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
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.
