RxLR effectors produced by Phytophthora pathogens have been proposed to bind to phosphatidylinositol 3-phosphate （Ptdlns（3）P） to mediate their translocation into host cells and/or to increase their stability in pl...RxLR effectors produced by Phytophthora pathogens have been proposed to bind to phosphatidylinositol 3-phosphate （Ptdlns（3）P） to mediate their translocation into host cells and/or to increase their stability in planta. Since the levels of Ptdlns（3）P in plants are low, we examined whether Phytophthora species may produce Ptdlns（3）P to pro- mote infection. We observed that Ptdlns（3）P-specific GFP biosensors could bind to P. parasitica and P. sojae hyphae dur- ing infection of Nicotiana benthamiana leaves transiently secreting the biosensors, suggesting that the hyphae exposed Ptdlns（3）P on their plasma membrane and/or secreted Ptdlns（3）R Silencing of the phosphatidylinositol 3-kinases （PI3K） genes, treatment with LY294002, or expression of Ptdlns（3）pobinding proteins by P. sojae reduced the virulence of the pathogen on soybean, indicating that pathogen-synthesized Ptdlns（3）P was required for full virulence. Secretion of Ptdlns（3）P-binding proteins or of a PI3P-5-kinase by N. benthamiana leaves significantly increased the level of resist- ance to infection by P. parasitica and P. capsici. Together, our results support the hypothesis that Phytophthora species produce external Ptdlns（3）P to aid in infection, such as to promote entry of RxLR effectors into host cells. Our results derived from P. sojae RxLR effector Avrlb confirm that both the N-terminus and the C-terminus of this effector can bind Ptdlns（3）P.展开更多
Escherichia coli 3-Deoxy-D-manno-octulosonate 8-phosphate（KDO8P） synthase catalyzed the condensation reaction between D-arabinose 5-phosphate（A5P） and phosphoenolpyruvate（PEP） to form KDO8P and inorganic phosph...Escherichia coli 3-Deoxy-D-manno-octulosonate 8-phosphate（KDO8P） synthase catalyzed the condensation reaction between D-arabinose 5-phosphate（A5P） and phosphoenolpyruvate（PEP） to form KDO8P and inorganic phosphate（Pi）. The noncovalent tetrameric association ofKDO8P synthase was observed and dissociated in gas phase by means of electrospray ionization mass spectrometry under the very ＂soft＂ conditions. The results indicate that PEP-bound enzyme generated abundant tetrameric species as well as monomeric species at the ＂soft＂ conditions, whereas, the unbound enzyme favored the formation of a dimeric species. The mass spectra of the mixture of the enzyme with one of substrates, PEP, and A5P or one of products, KDO8P and Pi show that the complex of the unbound enzyme with PEP or Pi was prone to the formation of a monomeric species, whereas, that of the unbound enzyme with A5P or KDO8P was similar to the unbound enzyme. The intensity of the dimeric species increased with the increase of temperature at a collision voltage of 10 V. Taken together, the results presented here suggest that mass spectrometry will be a powerful tool to explore subtile conformational changes and/or subunit-subunit interactions of multiprotein assembly induced by ligand-binding and/or the changes of environmental conditions.展开更多
The mechanisms underlying autophagic defects in nonalcoholic steatohepatitis(NASH)remain largely unknown.We aimed to elucidate the roles of hepatic cyclooxygenase 1(COX1)in autophagy and the pathogenesis of diet-induc...The mechanisms underlying autophagic defects in nonalcoholic steatohepatitis(NASH)remain largely unknown.We aimed to elucidate the roles of hepatic cyclooxygenase 1(COX1)in autophagy and the pathogenesis of diet-induced steatohepatitis in mice.Human nonalcoholic fatty liver disease(NAFLD)liver samples were used to examine the protein expression of COX1 and the level of autophagy.Cox1^(Δhepa)mice and their wildtype littermates were generated and fed with 3 different NASH models.We found that hepatic COX1 expression was increased in patients with NASH and diet induced NASH mice models accompanied by impaired autophagy.COX1 was required for basal autophagy in hepatocytes and liver specific COX1 deletion exacerbated steatohepatitis by inhibiting autophagy.Mechanistically,COX1 directly interacted with WD repeat domain,phosphoinositide interacting 2(WIPI2),which was crucial for autophagosome maturation.Adeno-associated virus(AAV)-mediated rescue of WIPI2 reversed the impaired autophagic flux and improved NASH phenotypes in Cox1^(Δhepa)mice,indicating that COX1 deletion-mediated steatohepatitis was partially dependent on WIPI2-mediated autophagy.In conclusion,we demonstrated a novel role of COX1 in hepatic autophagy that protected against NASH by interacting with WIPI2.Targeting the COX1 WIPI2 axis may be a novel therapeutic strategy for NASH.展开更多
Insulin stimulates the production of PI(3,4,5)P3 in muscle cells, and this is required to stimulate GLUT4 fusion with the plasma membrane. Introduction of exogenous PI(3,4,5)P3 to muscle cells recapitulates insulin...Insulin stimulates the production of PI(3,4,5)P3 in muscle cells, and this is required to stimulate GLUT4 fusion with the plasma membrane. Introduction of exogenous PI(3,4,5)P3 to muscle cells recapitulates insulin's effects on GLUT4 fusion with the plasma membrane, but not glucose uptake. This study aims to explore the mechanism behind this difference. In L6-GLUT4myc muscle cells, the availability of the GLUT4 intracellular C-terminus and extracellular myc epitopes for immunoreactivity on plasma membrane lawns was detected with the corresponding antibody. The availability of the active site of GLUT4 from extracellular medium was assessed by affinity photolabeling with the cell impermeant compound Bio-LC-ATB-BMPA. 100nmol/L insulin and 10μmol/L PI(3,4,5)P3 caused myc signal gain on the plasma membrane lawns by 1.64-fold and 1.58-fold over basal, respectively. Insulin, but not PI(3,4,5)P3, increased photolabeling of GLUT4 and immunolabeling with C-terminus antibody by 2.47-fold and 2.04-fold over basal, respectively. Upon insulin stimulation, the C-terminus signal gain was greater than myc signal gain (2.04-fold vs. 1.64-fold over basal, respectively) in plasma membrane lawns. These results indicate that (i) PI(3,4,5)P3 does not make the active site of GLUT4 available from the extracellular surface despite causing GLUT4 fusion with the plasma membrane; (ii) the availability of the active site of GLUT4 from the extracellular medium and availability of the C-terminus from the cytosolic site are correlated; (iii) in addition to stimulating GLUT4 translocation, insulin stimulation displaces a protein which masks the GLUT4 C-terminus. We propose that a protein which masks the C-terminus also prevents the active site from being available for photolabelling and possibly glucose uptake after treatment with PI(3,4,5)P3.展开更多
Objective:To investigate whether peroxisome proliferator-activated receptorγ(PPARγ)agonists,rosiglitazone and GW1929,activate the phosphatidylinositol 3-kinase(PI3K)-AKT/protein kinase B pathway and the mitogen-acti...Objective:To investigate whether peroxisome proliferator-activated receptorγ(PPARγ)agonists,rosiglitazone and GW1929,activate the phosphatidylinositol 3-kinase(PI3K)-AKT/protein kinase B pathway and the mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase1/2(ERK1/2)pathway by upgrading the expression of chemerin.Methods:The HTR-8/SVneo trophoblastic cells were cultured in vitro in high glucose concentration(25 mmol/L)to mimic gestational diabetic phenotypes.We transfected small interfering RNA into HTR-8/SVneo cells to silence two receptors of chemerin,that are chemokine-like receptor 1(CMKLR1)and G protein-coupled receptor1(GPR1).And recombinant human chemerin,PPARγagonists(rosiglitazone,10μmol/L and GW1929,10μmol/L)and PPARγinhibitor(GW9662,5μmol/L)were additionally added to the medium,respectively.The existence of chemerin was verified by immunocytochemistry,and the expressions of PPARγ,chemerin,and its receptors as well as insulin signaling-related factors PI3K,AKT2,and MAPK(ERK1/2)were detected by real time quantitative-polymerase chain reaction and western blot.Results:Chemerin existed in the HTR-8/SVneo cells.Effects of chemerin on PI3K-AKT pathway and MAPK(ERK1/2)pathway were dependent on the density of chemerin.When rosiglitazone and GW1929 were added to the medium,the mRNA levels of PI3K,AKT2,and MAPK1 were upregulated(P<0.05).Conversely,GW9662 downregulated the mRNA levels of AKT2 and MAPK1(P<0.05).Rosiglitazone and GW1929 increased the protein levels of PPARγ,chemerin,CMKLR1 and GPR1(P<0.05).Rosiglitazone and GW1929 had no effect on the expression of PI3K p110βand phospho-AKT2 without CMKLR1(P>0.05).Meanwhile,the expression of phospho-ERK2 remained unaffected in the absence of GPR1(P>0.05).Conclusion:Both rosiglitazone and GW1929 have the effect of improving insulin signaling pathways via upgrading the level of chemerin in high glucose treated HTR-8/SVneo cells.展开更多
Sorting nexins (SNXs) are PX domain containing proteins and essential for intracellular protein sorting, trafficking and signal transduction. The PX domains of SNXs can bind to various phosphorelated phosphoinositides...Sorting nexins (SNXs) are PX domain containing proteins and essential for intracellular protein sorting, trafficking and signal transduction. The PX domains of SNXs can bind to various phosphorelated phosphoinositides (PIs) and target the host proteins to endosomes. Recently, we have reported that overexpression of SNX10 in mammalian cells could induce giant vacuoles. In this study, we aimed to identify regions in SNX10 critical for the vacuolation activity. We found that both the PX domain and the CD1 region were essential for vacuolation. We provided evidence that the PX domain was able to specifically bind to PtdIns(3)P and target SNX10 to endosomes. A mutation in the β1 region of the PX domain (V15A) disrupted the PtdIns(3)P binding ability and the endosomal localization of SNX10. However, correct subcellular localization alone was not sufficient for SNX10 to induce vacuoles. We found that the CD1 region, which was not required for the localization, was indispensable for the vacuolation activity of SNX10. In summary, both the PX domain and the CD1 region are necessary for SNX10 to induce vacuoles but they play different roles in this process.展开更多
基金This work was supported by grants from the National Natural Science Foundation of China （No. 30971889）, National Science and Technology Major Projects （No. 2009ZX08005-003B）, and the Natural Science Foundation of Jiangsu Province （No. BK2012027）.We appreciate Prof. Brett Tyler （Oregon State） for manuscript preparation, and Yuanming Zhang （Nanjing Agricultural University） for suggestions in data analysis. No conflict of interest declared.
文摘RxLR effectors produced by Phytophthora pathogens have been proposed to bind to phosphatidylinositol 3-phosphate （Ptdlns（3）P） to mediate their translocation into host cells and/or to increase their stability in planta. Since the levels of Ptdlns（3）P in plants are low, we examined whether Phytophthora species may produce Ptdlns（3）P to pro- mote infection. We observed that Ptdlns（3）P-specific GFP biosensors could bind to P. parasitica and P. sojae hyphae dur- ing infection of Nicotiana benthamiana leaves transiently secreting the biosensors, suggesting that the hyphae exposed Ptdlns（3）P on their plasma membrane and/or secreted Ptdlns（3）R Silencing of the phosphatidylinositol 3-kinases （PI3K） genes, treatment with LY294002, or expression of Ptdlns（3）pobinding proteins by P. sojae reduced the virulence of the pathogen on soybean, indicating that pathogen-synthesized Ptdlns（3）P was required for full virulence. Secretion of Ptdlns（3）P-binding proteins or of a PI3P-5-kinase by N. benthamiana leaves significantly increased the level of resist- ance to infection by P. parasitica and P. capsici. Together, our results support the hypothesis that Phytophthora species produce external Ptdlns（3）P to aid in infection, such as to promote entry of RxLR effectors into host cells. Our results derived from P. sojae RxLR effector Avrlb confirm that both the N-terminus and the C-terminus of this effector can bind Ptdlns（3）P.
基金Supported by National Hi-tech Research and Development Program of China(No.2006AA02Z154)the National Natural Science Foundation of China(No.20675088) and SRF for ROCS, SEM, China
文摘Escherichia coli 3-Deoxy-D-manno-octulosonate 8-phosphate（KDO8P） synthase catalyzed the condensation reaction between D-arabinose 5-phosphate（A5P） and phosphoenolpyruvate（PEP） to form KDO8P and inorganic phosphate（Pi）. The noncovalent tetrameric association ofKDO8P synthase was observed and dissociated in gas phase by means of electrospray ionization mass spectrometry under the very ＂soft＂ conditions. The results indicate that PEP-bound enzyme generated abundant tetrameric species as well as monomeric species at the ＂soft＂ conditions, whereas, the unbound enzyme favored the formation of a dimeric species. The mass spectra of the mixture of the enzyme with one of substrates, PEP, and A5P or one of products, KDO8P and Pi show that the complex of the unbound enzyme with PEP or Pi was prone to the formation of a monomeric species, whereas, that of the unbound enzyme with A5P or KDO8P was similar to the unbound enzyme. The intensity of the dimeric species increased with the increase of temperature at a collision voltage of 10 V. Taken together, the results presented here suggest that mass spectrometry will be a powerful tool to explore subtile conformational changes and/or subunit-subunit interactions of multiprotein assembly induced by ligand-binding and/or the changes of environmental conditions.
基金partly supported by National Natural Science Foundation of China(82125026 and 82122009)Natural Science Foundation of Shandong Province(ZR2022QH241 and ZR2020ZD11,China)Seed Fund for Basic Research of University Research Committee of The University of Hong Kong(20161159263,Hong Kong,China)。
文摘The mechanisms underlying autophagic defects in nonalcoholic steatohepatitis(NASH)remain largely unknown.We aimed to elucidate the roles of hepatic cyclooxygenase 1(COX1)in autophagy and the pathogenesis of diet-induced steatohepatitis in mice.Human nonalcoholic fatty liver disease(NAFLD)liver samples were used to examine the protein expression of COX1 and the level of autophagy.Cox1^(Δhepa)mice and their wildtype littermates were generated and fed with 3 different NASH models.We found that hepatic COX1 expression was increased in patients with NASH and diet induced NASH mice models accompanied by impaired autophagy.COX1 was required for basal autophagy in hepatocytes and liver specific COX1 deletion exacerbated steatohepatitis by inhibiting autophagy.Mechanistically,COX1 directly interacted with WD repeat domain,phosphoinositide interacting 2(WIPI2),which was crucial for autophagosome maturation.Adeno-associated virus(AAV)-mediated rescue of WIPI2 reversed the impaired autophagic flux and improved NASH phenotypes in Cox1^(Δhepa)mice,indicating that COX1 deletion-mediated steatohepatitis was partially dependent on WIPI2-mediated autophagy.In conclusion,we demonstrated a novel role of COX1 in hepatic autophagy that protected against NASH by interacting with WIPI2.Targeting the COX1 WIPI2 axis may be a novel therapeutic strategy for NASH.
基金the National Natural Science Foundation of China (Grant No. 30570912)the National Natural Science Foundation of China (China-Canada Joint Health Research) (Grant No. 30611120532)+1 种基金the Foundation of Tianjin Education Bureau, China to Niu Wenyan (Grant No. 20040106)the Tianjin Municipal Science and Technology Commission, China (Grant Nos. 06YFGPSH03300 and 07JCZDJC07900)
文摘Insulin stimulates the production of PI(3,4,5)P3 in muscle cells, and this is required to stimulate GLUT4 fusion with the plasma membrane. Introduction of exogenous PI(3,4,5)P3 to muscle cells recapitulates insulin's effects on GLUT4 fusion with the plasma membrane, but not glucose uptake. This study aims to explore the mechanism behind this difference. In L6-GLUT4myc muscle cells, the availability of the GLUT4 intracellular C-terminus and extracellular myc epitopes for immunoreactivity on plasma membrane lawns was detected with the corresponding antibody. The availability of the active site of GLUT4 from extracellular medium was assessed by affinity photolabeling with the cell impermeant compound Bio-LC-ATB-BMPA. 100nmol/L insulin and 10μmol/L PI(3,4,5)P3 caused myc signal gain on the plasma membrane lawns by 1.64-fold and 1.58-fold over basal, respectively. Insulin, but not PI(3,4,5)P3, increased photolabeling of GLUT4 and immunolabeling with C-terminus antibody by 2.47-fold and 2.04-fold over basal, respectively. Upon insulin stimulation, the C-terminus signal gain was greater than myc signal gain (2.04-fold vs. 1.64-fold over basal, respectively) in plasma membrane lawns. These results indicate that (i) PI(3,4,5)P3 does not make the active site of GLUT4 available from the extracellular surface despite causing GLUT4 fusion with the plasma membrane; (ii) the availability of the active site of GLUT4 from the extracellular medium and availability of the C-terminus from the cytosolic site are correlated; (iii) in addition to stimulating GLUT4 translocation, insulin stimulation displaces a protein which masks the GLUT4 C-terminus. We propose that a protein which masks the C-terminus also prevents the active site from being available for photolabelling and possibly glucose uptake after treatment with PI(3,4,5)P3.
基金This study was supported by the National Key R&D Program of China(No.2016YFC1000405 and No.2018YFC1002903)
文摘Objective:To investigate whether peroxisome proliferator-activated receptorγ(PPARγ)agonists,rosiglitazone and GW1929,activate the phosphatidylinositol 3-kinase(PI3K)-AKT/protein kinase B pathway and the mitogen-activated protein kinase(MAPK)/extracellular signal-regulated kinase1/2(ERK1/2)pathway by upgrading the expression of chemerin.Methods:The HTR-8/SVneo trophoblastic cells were cultured in vitro in high glucose concentration(25 mmol/L)to mimic gestational diabetic phenotypes.We transfected small interfering RNA into HTR-8/SVneo cells to silence two receptors of chemerin,that are chemokine-like receptor 1(CMKLR1)and G protein-coupled receptor1(GPR1).And recombinant human chemerin,PPARγagonists(rosiglitazone,10μmol/L and GW1929,10μmol/L)and PPARγinhibitor(GW9662,5μmol/L)were additionally added to the medium,respectively.The existence of chemerin was verified by immunocytochemistry,and the expressions of PPARγ,chemerin,and its receptors as well as insulin signaling-related factors PI3K,AKT2,and MAPK(ERK1/2)were detected by real time quantitative-polymerase chain reaction and western blot.Results:Chemerin existed in the HTR-8/SVneo cells.Effects of chemerin on PI3K-AKT pathway and MAPK(ERK1/2)pathway were dependent on the density of chemerin.When rosiglitazone and GW1929 were added to the medium,the mRNA levels of PI3K,AKT2,and MAPK1 were upregulated(P<0.05).Conversely,GW9662 downregulated the mRNA levels of AKT2 and MAPK1(P<0.05).Rosiglitazone and GW1929 increased the protein levels of PPARγ,chemerin,CMKLR1 and GPR1(P<0.05).Rosiglitazone and GW1929 had no effect on the expression of PI3K p110βand phospho-AKT2 without CMKLR1(P>0.05).Meanwhile,the expression of phospho-ERK2 remained unaffected in the absence of GPR1(P>0.05).Conclusion:Both rosiglitazone and GW1929 have the effect of improving insulin signaling pathways via upgrading the level of chemerin in high glucose treated HTR-8/SVneo cells.
基金Supported by the National Natural Science Foundation of China (Grant No. 30700410)
文摘Sorting nexins (SNXs) are PX domain containing proteins and essential for intracellular protein sorting, trafficking and signal transduction. The PX domains of SNXs can bind to various phosphorelated phosphoinositides (PIs) and target the host proteins to endosomes. Recently, we have reported that overexpression of SNX10 in mammalian cells could induce giant vacuoles. In this study, we aimed to identify regions in SNX10 critical for the vacuolation activity. We found that both the PX domain and the CD1 region were essential for vacuolation. We provided evidence that the PX domain was able to specifically bind to PtdIns(3)P and target SNX10 to endosomes. A mutation in the β1 region of the PX domain (V15A) disrupted the PtdIns(3)P binding ability and the endosomal localization of SNX10. However, correct subcellular localization alone was not sufficient for SNX10 to induce vacuoles. We found that the CD1 region, which was not required for the localization, was indispensable for the vacuolation activity of SNX10. In summary, both the PX domain and the CD1 region are necessary for SNX10 to induce vacuoles but they play different roles in this process.
