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
Objective The nucleolar protein PES1(Pescadillo homolog 1)plays critical roles in ribosome biogenesis and cell cycle regulation,yet its involvement in cellular senescence remains poorly understood.This study aimed to ...Objective The nucleolar protein PES1(Pescadillo homolog 1)plays critical roles in ribosome biogenesis and cell cycle regulation,yet its involvement in cellular senescence remains poorly understood.This study aimed to comprehensively investigate the functional consequences of PES1 suppression in cellular senescence and elucidate the molecular mechanisms underlying its regulatory role.Methods Initially,we assessed PES1 expression patterns in two distinct senescence models:replicative senescent mouse embryonic fibroblasts(MEFs)and doxorubicin-induced senescent human hepatocellular carcinoma HepG2 cells.Subsequently,PES1 expression was specifically downregulated using siRNA-mediated knockdown in these cell lines as well as additional relevant cell types.Cellular proliferation and senescence were assessed by EdU incorporation and SA-β-gal staining assays,respectively.The expression of senescence-associated proteins(p53,p21,and Rb)and SASP factors(IL-6,IL-1β,and IL-8)were analyzed by Western blot or qPCR.Furthermore,Northern blot and immunofluorescence were employed to evaluate pre-rRNA processing and nucleolar morphology.Results PES1 expression was significantly downregulated in senescent MEFs and HepG2 cells.PES1 knockdown resulted in decreased EdU-positive cells and increased SA-β-gal-positive cells,indicating proliferation inhibition and senescence induction.Mechanistically,PES1 suppression activated the p53-p21 pathway without affecting Rb expression,while upregulating IL-6,IL-1β,and IL-8 production.Notably,PES1 depletion impaired pre-rRNA maturation and induced nucleolar stress,as evidenced by aberrant nucleolar morphology.Conclusion Our findings demonstrate that PES1 deficiency triggers nucleolar stress and promotes p53-dependent(but Rb-independent)cellular senescence,highlighting its crucial role in maintaining nucleolar homeostasis and regulating senescence-associated pathways.展开更多
The growth of Caenorhabditis elegans involves multiple molting processes,during which old cuticles are shed and new cuticles are rapidly formed.This process requires the regulated bulk secretion of cuticle components....The growth of Caenorhabditis elegans involves multiple molting processes,during which old cuticles are shed and new cuticles are rapidly formed.This process requires the regulated bulk secretion of cuticle components.The transmembrane protein-39(TMEM-39)mutant exhibits distinct dumpy and ruptured phenotypes characterized by notably thin cuticles.TMEM-39 primarily co-localizes with the coat protein II complex(COPII)in large vesicles rather than small COPII vesicles.These TMEM-39-associated large vesicles(TMEM-39-LVs)form robustly during the molting period and co-localize with various extracellular matrix components,including BLI-1 collagen,BLI-3 dual oxidase,and carboxypeptidases.Through immunoprecipitation using TMEM39A-FLAG and proteomics analysis in human sarcoma cells,we identify TMEM39A-associated proteins,including TMEM131.Knockdown of TMEM131 results in reduced TMEM39A-LV formation and collagen secretion in both C.elegans and human sarcoma cells,indicating a cooperative role between TMEM39A and TMEM131 in the secretion of extracellular components through the formation of large COPII vesicles.Given the conservation of TMEM39A and its associated proteins between C.elegans and humans,TMEM39A-LVs may represent a fundamental machinery for rapid and extensive secretion across metazoans.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Objective The nucleolar protein PES1(Pescadillo homolog 1)plays critical roles in ribosome biogenesis and cell cycle regulation,yet its involvement in cellular senescence remains poorly understood.This study aimed to comprehensively investigate the functional consequences of PES1 suppression in cellular senescence and elucidate the molecular mechanisms underlying its regulatory role.Methods Initially,we assessed PES1 expression patterns in two distinct senescence models:replicative senescent mouse embryonic fibroblasts(MEFs)and doxorubicin-induced senescent human hepatocellular carcinoma HepG2 cells.Subsequently,PES1 expression was specifically downregulated using siRNA-mediated knockdown in these cell lines as well as additional relevant cell types.Cellular proliferation and senescence were assessed by EdU incorporation and SA-β-gal staining assays,respectively.The expression of senescence-associated proteins(p53,p21,and Rb)and SASP factors(IL-6,IL-1β,and IL-8)were analyzed by Western blot or qPCR.Furthermore,Northern blot and immunofluorescence were employed to evaluate pre-rRNA processing and nucleolar morphology.Results PES1 expression was significantly downregulated in senescent MEFs and HepG2 cells.PES1 knockdown resulted in decreased EdU-positive cells and increased SA-β-gal-positive cells,indicating proliferation inhibition and senescence induction.Mechanistically,PES1 suppression activated the p53-p21 pathway without affecting Rb expression,while upregulating IL-6,IL-1β,and IL-8 production.Notably,PES1 depletion impaired pre-rRNA maturation and induced nucleolar stress,as evidenced by aberrant nucleolar morphology.Conclusion Our findings demonstrate that PES1 deficiency triggers nucleolar stress and promotes p53-dependent(but Rb-independent)cellular senescence,highlighting its crucial role in maintaining nucleolar homeostasis and regulating senescence-associated pathways.
基金supported by the National Institutes of Health-Office of Research Infrastructure Programs(P40 OD010440)supported in part by grants from the National Cancer Center of Korea(NCC-2110160,NCC-2110263,and NCC-2310750)supported by the Basic Science Research Program of the National Research Foundation of Korea,funded by the Ministry of Science,ICT,and Future Planning(NRF-2015R1C1A1A01053611).
文摘The growth of Caenorhabditis elegans involves multiple molting processes,during which old cuticles are shed and new cuticles are rapidly formed.This process requires the regulated bulk secretion of cuticle components.The transmembrane protein-39(TMEM-39)mutant exhibits distinct dumpy and ruptured phenotypes characterized by notably thin cuticles.TMEM-39 primarily co-localizes with the coat protein II complex(COPII)in large vesicles rather than small COPII vesicles.These TMEM-39-associated large vesicles(TMEM-39-LVs)form robustly during the molting period and co-localize with various extracellular matrix components,including BLI-1 collagen,BLI-3 dual oxidase,and carboxypeptidases.Through immunoprecipitation using TMEM39A-FLAG and proteomics analysis in human sarcoma cells,we identify TMEM39A-associated proteins,including TMEM131.Knockdown of TMEM131 results in reduced TMEM39A-LV formation and collagen secretion in both C.elegans and human sarcoma cells,indicating a cooperative role between TMEM39A and TMEM131 in the secretion of extracellular components through the formation of large COPII vesicles.Given the conservation of TMEM39A and its associated proteins between C.elegans and humans,TMEM39A-LVs may represent a fundamental machinery for rapid and extensive secretion across metazoans.
