摘要
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.
目的表征一条三酰甘油(TAG)生物合成替代途径中的跨膜蛋白68(TMEM68),并确定TMEM68与经典TAG合成酶酰基辅酶A∶二酰甘油酰基转移酶(DGAT)之间的相互作用。方法通过比较DGAT抑制剂处理与否的脂滴形态、油红O染色面积以及TAG水平,研究外源脂肪酸和单酰甘油对TMEM68过表达和敲除细胞的TAG合成和脂滴形成的影响。脂滴采用荧光染料染色并使用共聚焦荧光显微术观察。TAG水平使用基于酶法的脂肪检测试剂盒测定。TMEM68和DGAT1的共定位通过共表达和共聚焦荧光显微术检测,并采用免疫共沉淀检测它们的相互作用。采用逆转录定量聚合酶链反应和免疫印迹法检测DGAT1的表达。结果TMEM68催化的TAG合成不依赖DGAT活性。在人神经母细胞瘤细胞中,外源脂肪酸和单酰基甘油主要通过DGAT促进TAG合成。TMEM68与DGAT形成的脂滴具有不同的形态。此外,TMEM68和DGAT1在同一内质网(ER)区室共定位,但不发生物理相互作用。TMEM68的过表达降低了参与TAG合成的主要DGAT酶DGAT1的表达,然而TMEM68敲除没有影响。结论TMEM68介导的TAG合成途径具有不同于经典DGAT通路的特征,然而TMEM68和DGAT可能共同调控细胞内TAG水平。
出处
《生物化学与生物物理进展》
北大核心
2025年第3期705-715,共11页
Progress In Biochemistry and Biophysics
基金
重庆市自然科学基金(CSTB2022NSCQ-MSX0957)
重庆市教委科技研究项目(KJQN20230065)资助。
