulation of Neuritogenesis by Ganglioside-Specific Sialidase (Neu 3) in Human Neuroblastoma NB-1 Cells 总被引:3,自引:0,他引:3
Plasma membrane-associated sialidase (Neu 3), which specifically hydrolyzes gangliosides, is relatively abundantly present in the nervous system. To understand the role of Neu 3 in neuronal differentiation, we studied the relationship between neurite outgrowth and Neu 3 expression in human neuroblastoma NB-1 cells. The expression of Neu 3 in NB-1 cells increased when neurite outgrowth in these cells was induced by dibutyryl cAMP. While treatment with dibutyryl cAMP alone enhanced the outgrowth of dendrite-like processes, transfection of the Neu 3 gave rise to a more prominent outgrowth of neurites with axon-like characteristics, even in the absence of dibutyryl cAMP. Neu 3 induction by dibutyryl cAMP is probably attributable, in part, to transactivation of the Neu 3 gene through cAMP responsive elements in the 5-upstream region, as revealed by the promotor activity assay using Neu 3 promotor expression plasmid. These results indicate that Neu 3 regulates neurite formation in NB-1 cells, and suggest that this effect may be enhanced by dibutyryl cAMP via a cAMP-dependent pathway. 相似文献
4.
Sialidase Activity in Nuclear Membranes of Rat Brain 总被引:1,自引:1,他引:0
Abstract: A highly purified nuclear membrane preparation was obtained from adult rat brain and examined for sialidase activity using GM3, GD1a, GD1b, or N -acetylneuramin lactitol as the substrate. The nuclear membranes contained an appreciable level of sialidase activity; the specific activities toward GM3 and N -acetylneuramin lactitol were 20.5 and 23.8% of the activities in the total brain homogenate, respectively. The sialidase activity in nuclear membranes showed substrate specificity distinct from other membrane-bound sialidases localized in lysosomal membranes, synaptosomal plasma membranes, or myelin membranes. These results strongly suggest the existence of a sialidase activity associated with the nuclear membranes from rat brain. 相似文献
5.
The mechanism(s) behind GPCR transactivation of TLR receptors independent of TLR ligands is unknown. Here, GPCR agonists bombesin, bradykinin, lysophosphatidic acid (LPA), cholesterol, angiotensin-1 and -2, but not thrombin induce Neu1 activity in live macrophage cell lines and primary bone marrow macrophage cells from wild-type (WT) mice but not from Neu1-deficient mice. Using immunocytochemistry and NFκB-dependent secretory alkaline phosphatase (SEAP) analyses, bombesin induced NFκB activation in BMC-2 and RAW-blue macrophage cells, which was inhibited by MyD88 homodimerization inhibitor, Tamiflu, galardin, piperazine and anti-MMP-9 antibody. Bombesin receptor, neuromedin B (NMBR), forms a complex with TLR4 and MMP9. Silencing MMP9 mRNA using siRNA transfection of RAW-blue macrophage cells markedly reduced Neu1 activity associated with bombesin-, bradykinin- and LPA-treated cells to the untreated controls. These findings uncover a molecular organizational GPCR signaling platform to potentiate Neu1 and MMP-9 cross-talk on the cell surface that is essential for the transactivation of TLR receptors and subsequent cellular signaling. 相似文献
6.
Caroline Bull Melsom ?ivind ?rstavik Jan-Bj?rn Osnes Tor Skomedal Finn Olav Levy Kurt Allen Krobert 《PloS one》2014,9(9)
Background and purpose
Despite the view that only β2- as opposed to β1-adrenoceptors (βARs) couple to Gi, some data indicate that the β1AR-evoked inotropic response is also influenced by the inhibition of Gi. Therefore, we wanted to determine if Gi exerts tonic receptor-independent inhibition upon basal adenylyl cyclase (AC) activity in cardiomyocytes.Experimental approach
We used the Gs-selective (R,R)- and the Gs- and Gi-activating (R,S)-fenoterol to selectively activate β2ARs (β1AR blockade present) in combination with Gi inactivation with pertussis toxin (PTX). We also determined the effect of PTX upon basal and forskolin-mediated responses. Contractility was measured ex vivo in left ventricular strips and cAMP accumulation was measured in isolated ventricular cardiomyocytes from adult Wistar rats.Key results
PTX amplified both the (R,R)- and (R,S)-fenoterol-evoked maximal inotropic response and concentration-dependent increases in cAMP accumulation. The EC50 values of fenoterol matched published binding affinities. The PTX enhancement of the Gs-selective (R,R)-fenoterol-mediated responses suggests that Gi regulates AC activity independent of receptor coupling to Gi protein. Consistent with this hypothesis, forskolin-evoked cAMP accumulation was increased and inotropic responses to forskolin were potentiated by PTX treatment. In non-PTX-treated tissue, phosphodiesterase (PDE) 3 and 4 inhibition or removal of either constitutive muscarinic receptor activation of Gi with atropine or removal of constitutive adenosine receptor activation with CGS 15943 had no effect upon contractility. However, in PTX-treated tissue, PDE3 and 4 inhibition alone increased basal levels of cAMP and accordingly evoked a large inotropic response.Conclusions and implications
Together, these data indicate that Gi exerts intrinsic receptor-independent inhibitory activity upon AC. We propose that PTX treatment shifts the balance of intrinsic Gi and Gs activity upon AC towards Gs, enhancing the effect of all cAMP-mediated inotropic agents. 相似文献7.
Jeroen F. J. Bogie Silke Timmermans Van Anh Huynh-Thu Alexandre Irrthum Hubert J. M. Smeets Jan-?ke Gustafsson Knut R. Steffensen Monique Mulder Piet Stinissen Niels Hellings Jerome J. A. Hendriks 《PloS one》2012,7(9)
Multiple sclerosis is a chronic, inflammatory, demyelinating disease of the central nervous system in which macrophages and microglia play a central role. Foamy macrophages and microglia, containing degenerated myelin, are abundantly found in active multiple sclerosis lesions. Recent studies have described an altered macrophage phenotype after myelin internalization. However, it is unclear by which mechanisms myelin affects the phenotype of macrophages and how this phenotype can influence lesion progression. Here we demonstrate, by using genome wide gene expression analysis, that myelin-phagocytosing macrophages have an enhanced expression of genes involved in migration, phagocytosis and inflammation. Interestingly, myelin internalization also induced the expression of genes involved in liver-X-receptor signaling and cholesterol efflux. In vitro validation shows that myelin-phagocytosing macrophages indeed have an increased capacity to dispose intracellular cholesterol. In addition, myelin suppresses the secretion of the pro-inflammatory mediator IL-6 by macrophages, which was mediated by activation of liver-X-receptor β. Our data show that myelin modulates the phenotype of macrophages by nuclear receptor activation, which may subsequently affect lesion progression in demyelinating diseases such as multiple sclerosis. 相似文献
8.
The precise mechanism(s) by which intracellular TOLL-like receptors (TLRs) become activated by their ligands remains unclear. Here, we report a molecular organizational G-protein coupled receptor (GPCR) signaling platform to potentiate a novel mammalian neuraminidase-1 (Neu1) and matrix metalloproteinase-9 (MMP-9) cross-talk in alliance with neuromedin B GPCR, all of which form a tripartite complex with TLR-7 and -9. siRNA silencing Neu1, MMP-9 and neuromedin-B GPCR in RAW-blue macrophage cells significantly reduced TLR7 imiquimod- and TLR9 ODN1826-induced NF-κB (NF-κB-pSer536) activity. Tamiflu, specific MMP-9 inhibitor, neuromedin B receptor specific antagonist BIM23127, and the selective inhibitor of whole heterotrimeric G-protein complex BIM-46174 significantly block nucleic acid-induced TLR-7 and -9 MyD88 recruitment, NF-κB activation and proinflammatory TNFα and MCP-1 cytokine responses. For the first time, Neu1 clearly plays a central role in mediating nucleic acid-induced intracellular TLR activation, and the interactions involving NMBR–MMP9–Neu1 cross-talk constitute a novel intracellular TLR signaling platform that is essential for NF-κB activation and pro-inflammatory responses. 相似文献
9.
Dario Bonardi Viola Ravasio Giuseppe Borsani Alessandra d'Azzo Roberto Bresciani Eugenio Monti Edoardo Giacopuzzi 《PloS one》2014,9(8)
The NEU1 gene is the first identified member of the human sialidases, glycohydrolitic enzymes that remove the terminal sialic acid from oligosaccharide chains. Mutations in NEU1 gene are causative of sialidosis (MIM 256550), a severe lysosomal storage disorder showing autosomal recessive mode of inheritance. Sialidosis has been classified into two subtypes: sialidosis type I, a normomorphic, late-onset form, and sialidosis type II, a more severe neonatal or early-onset form. A total of 50 causative mutations are reported in HGMD database, most of which are missense variants. To further characterize the NEU1 gene and identify new functionally relevant protein isoforms, we decided to study its genetic variability in the human population using the data generated by two large sequencing projects: the 1000 Genomes Project (1000G) and the NHLBI GO Exome Sequencing Project (ESP). Together these two datasets comprise a cohort of 7595 sequenced individuals, making it possible to identify rare variants and dissect population specific ones. By integrating this approach with biochemical and cellular studies, we were able to identify new rare missense and frameshift alleles in NEU1 gene. Among the 9 candidate variants tested, only two resulted in significantly lower levels of sialidase activity (p<0.05), namely c.650T>C and c.700G>A. These two mutations give rise to the amino acid substitutions p.V217A and p.D234N, respectively. NEU1 variants including either of these two amino acid changes have 44% and 25% residual sialidase activity when compared to the wild-type enzyme, reduced protein levels and altered subcellular localization. Thus they may represent new, putative pathological mutations resulting in sialidosis type I. The in silico approach used in this study has enabled the identification of previously unknown NEU1 functional alleles that are widespread in the population and could be tested in future functional studies. 相似文献
10.
Activation of Neu (ErbB-2) Mediated by Disulfide Bond-Induced Dimerization Reveals a Receptor Tyrosine Kinase Dimer Interface 总被引:6,自引:2,他引:4
下载免费PDF全文
![点击此处可从《Molecular and cellular biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Receptor dimerization is a crucial intermediate step in activation of signaling by receptor tyrosine kinases (RTKs). However, dimerization of the RTK Neu (also designated ErbB-2, HER-2, and p185neu), while necessary, is not sufficient for signaling. Earlier work in our laboratory had shown that introduction of an ectopic cysteine into the Neu juxtamembrane domain induces Neu dimerization but not signaling. Since Neu signaling does require dimerization, we hypothesized that there are additional constraints that govern signaling ability. With the importance of the interreceptor cross-phosphorylation reaction, a likely constraint was the relative geometry of receptors within the dimer. We have tested this possibility by constructing a consecutive series of cysteine substitutions in the Neu juxtamembrane domain in order to force dimerization along a series of interreceptor faces. Within the group that dimerized constitutively, a subset had transforming activity. The substitutions in this subset all mapped to the same face of a predicted alpha helix, the most likely conformation for the intramembrane domain. Furthermore, this face of interaction aligns with the projected Neu* V664E substitution and with a predicted amphipathic interface in the Neu juxtamembrane domain. We propose that these results identify an RTK dimer interface and that dimerization of this RTK induces an extended contact between juxtamembrane and intramembrane alpha helices. 相似文献
11.
Developmental Change of Sialidase Neu4 Expression in Murine Brain and Its Involvement in the Regulation of Neuronal Cell Differentiation 总被引:1,自引:0,他引:1
Kazuhiro Shiozaki Koichi Koseki Kazunori Yamaguchi Momo Shiozaki Hisashi Narimatsu Taeko Miyagi 《The Journal of biological chemistry》2009,284(32):21157-21164
Sialidase Neu4 is reported to be dominantly expressed in the mouse brain, but its functional significance is not fully understood. We previously demonstrated that sialidase Neu3, also rich in mouse brain, is up-regulated during neuronal differentiation with involvement in acceleration of neurite formation. To elucidate physiological functions of Neu4, as well as Neu3, we determined expression during mouse brain development by quantitative RT-PCR. Expression was relatively low in the embryonic stage and then rapidly increased at 3–14 days after birth, whereas Neu3 demonstrated high levels in the embryonic stage and down-regulation after birth. Murine Neu4 was found to possess two isoforms differing in expression levels, developmental pattern, and enzymatic character. Distinct from the human isoforms, the murine forms, to a different extent, both catalyzed the removal of sialic acid from gangliosides as well as glycoproteins, and one isoform seemed to act on polysialylated NCAM efficiently, despite the low activity toward ordinary substrates. In situ hybridization demonstrated Neu4 mRNA to be present mainly in the hippocampus in which NCAM is rich and decreases after birth. During retinoic acid-induced differentiation, Neu4 expression was down-regulated in Neuro2a cells. Overexpression of Neu4 resulted in suppression of neurite formation, and its knockdown showed the acceleration. Thin layer chromatography of the glycolipids from Neu4-transfected cells showed ganglioside compositions to be only slightly affected, although lectin blot analysis revealed increased binding to Ricinus communis agglutinin (RCA) lectin of a ∼95-kDa glycoprotein, which decreased with cell differentiation. These results suggest that mouse Neu4 plays an important regulatory role in neurite formation, possibly through desialylation of glycoproteins.Sialidases catalyze the removal of sialic acid from non-reducing ends of glycoproteins and glycolipids. In mammals, four types of sialidases have so far been cloned, classified according to their subcellular localization and enzymatic properties (abbreviated to Neu1, Neu2, Neu3, and Neu4) (1–3). Studies have provided strong evidence that these sialidases play crucial roles in various physiological functions such as cell differentiation, cell growth, and malignant transformation. Among these sialidases, Neu4 is unique in its tissue expression pattern and enzymatic properties. In the mouse, it is dominantly expressed in brain, but its sialidase activity is very weak compared with other mouse sialidases (4). In contrast, human NEU4 is expressed not only in brain, but also in liver, kidney, and colon (5–7). We have demonstrated that NEU4 has two isoforms, differing in the N-terminal 12-amino acid residues that act as a mitochondrial-targeting sequence (7). Except for the subcellular localization, enzymatic properties are very similar. The short form of NEU4 (NEU4S) suppresses malignancy in colon cancer cells, mainly through desialylation of some glycoproteins, whereas the long form of NEU4 (NEU4L) may be involved in apoptosis with hydrolysis of ganglioside GD3 in mitochondria (8). Recently, Neu4 knockout mice (Neu4−/−) were generated for pathological analysis (9). Neu4−/− grew normally with a normal lifespan and proved fertile, but vacuolization of the lung and spleen was observed with a lysosomal storage phenotype, and the GM1/GD1a ratio was decreased in the brain. The observations on Neu4−/− are very interesting, but there is some ambiguity in the available previous reports, because, as mentioned above, mouse Neu4 has been reported to have weak sialidase activity in vitro, and its expression is restricted in brain. To clarify this ambiguity and further understand the physiological functions of Neu4, we examined expression in the mouse brain and observed a possible involvement in neural differentiation in connection with another sialidase, Neu3, which greatly increases during differentiation of neuroblastoma cells (10, 11) and causes acceleration of neurite formation (10–13).In the GenBankTM data base, nucleotide sequences of mouse Neu4 have been submitted as and AY258421. The former contains a complete coding sequence of 1506 bp, with two ATGs at positions 1 and 70, and AK034236 encodes only the second ATG ( AK0342364). The gene from has been reported to encode Neu4, showing weak sialidase activity, but there is no information on whether the gene based on AY258421 encodes Neu4 with sialidase activity toward natural substrates. We have now extended our studies to the existence of different mouse Neu4 isoforms, focusing on their significance in neuronal cells by measuring expression levels during cell differentiation. We present, here, evidence that two murine Neu4 isoforms contribute to neurite formation. AK034236相似文献
12.
Abstract: Density-dependent changes in ganglioside composition, Vibrio cholerae neuraminidase (VCN)-susceptible sialyl residues, and membrane- associated sialidase activity were determined for the cholinergic murine neuroblastoma cell line S20Y. A decrease in total ganglioside sialic acid and VCN-releasable sialic acid was observed with increasing cell density. GM3 was the major ganglioside component of preconfluent S20Y cells, whereas GDIA was predominant in postconfluent cells. Sialidase activity increased in confluent and postconfluent cells and may account for the reduction in total ganglioside sialic acid observed with increasing cell density. In contrast, while adrenergic N115 cells showed a decrease in VCN-susceptible sialic acid residues with increasing cell density, there was no significant change in ganglioside composition or ganglioside sialic acid levels. 相似文献
13.
Erik P. Lillehoj Sang Won Hyun Anguo Liu Wei Guang Avelino C. Verceles Irina G. Luzina Sergei P. Atamas K. Chul Kim Simeon E. Goldblum 《The Journal of biological chemistry》2015,290(30):18316-18331
Airway epithelia express sialylated receptors that recognize exogenous danger signals. Regulation of receptor responsiveness to these signals remains incompletely defined. Here, we explore the mechanisms through which the human sialidase, neuraminidase-1 (NEU1), promotes the interaction between the sialoprotein, mucin 1 (MUC1), and the opportunistic pathogen, Pseudomonas aeruginosa. P. aeruginosa flagellin engaged the MUC1 ectodomain (ED), increasing NEU1 association with MUC1. The flagellin stimulus increased the association of MUC1-ED with both NEU1 and its chaperone/transport protein, protective protein/cathepsin A. Scatchard analysis demonstrated NEU1-dependent increased binding affinity of flagellin to MUC1-expressing epithelia. NEU1-driven MUC1-ED desialylation rapidly increased P. aeruginosa adhesion to and invasion of the airway epithelium. MUC1-ED desialylation also increased its shedding, and the shed MUC1-ED competitively blocked P. aeruginosa adhesion to cell-associated MUC1-ED. Levels of desialylated MUC1-ED were elevated in the bronchoalveolar lavage fluid of mechanically ventilated patients with P. aeruginosa airway colonization. Preincubation of P. aeruginosa with these same ex vivo fluids competitively inhibited bacterial adhesion to airway epithelia, and MUC1-ED immunodepletion completely abrogated their inhibitory activity. These data indicate that a prokaryote, P. aeruginosa, in a ligand-specific manner, mobilizes eukaryotic NEU1 to enhance bacterial pathogenicity, but the host retaliates by releasing MUC1-ED into the airway lumen as a hyperadhesive decoy receptor. 相似文献
14.
Amandine Scandolera Fanja Rabenoelina Carine Chaintreuil Anthony Rusciani Pascal Maurice Sébastien Blaise Béatrice Romier-Crouzet Hassan El Btaouri Laurent Martiny Laurent Debelle Laurent Duca 《PloS one》2015,10(6)
Degradation of elastin leads to the production of elastin-derived peptides (EDP), which exhibit several biological effects, such as cell proliferation or protease secretion. Binding of EDP on the elastin receptor complex (ERC) triggers lactosylceramide (LacCer) production and ERK1/2 activation following ERC Neu-1 subunit activation. The ability for ERC to transduce signals is lost during aging, but the mechanism involved is still unknown. In this study, we characterized an in vitro model of aging by subculturing human dermal fibroblasts. This model was used to understand the loss of EDP biological activities during aging. Our results show that ERC uncoupling does not rely on Neu-1 or PPCA mRNA or protein level changes. Furthermore, we observe that the membrane targeting of these subunits is not affected with aging. However, we evidence that Neu-1 activity and LacCer production are altered. Basal Neu-1 catalytic activity is strongly increased in aged cells. Consequently, EDP fail to promote Neu-1 catalytic activity and LacCer production in these cells. In conclusion, we propose, for the first time, an explanation for ERC uncoupling based on the age-related alterations of Neu-1 activity and LacCer production that may explain the loss of EDP-mediated effects occurring during aging. 相似文献
15.
《Cell cycle (Georgetown, Tex.)》2013,12(6):549-551
The extrinsic pathway of apoptosis originates at the membrane and engages membrane death receptors. Tumor necrosis factor receptor 1 (TNF-R1) is a death receptor that transduces both the death and survival signals but the molecular mechanisms via which TNF-R1 mediates these signals remain poorly understood. Recently, it has been reported that the TNF-R1 transduces these signals via two signaling complexes. The first complex (complex I) is formed at the membrane by TNF-R1, TRADD, RIP, TRAF2 and c-IAP1, while the second complex (complex II), formed in the cytosol, predominantly contains FADD and pro-caspases 8/10 but lacks TNF-R1. Complex I is responsible for activating NF-kB and thus, the transduction of survival signals. Complex II, on the other hand, is reported to transduce the apoptotic signals and it does so only if NF-kB is unable to promote upregulation of the anti-apoptotic FLIPL. These findings highlighting the complexities of TNF-R1-mediated signaling events are likely to further the progress in the constantly evolving area of death receptor-dependent signaling pathways. 相似文献
16.
Lyudmyla Dorosh Olesya A. Kharenko Nandhakishore Rajagopalan Michele C. Loewen Maria Stepanova 《PLoS computational biology》2013,9(6)
The pyrabactin resistance 1 (PYR1)/PYR1-like (PYL)/regulatory component of abscisic acid (ABA) response (RCAR) proteins comprise a well characterized family of ABA receptors. Recent investigations have revealed two subsets of these receptors that, in the absence of ABA, either form inactive homodimers (PYR1 and PYLs 1–3) or mediate basal inhibition of downstream target type 2C protein phosphatases (PP2Cs; PYLs 4–10) respectively in vitro. Addition of ABA has been shown to release the apo-homodimers yielding ABA-bound monomeric holo-receptors that can interact with PP2Cs; highlighting a competitive-interaction process. Interaction selectivity has been shown to be mediated by subtle structural variations of primary sequence and ligand binding effects. Now, the dynamical contributions of ligand binding on interaction selectivity are investigated through extensive molecular dynamics (MD) simulations of apo and holo-PYR1 in monomeric and dimeric form as well as in complex with a PP2C, homology to ABA insensitive 1 (HAB1). Robust comparative interpretations were enabled by a novel essential collective dynamics approach. In agreement with recent experimental findings, our analysis indicates that ABA-bound PYR1 should efficiently bind to HAB1. However, both ABA-bound and ABA-extracted PYR1-HAB1 constructs have demonstrated notable similarities in their dynamics, suggesting that apo-PYR1 should also be able to make a substantial interaction with PP2Cs, albeit likely with slower complex formation kinetics. Further analysis indicates that both ABA-bound and ABA-free PYR1 in complex with HAB1 exhibit a higher intra-molecular structural stability and stronger inter-molecular dynamic correlations, in comparison with either holo- or apo-PYR1 dimers, supporting a model that includes apo-PYR1 in complex with HAB1. This possibility of a conditional functional apo-PYR1-PP2C complex was validated in vitro. These findings are generally consistent with the competitive-interaction model for PYR1 but highlight dynamical contributions of the PYR1 structure in mediating interaction selectivity suggesting added degrees of complexity in the regulation of the competitive-inhibition. 相似文献
17.
Wen Yi Jiang Yuan Li Zhi Ying Li Zong Jie Cui 《Cellular and molecular neurobiology》2018,38(6):1283-1292
The G protein-coupled cholecystokinin 1 receptor (CCK1R) is activated permanently by type II photodynamic action (i.e., by singlet oxygen) in the freshly isolated rat pancreatic acini, in contrast to reversible activation by CCK. But how CCK1R is photodynamically activated is not known. Therefore, in the present work, we subjected membrane proteins extracted from isolated rat pancreatic acini to photodynamic action with photosensitiser sulphonated aluminium phthalocyanine (SALPC), and used reducing gel electrophoresis and Western blot to detect possible changes in CCK1R oligomerization status. Photodynamic action (SALPC 1 µM, light 36.7 mW cm??2 × 10 min) was found to convert dimeric CCK1R nearly quantitatively to monomers. Such conversion was dependent on both irradiance (8.51–36.7 mW cm??2) and irradiation time (1–20 min). Minimum effective irradiance was found to be 11.1 mW cm??2 (× 10 min, with SALPC 1 µM), and brief photodynamic action (SALPC 1 µM, 36.7 mW cm??2 × 1 min) was effective. Whilst CCK stimulation of purified membrane proteins alone had no effect on CCK1R dimer/monomer balance, sub-threshold photodynamic action (SALPC 100 nM, 36.7 mW cm??2 × 10 min) plus CCK revealed a bell-shaped CCK dose response curve for CCK1R monomerization, which was remarkably similar to the dose response curve for CCK-stimulated amylase secretion in isolated rat pancreatic acini. These two lines of evidence together suggest that during photodynamic CCK1R activation, CCK1R is permanently monomerized, thus providing a unique approach for permanent G protein-coupled receptor (GPCR) activation which has not been achieved before. 相似文献
18.
Taisuke Ohira Makoto Arita Kazuhiro Omori Antonio Recchiuti Thomas E. Van Dyke Charles N. Serhan 《The Journal of biological chemistry》2010,285(5):3451-3461
Resolvins are endogenous lipid mediators that actively regulate the resolution of acute inflammation. Resolvin E1 (RvE1; (5S,12R,18R)-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) is an endogenous anti-inflammatory and pro-resolving mediator derived from eicosapentaenoic acid that regulates leukocyte migration and enhances macrophage phagocytosis of apoptotic neutrophils to resolve inflammation. In the inflammatory milieu, RvE1 mediates counter-regulatory actions initiated via specific G protein-coupled receptors. Here, we have identified RvE1-specific signaling pathways initiated by the RvE1 receptor ChemR23. RvE1 stimulated phosphorylation of Akt that was both ligand- and receptor-dependent. RvE1 regulated Akt phosphorylation in a time (0–15 min)- and dose-dependent (0.01–100 nm) manner in human ChemR23-transfected Chinese hamster ovary cells. RvE1 stimulated phosphorylation of both Akt and a 30-kDa protein, a downstream target of Akt, identified using a phospho-Akt substrate antibody. The 30-kDa protein was identified as ribosomal protein S6, a translational regulator, and its phosphorylation was inhibited by a phosphatidylinositol 3-kinase (PI3K) inhibitor (wortmannin) and an ERK inhibitor (PD98059) but not by a p38-MAPK inhibitor (SB203580). Ribosomal protein S6 is a downstream target of the PI3K/Akt signaling pathway as well as the Raf/ERK pathway. In ChemR23-expressing differentiated HL60 cells, RvE1 also stimulated the phosphorylation of ribosomal protein S6. In addition, RvE1 enhanced phagocytosis of zymosan A by human macrophages, which are inhibited by PD98059 and rapamycin (mTOR inhibitor). These results indicate that RvE1 initiates direct activation of ChemR23 and signals receptor-dependent phosphorylation. These phosphorylation-signaling pathways identified for RvE1 receptor-ligand interactions underscore the importance of endogenous pro-resolving agonists in resolving acute inflammation. 相似文献
19.
Casey Trimmer Lindsey L. Snyder Joel D. Mainland 《Journal of visualized experiments : JoVE》2014,(88)
Odorants create unique and overlapping patterns of olfactory receptor activation, allowing a family of approximately 1,000 murine and 400 human receptors to recognize thousands of odorants. Odorant ligands have been published for fewer than 6% of human receptors1-11. This lack of data is due in part to difficulties functionally expressing these receptors in heterologous systems. Here, we describe a method for expressing the majority of the olfactory receptor family in Hana3A cells, followed by high-throughput assessment of olfactory receptor activation using a luciferase reporter assay. This assay can be used to (1) screen panels of odorants against panels of olfactory receptors; (2) confirm odorant/receptor interaction via dose response curves; and (3) compare receptor activation levels among receptor variants. In our sample data, 328 olfactory receptors were screened against 26 odorants. Odorant/receptor pairs with varying response scores were selected and tested in dose response. These data indicate that a screen is an effective method to enrich for odorant/receptor pairs that will pass a dose response experiment, i.e. receptors that have a bona fide response to an odorant. Therefore, this high-throughput luciferase assay is an effective method to characterize olfactory receptors—an essential step toward a model of odor coding in the mammalian olfactory system. 相似文献