Xenophagy plays a crucial role in restraining the growth of intracellular bacteria in macrophages.However,the machinery governing autophagosome‒lysosome fusion during bacterial infection remains incompletely understoo...Xenophagy plays a crucial role in restraining the growth of intracellular bacteria in macrophages.However,the machinery governing autophagosome‒lysosome fusion during bacterial infection remains incompletely understood.Here,we utilize leprosy,an ideal model for exploring the interactions between host defense mechanisms and bacterial infection.We highlight the glycoprotein nonmetastatic melanoma protein B(GPNMB),which is highly expressed in macrophages from lepromatous leprosy(L-Lep)patients and interferes with xenophagy during bacterial infection.Upon infection,GPNMB interacts with autophagosomal-localized STX17,leading to a reduced N-glycosylation level at N296 of GPNMB.This modification promotes the degradation of SNAP29,thus preventing the assembly of the STX17-SNAP29-VAMP8 SNARE complex.Consequently,the fusion of autophagosomes with lysosomes is disrupted,resulting in inhibited cellular autophagic flux.In addition to Mycobacterium leprae,GPNMB deficiency impairs the proliferation of various intracellular bacteria in human macrophages,suggesting a universal role of GPNMB in intracellular bacterial infection.Furthermore,compared with their counterparts,Gpnmb^(fl/fl) Lyz2-Cre mice presented decreased Mycobacterium marinum amplification.Overall,our study reveals a previously unrecognized role of GPNMB in host antibacterial defense and provides insights into its regulatory mechanism in SNARE complex assembly.展开更多
Delivery of proteins to the plasma membrane occurs via secretion,which requires tethering,docking,priming,and fusion of vesicles.In yeast and mammalian cells,an evolutionarily conserved RAB GTPase activation cascade f...Delivery of proteins to the plasma membrane occurs via secretion,which requires tethering,docking,priming,and fusion of vesicles.In yeast and mammalian cells,an evolutionarily conserved RAB GTPase activation cascade functions together with the exocyst and SNARE proteins to coordinate vesicle transport with fusion at the plasma membrane.However,it is unclear whether this is the case in plants.In this study,we show that the small GTPase RABA2a recruits and interacts with the VAMP721/722-SYP121-SNAP33 SNARE ternary complex for membrane fusion.Through immunoprecipitation coupled with mass spectrometry analysis followed by the validatation with a series of biochemical assays,we identified the SNARE proteins VAMP721 and SYP121 as the interactors and downstream effectors of RABA2a.Further expreiments showed that RABA2a interacts with all members of the SNARE complex in its GTP-bound form and modulates the assembly of the VAMP721/722-SYP121-SNAP33 SNARE ternary complex.Intriguingly,we did not observe the interaction of the exocyst subunits with either RABA2a or theSNARE proteins in several different experiments.Neither RABA2a inactivation affects the subcellular localization or assembly of the exocystnor the exocyst subunit mutant exo84b shows the disrupted RABA2a-SNARE association or SNARE assembly,suggesting that the RABA2a-SNARE-and exocyst-mediated secretory pathways are largely independent.Consistently,our live imaging experiments reveal that the two sets of proteins follow non-overlapping trafficking routes,and genetic and cell biologyanalyses indicate that the two pathways select different cargos.Finally,we demonstrate that the plant-specific RABA2a-SNARE pathway is essential for the maintenance of potassium homeostasis in Arabisopsis seedlings.Collectively,our findings imply that higher plants might have generated different endomembrane sorting pathways during evolution and may enable the highly conserved endomembrane proteins to participate in plant-specific trafficking mechanisms for adaptation to the changing environment.展开更多
目的探讨钙蛋白酶拮抗剂Calpeptin对锰干扰突触体囊泡融合的保护作用。方法将24只小鼠随机分成4组,每组6只。对照组(腹腔注射0.9%氯化钠),低、高剂量染锰组(腹腔注射25μmol/kg、100μmol/kg氯化锰),Calpeptin预处理组(皮下注射Calpepti...目的探讨钙蛋白酶拮抗剂Calpeptin对锰干扰突触体囊泡融合的保护作用。方法将24只小鼠随机分成4组,每组6只。对照组(腹腔注射0.9%氯化钠),低、高剂量染锰组(腹腔注射25μmol/kg、100μmol/kg氯化锰),Calpeptin预处理组(皮下注射Calpeptin 100μg/kg,30 min后腹腔注射100μmol/kg氯化锰),注射容量为2 ml/kg,每周5次,共4周。处死小鼠后分离基底核,制备突触体,测量小鼠基底核内锰含量,神经细胞内钙离子浓度和钙蛋白活力,突触体SNARE复合物及其相关蛋白的变化和囊泡融合情况。结果与对照组比较,高剂量染锰组小鼠基底核锰含量,神经细胞内钙离子浓度和钙蛋白酶活力显著增加;SNAP25蛋白表达下降,并出现裂解碎片,SNARE复合物形成减少,FM1-43荧光强度的下降;Calpeptin预处理可以明显抑制神经细胞内钙蛋白酶活力,缓解钙蛋白酶对SNAP25的裂解,100 k Da SNARE复合物也明显增多,而且SNARE复合物介导的突触囊泡融合有所上升。结论钙蛋白酶拮抗剂Calpeptin可以对锰干扰突触体囊泡融合起到有效的保护作用。展开更多
基金supported by grants from the National Natural Science Foundation of China(82230107,82273545,and 82304038)the Natural Science Foundation of Shandong Province(ZR2023QH435,ZR2022MH258,and ZR2023MH046)+1 种基金the Shandong Province Taishan Scholar Project(tspd20230608)the Joint Innovation Team for Clinical&Basic Research(202410).
文摘Xenophagy plays a crucial role in restraining the growth of intracellular bacteria in macrophages.However,the machinery governing autophagosome‒lysosome fusion during bacterial infection remains incompletely understood.Here,we utilize leprosy,an ideal model for exploring the interactions between host defense mechanisms and bacterial infection.We highlight the glycoprotein nonmetastatic melanoma protein B(GPNMB),which is highly expressed in macrophages from lepromatous leprosy(L-Lep)patients and interferes with xenophagy during bacterial infection.Upon infection,GPNMB interacts with autophagosomal-localized STX17,leading to a reduced N-glycosylation level at N296 of GPNMB.This modification promotes the degradation of SNAP29,thus preventing the assembly of the STX17-SNAP29-VAMP8 SNARE complex.Consequently,the fusion of autophagosomes with lysosomes is disrupted,resulting in inhibited cellular autophagic flux.In addition to Mycobacterium leprae,GPNMB deficiency impairs the proliferation of various intracellular bacteria in human macrophages,suggesting a universal role of GPNMB in intracellular bacterial infection.Furthermore,compared with their counterparts,Gpnmb^(fl/fl) Lyz2-Cre mice presented decreased Mycobacterium marinum amplification.Overall,our study reveals a previously unrecognized role of GPNMB in host antibacterial defense and provides insights into its regulatory mechanism in SNARE complex assembly.
基金This work was supported by the Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher Education Institutes(2019KSYS006)was also financially supported by grants from the Natural Science Foundation of China(31770306)+7 种基金the Natural Science Foundation of Guangdong Province(2020A1515010966)the Guangdong Innovation Research Team Fund(2016ZT06S172)the Shenzhen Sci-Tech Fund(KYTDPT20181011104005)Z.M.and Y.M.were supported financially by the Singapore Ministry of Education(MOE)Tier 1(RG32/20)and Tier 3(MOE2019-T3-1-012)X.Z.was supported financially by the National Science Foundation for Young Scientists of China(32000558)the China Postdoctoral Science Foundation(grant no.2019M660494)R.L.L.was supported financially by the Natural Science Foundation of China(31970182,31670182)the Fundamental Research Funds for the Central Universities(2019ZY29).
文摘Delivery of proteins to the plasma membrane occurs via secretion,which requires tethering,docking,priming,and fusion of vesicles.In yeast and mammalian cells,an evolutionarily conserved RAB GTPase activation cascade functions together with the exocyst and SNARE proteins to coordinate vesicle transport with fusion at the plasma membrane.However,it is unclear whether this is the case in plants.In this study,we show that the small GTPase RABA2a recruits and interacts with the VAMP721/722-SYP121-SNAP33 SNARE ternary complex for membrane fusion.Through immunoprecipitation coupled with mass spectrometry analysis followed by the validatation with a series of biochemical assays,we identified the SNARE proteins VAMP721 and SYP121 as the interactors and downstream effectors of RABA2a.Further expreiments showed that RABA2a interacts with all members of the SNARE complex in its GTP-bound form and modulates the assembly of the VAMP721/722-SYP121-SNAP33 SNARE ternary complex.Intriguingly,we did not observe the interaction of the exocyst subunits with either RABA2a or theSNARE proteins in several different experiments.Neither RABA2a inactivation affects the subcellular localization or assembly of the exocystnor the exocyst subunit mutant exo84b shows the disrupted RABA2a-SNARE association or SNARE assembly,suggesting that the RABA2a-SNARE-and exocyst-mediated secretory pathways are largely independent.Consistently,our live imaging experiments reveal that the two sets of proteins follow non-overlapping trafficking routes,and genetic and cell biologyanalyses indicate that the two pathways select different cargos.Finally,we demonstrate that the plant-specific RABA2a-SNARE pathway is essential for the maintenance of potassium homeostasis in Arabisopsis seedlings.Collectively,our findings imply that higher plants might have generated different endomembrane sorting pathways during evolution and may enable the highly conserved endomembrane proteins to participate in plant-specific trafficking mechanisms for adaptation to the changing environment.
文摘目的探讨钙蛋白酶拮抗剂Calpeptin对锰干扰突触体囊泡融合的保护作用。方法将24只小鼠随机分成4组,每组6只。对照组(腹腔注射0.9%氯化钠),低、高剂量染锰组(腹腔注射25μmol/kg、100μmol/kg氯化锰),Calpeptin预处理组(皮下注射Calpeptin 100μg/kg,30 min后腹腔注射100μmol/kg氯化锰),注射容量为2 ml/kg,每周5次,共4周。处死小鼠后分离基底核,制备突触体,测量小鼠基底核内锰含量,神经细胞内钙离子浓度和钙蛋白活力,突触体SNARE复合物及其相关蛋白的变化和囊泡融合情况。结果与对照组比较,高剂量染锰组小鼠基底核锰含量,神经细胞内钙离子浓度和钙蛋白酶活力显著增加;SNAP25蛋白表达下降,并出现裂解碎片,SNARE复合物形成减少,FM1-43荧光强度的下降;Calpeptin预处理可以明显抑制神经细胞内钙蛋白酶活力,缓解钙蛋白酶对SNAP25的裂解,100 k Da SNARE复合物也明显增多,而且SNARE复合物介导的突触囊泡融合有所上升。结论钙蛋白酶拮抗剂Calpeptin可以对锰干扰突触体囊泡融合起到有效的保护作用。
