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CaMKIIα and caveolin-1 cooperate to drive ATP-induced membrane delivery of the P2X3 receptor

Journal of Molecular Cell Biology 2014年第2期8卷 140-153页

作者： Xu-Qiao Chen Jing-Xiang Zhu Yan Wang Xu Zhang Lan Bao State Key Laboratory of Cell Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China Institute of Neuroscience and State Key Laboratory of Neuroscience Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai 200031 China

The P2X3 receptor plays a vital role in sensory processing and transmission. The assembly and trafficking of the P2X3 receptor are important for its function in primary sensory neurons. As an important inflammation mediator, ATP is released from different cell types around primary sensory neurons, especially under pathological pain conditions. Here, we showthat α, β-MeATP dramatically promoted membrane delivery of the P2X3 receptor both in HEK293T celts expressing recombinant P2X3 receptor and in rat primary sensory neurons. α, β-MeATP induced P2X3 receptor-mediated Ca^2＋ influx, which further activated Ca^2＋/calmodulin-dependent protein kinase Ilec （CaMKIIα）. The N terminus of the P2X3 receptor was responsible for CaMKIleα binding, whereas Thr38s in the C terminus was phosphorylated by CaMKIIα. Thr^388 phosphorylation increased P2X3 receptor binding to caveoUn-1. CaveoUn-1 knockdown abrogated the α, β-MeATP-induced membrane insertion of the P2X3 receptor. Moreover,α, β-MeATP drove the CaMKIlec-mediated membrane coinsertion of the P2X2 receptor with the P2X3 receptor. The increased P2X3 receptors on the cell membrane that are due to Thr388 phosphorytation facilitated P2X3 receptor-mediated signal transduction. Together, our data indicate that CaMKIIoL and caveoUn-1 cooperate to drive Ugand-induced membrane delivery of the P2X3 receptor and may provide a mechanism of P2X3 receptor sensitization in pain development.

关键词： P2X3 receptor ATP membrane delivery CaMKIIα caveolin-1

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Negative regulation of NLRP3 infl ammasome signaling

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Protein & Cell 2013年第4期4卷 251-258页

作者： Shuzhen Chen Bing Sun State Key Laboratory of Cell Biology Institute of Biochemistry and Cell BiologyShanghai Institutes of Biological SciencesChinese Academy of SciencesShanghai 200031China Key Laboratory of Molecular Virology&Immunology Institute Pasteur of ShanghaiShanghai Institutes of Biological SciencesChinese Academy of SciencesShanghai 200025China

Infl ammasomes are multiprotein complexes that serve as a platform for caspase-1 activation and interleukin-1β(IL-1β)maturation as well as *** a number of infl ammasomes have been described,the NLRP3 inflammasome is the most extensively ***3 inflammasome is triggered by a variety of stimuli,including infection,tissue damage and metabolic dysregulation,and then activated through an integrated cellular *** regulatory mechanisms have been identifi ed to attenuate NLRP3 infl ammasome signaling at multiple ***,we review the developments in the negative regulation of NLRP3 inflammasome that protect host from inflammatory damage.

关键词： infl ammasome NLRP3 negative regulation infl ammation

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Single amino-acid substitution in the N-terminal arm altered the tetramer stability of rat muscle lactate dehydrogenase A

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Science China(Life sciences) 2001年第6期44卷 576-584页

作者：袁翀胡红雨许根俊 Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China

Lactate dehydrogenase A (LDHA) is a well-characterized tetrameric enzyme. Its N-terminal arm, comprised of an α-helix and a p-strand, was suggested to be essential for subunit interactions. To examine the critical amino acid residues in the N-terminus involved in the subunit association, two single-point mutants, Leu3Pro (L3P) and IIe8GIu (I8E), have been constructed. We compared the stability of WT-LDHA (WT) and its variants by unfolding experiments. For WT, a dimeric but inactive intermediate was observed by size-exclusion chromatography at 0.6-0.8 mol/L GdmCI. Leu3Pro exists in an active tetrameric structure in aqueous solution as WT does, but it dissociates into dimers under lower concentration of GdmCI (0.2 mol/L). In aqueous solution, the lle8GIu variant exists predominantly in the dimeric form with increased KM and decreased kcat as compared with those of WT and L3P. However, the activity of lle8GIu increases significantly in the presence of sodium sulfate. In conclusion, two mutants are less stable than WT in oligomer structure. Results also support the fact that some residues in the N-terminal arm, especially the Leu8 in the p-structure, contribute the important binding energies to the dimerization of dimers, which might affect the assembly of the enzyme as well as the catalytic function.

关键词： lactate dehydrogenase A N-terminal residues size-exclusion chromatograph tetramer stability site-directed mutagenesis unfolding experiments subunit interaction.

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(5R)-5-hydroxytriptolide inhibits IFN-γ-related signaling

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Acta Pharmacologica Sinica 2006年第12期27卷 1616-1621页

作者： Ru ZHOU~2 Jun-xia WANG~2 Wei TANG~2 Pei-lan HE~2 Yi-fu YANG~2 Yuan-chao LI~3 Xiao-yu LI~2 Jian-ping ZUO~(2,4)2 Laboratory of Immunopharmacology and 3 Laboratory of Chemistry,State Key Laboratory of Drug Research,shanghai Institute of Materia Medica,shanghai institutes for biological sciences,Chinese Academy of sciences,shanghai 201203,China Laboratory of Immunopharmacology State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China Laboratory of Chemistry State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China

Aim:(5R)-5-hydroxytriptolide(LLDT-8)displayed anti-arthritis and anti-allogenictransplantation rejection activities in our previous ***,we aim to furtherclarify the effect of LLDT-8 on the pro-inflammatory cytokine IFN-γ.Methods:Tcells were activated with anti-CD3 antibody or concanavalin A(ConA).The ex-pression of cell surface molecules was detected with flow *** werelabeled with carboxyfluorescein diacetate succinimidyl ester(CFSE)to test ***-γ production was determined by enzyme-linked *** proliferation was evaluated by[~3H]-thymidine *** wereimmunized with ovalbumin to assess the in vivo immune ***-PCR andReal-time PCR were applied to determine the mRNA *** protein phos-phorylation levels were detected by Western immunoblot ***:LLDT-8 at 100 nmol/L did not change the CD25,CD69,and CD154 expressions in anti-CD3-stimulated T ***-8 markedly blocked the cell division of CD4 andCD8 T cells after ConA ***-8 inhibited T cell-derived IFN-γ***,LLDT-8 suppressed the ovalbumin-specific T cell prolif-eration and IFN-γ *** anti-CD3-activated T cells,LLDT-8 abrogated themRNA expression of signal transducer and activator of transcriptionl(STAT1),T-box transcription factor,IL-12 receptor β2,STAT4,and interferon regulatory fac-tor 1 in the IFN-γ expression *** blot analysis showed that LLDT-8 blocked the phosphorylation levels of extracellular signal-regulated kinase,stress-activated protein kinase(SAPK)/c-Jun N-terminal kinase,and p38 mitogen-activated protein kinase in anti-CD3 plus anti-CD28-activated T *** addition,LLDT-8 reduced the transcripts of macrophage inflammatory protein(Mip)-1α,Mip-1β,regulated upon activation normally T-cell expressed and secreted,induc-ible protein-10,IFN-inducible T cell a chemoattractant,and monokine induced byIFN-7 in IFN-γ-stimulated murine macrophage cell line Raw 264.7 ***:LLD

关键词： (5R)-5-hydroxytriptolide IFN-γ MAPK chemokine

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Effects of Huangqi (Hex) on Inducing Cell Differentiation and Cell Death in K562 and HEL Cells

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Acta Biochimica et Biophysica Sinica 2004年第3期36卷 211-217页

作者： Xiao-Dong CHENG1, Chun-Hui HOU1, Xue-Jun ZHANG2, Heng-Yue XIE1, Wei-Ying ZHOU1, Lei YANG2, Shu-Bing ZHANG1, and Ruo-Lan QIAN1 State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, shanghai institutes for biological sciences, the Chinese Academy of sciences, 320 Yueyang Road, shanghai 200031, China Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, shanghai institutes for biological sciences, the Chinese Academy of sciences,320 Yueyang Road shanghai 200031,China the Chinese Academy of sciences, 320 Yueyang Road, shanghai 200031, China State Key Laboratory of Molecular Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China Laboratory of Molecular Cell Biology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences the Chinese Academy of Sciences 320 Yueyang Road Shanghai 200031 China

Huangqi (Astragalus membranaceus), a traditional Chinese medicine, has been used to ameliorate side effects of cancer chemotherapy in China. However, little is known about its molecular mechanisms. Here we show that induction of K562 or HEL cells with 1.5 mg/ml of Huangqi (Hex) (Components extracted from Huangqi) for 3–5 d results in the expression of β-globin gene in both cell lines and leads to terminal differentiation. Moreover, the apoptosis in HEL cells can be induced by increasing concentration of Huangqi (Hex) to 4.5 mg/ml for 3–5 d. Upregulation of Apaf-1, caspase-3 and acetylcholinesterase (AChE) in HEL cells may play a crucial role in the process of apoptosis. The prospect of inducing expression of adult (β) globin gene and apoptosis selectively in cancer cells is obviously attractive from a therapeutic point of view.

关键词： cell death and differentiation Huangqi (Hex) human erythroleukemia cell lines

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The inhibition of lung cancer cell growth by intracellular immunization with LC-1 ScFv

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Cell Research 2002年第1期12卷 47-54页

作者： LIANG CHEN, GANG LI, LEI TANG, JUE WANG, XI, RUI GE Laboratory of Cellular Immunology, Institute of Biochemistry and Cell Biology, shanghai institutes for biological sciences, Chinese Academy of sciences, 320 Yueyang Road, shanghai 200031, China Laboratory of Cellular Immunology Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China

A monoclonal antibody, LC-1, recognizing lung cancer associated common antigens was obtained in authors’ laboratory. Its single chain Fv fragment (ScFv) named LC-1 ScFv was constructed based on recombinant phage displayed techniques. For expression on cell membrane, LC-1 ScFv was cloned into pDisplay vector, which directed the cloned gene to express as cell membrane bound protein. The resulting plasmid was sequenced and then introduced by the lipofectin method into a lung adenocarcinoma cell line SPC-A-1. G418 resistant cells were obtained by G418 selection. After transfection, LC-1 ScFv expression was observed by Western blot analysis and the expression of cognate antigens was down-regulated as shown in ELISA assay. SPC-A-1-pDisplay-ScFv cells grew in vitro at lower speed than the control intact cells and the cells transfected with vacant vector. Flow cytometry analysis detected a substantial increase in G1 phase and decrease in S phase in population of SPC-A-1-pDisplay-ScFv cells compared to SPC-A -1 and SPC-A1-pDisplay cells. Semi-quantitative RT-PCR analysis showed that c-myc expression was down-regulated in SPC-A-1-pDisplay-ScFv cells. It seems that the antigens recognized by LC-1 may be in some way involved in a growth stimulating pathway and the antibody blocking of the function of the antigens shut down the pathway and thus down-regulate the expression of c-myc and growth of the cells.

关键词： LC-1 LC-1 ScFv SPC-A-1 pDisplay lung cancer.

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Overexpression of γ-aminobutyric acid transporter subtype I leads to susceptibility to Kainic acid-induced seizure in transgenic mice

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Cell Research 2001年第1期11卷 61-67页

作者： MA YINGHUA, JIA HUA HU, WEN JUAN ZHAO, JIAN FEI, YUN YU, XIAO GANG ZHOU, ZHEN TONG MEI, LI HE GUO (Institute of Biochemistry and Cell Biology, shanghai institutes for biological sciences, Chinese Academy of sciences, shanghai 200031, China) (shanghai Inst Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China shanghai Institute of Physiology Chinese Academy of Sciences Shanghai China

γ-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter, and the GABAergic synaptic transmission is normally terminated by the rapid uptake through GABA transporters. With transgenic mice ubiquitously overexpressing GABA transporter subtype I (GAT1), the present study explored the pathophysiological role of GAT1 in epileptogenesis. Though displaying no spontaneous seizure activity, these mice exhibit altered electroencephalographic patterns and increased susceptibility to seizure induced by kainic acid. In addition, the GABAA receptor and glutamate transporters are up-regulated in transgenic mice, which perhaps reflects a compensatory or corrective change to the elevated level of GAT1. These preliminary findings support the hypothesis that excitatory and inhibitory neurotransmission, and seizure susceptibility can be altered by neurotransmitter transporters.

关键词： γ-aminobutyric acid transporter seizure susceptibility kainic acid electroencephalography transgenic mice

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Induction of leukemia cell apoptosis by cheliensisin A involves downregulation of Bcl-2 expression

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Acta Pharmacologica Sinica 2005年第5期26卷 623-628页

作者： Li ZHONG,Chao-ming LI~2,Xiao-jiang HAO~2,Li-guang LOU~3 shanghai Institute of Materia Medica,shanghai institutes for biological sciences,shanghai 201203,China ~2Kunming Institute of Botany, Chinese Academy of sciences,Kunming 650204,China shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Shanghai China Kunming Institute of Botany Chinese Academy of Sciences Kunming China

Aim:To investigate the apoptosis-inducing effect of cheliensisin A（GC-51）,anovel styryl-lactone isolated from Goniothalamus cheliensis,on humanpromyelocytic leukemia HL-60 cells and the mechanism of action ***:Apoptotic cell death was determined by morphological examination andDNA agarose gel *** activity of caspase-3 was assessed usingWestern blotting and the expression of Bcl-2 and Bax genes was analyzed usingthe reverse transcription-polymerase chain reaction（RT-PCR）***:GC-51 significantly inhibited the proliferation of HL-60 cells with an IC50of 2.4±0.2μmol/L and effectively induced apoptosis in HL-60 *** of HL-60cells to 10μmol/L GC-51 for 8 h resulted in approximately 53% of the cells under-going ***-3 was activated in GC-51-treated cells,which was mani-fested by the appearance of the 17 kDa active form of caspase-3 and the cleavageof poly（ADP-ribose）polymerase（PARP）.Meanwhile,GC-51 markedly reducedthe expression of the anti-apoptotic gene Bcl-2 and increased the expression ofthe pro-apoptotic gene *** apoptosis-inducing effect of GC-51 was cAMP-dependent protein kinase（PKA）dependent because PKA,but not the proteinkinase C,specific inhibitor H-89,blocked the induction of apoptosis by GC-51 inHL-60 ***:The results demonstrate that GC-51 effectively inducesapoptosis in HL-60 cells and that this effect is PKA-dependent and involves thedownregulation of Bcl-2 expression and the activation of caspase-3.

关键词： Goniothalamus cheliensis cheliensisin A apoptosis HL-60 cells bcl-2 genes cyclic AMP-dependent protein kinases,caspases

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Substitutions of the Conserved Gly47 Affect the CF1 Inhibitor and Proton Gate Functions of the Chloroplast ATP Synthase ε Subunit

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Acta Biochimica et Biophysica Sinica 2005年第7期37卷 453-462页

作者： Hui DONG Zhang-Lin NI Jia-Mian WEI Institute of Plant Physiology & Ecology,shanghai institutes for biological sciences,Chinese Academy of sciences,shanghai 200032,China Institute of Plant Physiology & Ecology Shanghai Institutes for Biological Sciences Chinese Academy of SciencesShanghai 200032 China

The conserved residue Gly47 of the chloroplast ATP synthase ε subunit was substituted withLeu,Arg,Ala and Glu by site-directed *** process generated the mutants εG47L,εG47R,εG47A and εG47E,*** the ε variants showed lower inhibitory effects on the soluble CF1（-ε）Ca2+-ATPase compared with wild-type ε.In reduced conditions,εG47E and εG47R had a lower inhibitoryeffect on the oxidized CF1（-ε）Ca2+-ATPase compared with wild-type ε.In contrast,εG47L and εG47Aincreased the Ca2+-ATPase activity of soluble oxidized CF1（-ε）.The replacement of Gly47 significantlyimpaired the interaction between the subunit ε and γ in an in vitro binding *** study showed thatall ε variants were more effective in blocking proton leakage from the thylakoid *** enhancedATP synthesis of the chloroplast and restored ATP synthesis activity of the reconstituted membranes to alevel that was more efficient than that achieved by wild-type ε.These results indicate that the conservedGly47 residue of the ε subunit is very important for maintaining the structure and function of the ε subunitand may affect the interaction between the ε subunit,β subunit of CF1and subunit Ⅲ of CF_o,therebyregulating the ATP hydrolysis and synthesis,as well as the proton translocation role of the subunit Ⅲ of CF_o.

关键词： chloroplast ATP synthase ε subunit GST pull-down assay site-directed mutagenesis

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(5R)-5-hydroxytriptolide (LLDT-8) protects against bleomycin-induced lung fibrosis in mice

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Acta Pharmacologica Sinica 2007年第4期28卷 518-525页

作者： Yong-xin REN,Ru ZHOU~2,Wei TANG~2,Wen-hai WANG~2,Yuan-chao LI~3,Yi-fu YANG~2,Jian-ping ZUO~(2,4)~2Laboratory of Immunopharmacology and ~3Laboratory of Chemistry,State Key Laboratory of Drug Research,shanghai Institute of MateriaMedica,shanghai institutes for biological sciences,Chinese Academy of sciences,555 Zuchongzhi Road,shanghai 201203 China Laboratory of Immunopharmacology State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China Laboratory of Chemistry State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China

Aim:To study the protective effects of a triptolide-derived,novel compound,(5R)-5-hydroxytriptolide (LLDT-8),on bleomycin-induced lung ***:C57BL/6 mice received an intratracheal injection of bleomycin and were then treatedwith LLDT-8 (0.5,1,2 mg/kg,ip) once daily for 7 or 14 consecutive *** bodyweight loss and lung index augmentation was observed;the inflammatory re-sponse including differential cells counts of neutrophils,macrophages,and lym-phocytes in the bronchoalveolar lavage fluid (BALF),superoxide dismutase (SOD),and malondialdehyde (MDA) level in the lung homogenates was detected,andthe fibrosis extent was evaluated by hydroxyproline content and histopathologi-cal changes in the *** addition,the pro-inflammatory and pro-fibroticcytokines,tumor necrosis factor-α(TNF-α),interleukin-4 (IL-4),and transforminggrowth factor-α (TGF-α) production in the lungs were ***:LLDT-8alleviated the body weight loss and lung index increase caused by bleomycin,reduced neutrophils and lymphocytes in the BALF,promoted SOD activity,decreased MDA production,and inhibited the hydroxyproline level and the amelio-ration of lung tissue histological ***,LLDT-8 suppressed TNF-α,1L-4,and TGF-β production in the lung ***:LLDT-8 showedprotective effects against bleomycin-induced lung fibrosis,and the resultssuggested the potential role of LLDT-8 in the treatment of this disease.

关键词： (5R)-5-hydroxytriptolide bleomycin lung fibrosis cytokines

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