作为一种二酰甘油酰基转移酶,跨膜蛋白68(transmembrane protein 68,TMEM68)介导一条不依赖酰基辅酶A:二酰甘油酰基转移酶(acyl-CoA:diacylglycerol acyltransferase,DGAT)的三酰甘油生物合成新途径。然而TMEM68催化三酰甘油合成的酰基...作为一种二酰甘油酰基转移酶,跨膜蛋白68(transmembrane protein 68,TMEM68)介导一条不依赖酰基辅酶A:二酰甘油酰基转移酶(acyl-CoA:diacylglycerol acyltransferase,DGAT)的三酰甘油生物合成新途径。然而TMEM68催化三酰甘油合成的酰基供体尚不明确。本文通过比较超表达TMEM68对不同脂酰链饱和度的甘油酯、脂肪酸和甘油磷脂的作用,发现超表达TMEM68对不同饱和度的三酰甘油、二酰甘油、脂肪酸、磷脂酰胆碱和磷脂酰乙醇胺及其醚脂表现出不同的影响,并且这些脂质的变化存在一定的相关性;通过DGAT抑制剂处理,发现TMEM68不依赖DGAT活性合成三酰甘油,促进脂滴形成;通过分子对接分析,发现TMEM68与磷脂:二酰甘油酰基转移酶具有相似甚至更强的针对磷脂酰胆碱和磷脂酰乙醇胺及其醚脂的结合力。这些结果提示,TMEM68以二酰甘油为酰基受体,可能利用甘油磷脂作为酰基供体合成三酰甘油。展开更多
Objective To characterize transmembrane protein 68(TMEM68)in an alternative triacylglycerol(TAG)biosynthesis pathway,and determine the interplay between TMEM68 and the canonical TAG synthesis enzyme acyl-CoA:diacylgly...Objective To characterize transmembrane protein 68(TMEM68)in an alternative triacylglycerol(TAG)biosynthesis pathway,and determine the interplay between TMEM68 and the canonical TAG synthesis enzyme acyl-CoA:diacylglycerol acyltransferase(DGAT).Methods Effects of exogenous fatty acid and monoacylglycerol on TAG synthesis and lipid droplet(LD)formation in TMEM68 overexpression and knockout cells treated with DGAT inhibitor or not were investigated by comparing LD morphology,Oil Red O staining,and measurement of TAG levels.LDs were stained with fluorescence dye and observed by confocal fluorescence microscopy.TAG levels were determined with an enzyme-based triglyceride assay kit.Colocalization of TMEM68 and DGAT1 was detected by co-expression and confocal fluorescence microscopy and their interaction was determined by co-immunoprecipitation.RT-qPCR and immunoblotting assay were used to detect the expression of DGAT1.Results The synthesis of TAG catalyzed by TMEM68 was independent of DGAT activity.Surplus exogenous fatty acids and monoacylglycerol promoted TAG synthesis mainly through DGAT in human neuroblastoma cells.The LDs formed by TMEM68 were different in morphology from those by DGAT.In addition,TMEM68 and DGAT1 colocalized in the same endoplasmic reticulum(ER)compartment but did not interact physically.TMEM68 overexpression reduced the expression of DGAT1,the major DGAT enzyme involved in TAG synthesis,while TMEM68 knockout had little impact.Conclusion The TMEM68-mediated TAG synthesis pathway has distinct features from the canonical DGAT pathway,however,TMEM68 and DGAT may coregulate intracellular TAG levels.展开更多
目的了解结核分枝杆菌PPE68稳定片段,对稳定片段蛋白进行原核表达及纯化,为PPE68的结构解析奠定基础。方法使用Expasy-ProParam、MEME等生信网站对PPE68的基本性状和序列进化保守性进行分析;使用双酶切方法,PCR扩增PPE68基因,并克隆到pG...目的了解结核分枝杆菌PPE68稳定片段,对稳定片段蛋白进行原核表达及纯化,为PPE68的结构解析奠定基础。方法使用Expasy-ProParam、MEME等生信网站对PPE68的基本性状和序列进化保守性进行分析;使用双酶切方法,PCR扩增PPE68基因,并克隆到pGL01载体上;使用大肠埃希菌BL21(DE3)进行PPE68片段蛋白的原核表达,在IPTG(isopropylβ-D-thiogalactoside)诱导表达后,利用SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis)电泳分析表达产物并使用镍离子亲和层析及凝胶过滤层析的方法对表达产物进行体外纯化。后通过胰蛋白酶切实验、质谱分析确定PPE68最稳定片段。结果生物信息学分析初步确定了PPE68可能的稳定片段为第7-354个氨基酸并成功构建pGL01-PPE68(7-354aa)表达载体,蛋白表达纯化后获得2.4mg/mL的PPE68(7-354aa)片段蛋白但纯度较低;针对纯化的PPE68(7-354aa)蛋白较杂问题进行胰蛋白酶切和质谱分析找到稳定片段,为PPE68的第7-180个氨基酸,构建质粒后在大肠埃希菌中能稳定表达并获得2.9mg/mL的纯化蛋白,蛋白纯度可达95%以上。结论成功找到结核分枝杆菌PPE68的稳定氨基酸序列,为未来PPE68蛋白晶体结构解析以及进一步结核病诊断和疫苗开发奠定基础。展开更多
Rational tuning of crystallographic surface and metal doping were effective to enhance the catalytic performance of metal organic frameworks,but limited work has been explored for achieving modulation of crystal facet...Rational tuning of crystallographic surface and metal doping were effective to enhance the catalytic performance of metal organic frameworks,but limited work has been explored for achieving modulation of crystal facets and metal doping in a single system.MIL-68(In)was promising for photocatalytic applications due to its low toxicity and excellent photoresponsivity.However,its catalytic activity was constrained by severe carrier recombination and a lack of active sites.Herein,increased(001)facet ratio and active sites exposure were simultaneously realized by cobalt doping in MIL-68(In)through a one-pot solvothermal strategy.Optimized MIL-68(In/Co)-2.5 exhibited remarkable catalytic performance in comparison with pristine MIL-68(In)and other MIL-68(In/Co).The reaction kinetic constant and degradation efficiency of MIL-68(In/Co)were approximately twice and 17%higher than the pristine MIL-68(In)in 36 min reaction,respectively.Density functional theory calculations revealed that Co dopant could modulate the orientation of MIL-68(In)facets,facilitate the exchange of electrons and reduce the adsorption energy of peroxymonosulfate(PMS).This work provides a novel pathway for improvement of In-based MOFs in PMS/vis system,it also promotes the profound comprehension of the correlation between crystal facet regulation and catalytic activation in the PMS/vis system.展开更多
文摘作为一种二酰甘油酰基转移酶,跨膜蛋白68(transmembrane protein 68,TMEM68)介导一条不依赖酰基辅酶A:二酰甘油酰基转移酶(acyl-CoA:diacylglycerol acyltransferase,DGAT)的三酰甘油生物合成新途径。然而TMEM68催化三酰甘油合成的酰基供体尚不明确。本文通过比较超表达TMEM68对不同脂酰链饱和度的甘油酯、脂肪酸和甘油磷脂的作用,发现超表达TMEM68对不同饱和度的三酰甘油、二酰甘油、脂肪酸、磷脂酰胆碱和磷脂酰乙醇胺及其醚脂表现出不同的影响,并且这些脂质的变化存在一定的相关性;通过DGAT抑制剂处理,发现TMEM68不依赖DGAT活性合成三酰甘油,促进脂滴形成;通过分子对接分析,发现TMEM68与磷脂:二酰甘油酰基转移酶具有相似甚至更强的针对磷脂酰胆碱和磷脂酰乙醇胺及其醚脂的结合力。这些结果提示,TMEM68以二酰甘油为酰基受体,可能利用甘油磷脂作为酰基供体合成三酰甘油。
文摘Objective To characterize transmembrane protein 68(TMEM68)in an alternative triacylglycerol(TAG)biosynthesis pathway,and determine the interplay between TMEM68 and the canonical TAG synthesis enzyme acyl-CoA:diacylglycerol acyltransferase(DGAT).Methods Effects of exogenous fatty acid and monoacylglycerol on TAG synthesis and lipid droplet(LD)formation in TMEM68 overexpression and knockout cells treated with DGAT inhibitor or not were investigated by comparing LD morphology,Oil Red O staining,and measurement of TAG levels.LDs were stained with fluorescence dye and observed by confocal fluorescence microscopy.TAG levels were determined with an enzyme-based triglyceride assay kit.Colocalization of TMEM68 and DGAT1 was detected by co-expression and confocal fluorescence microscopy and their interaction was determined by co-immunoprecipitation.RT-qPCR and immunoblotting assay were used to detect the expression of DGAT1.Results The synthesis of TAG catalyzed by TMEM68 was independent of DGAT activity.Surplus exogenous fatty acids and monoacylglycerol promoted TAG synthesis mainly through DGAT in human neuroblastoma cells.The LDs formed by TMEM68 were different in morphology from those by DGAT.In addition,TMEM68 and DGAT1 colocalized in the same endoplasmic reticulum(ER)compartment but did not interact physically.TMEM68 overexpression reduced the expression of DGAT1,the major DGAT enzyme involved in TAG synthesis,while TMEM68 knockout had little impact.Conclusion The TMEM68-mediated TAG synthesis pathway has distinct features from the canonical DGAT pathway,however,TMEM68 and DGAT may coregulate intracellular TAG levels.
文摘目的了解结核分枝杆菌PPE68稳定片段,对稳定片段蛋白进行原核表达及纯化,为PPE68的结构解析奠定基础。方法使用Expasy-ProParam、MEME等生信网站对PPE68的基本性状和序列进化保守性进行分析;使用双酶切方法,PCR扩增PPE68基因,并克隆到pGL01载体上;使用大肠埃希菌BL21(DE3)进行PPE68片段蛋白的原核表达,在IPTG(isopropylβ-D-thiogalactoside)诱导表达后,利用SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis)电泳分析表达产物并使用镍离子亲和层析及凝胶过滤层析的方法对表达产物进行体外纯化。后通过胰蛋白酶切实验、质谱分析确定PPE68最稳定片段。结果生物信息学分析初步确定了PPE68可能的稳定片段为第7-354个氨基酸并成功构建pGL01-PPE68(7-354aa)表达载体,蛋白表达纯化后获得2.4mg/mL的PPE68(7-354aa)片段蛋白但纯度较低;针对纯化的PPE68(7-354aa)蛋白较杂问题进行胰蛋白酶切和质谱分析找到稳定片段,为PPE68的第7-180个氨基酸,构建质粒后在大肠埃希菌中能稳定表达并获得2.9mg/mL的纯化蛋白,蛋白纯度可达95%以上。结论成功找到结核分枝杆菌PPE68的稳定氨基酸序列,为未来PPE68蛋白晶体结构解析以及进一步结核病诊断和疫苗开发奠定基础。
基金supported by the National Natural Science Foundation of China(Nos.52100087,52170079,U20A20322)Science and Technology Development Program of Jilin Province,China(Nos.20220508100RC,20230402035GH).
文摘Rational tuning of crystallographic surface and metal doping were effective to enhance the catalytic performance of metal organic frameworks,but limited work has been explored for achieving modulation of crystal facets and metal doping in a single system.MIL-68(In)was promising for photocatalytic applications due to its low toxicity and excellent photoresponsivity.However,its catalytic activity was constrained by severe carrier recombination and a lack of active sites.Herein,increased(001)facet ratio and active sites exposure were simultaneously realized by cobalt doping in MIL-68(In)through a one-pot solvothermal strategy.Optimized MIL-68(In/Co)-2.5 exhibited remarkable catalytic performance in comparison with pristine MIL-68(In)and other MIL-68(In/Co).The reaction kinetic constant and degradation efficiency of MIL-68(In/Co)were approximately twice and 17%higher than the pristine MIL-68(In)in 36 min reaction,respectively.Density functional theory calculations revealed that Co dopant could modulate the orientation of MIL-68(In)facets,facilitate the exchange of electrons and reduce the adsorption energy of peroxymonosulfate(PMS).This work provides a novel pathway for improvement of In-based MOFs in PMS/vis system,it also promotes the profound comprehension of the correlation between crystal facet regulation and catalytic activation in the PMS/vis system.
