Tumor necrosis factor alpha-induced protein 1(TNFAIP1)modulates a plethora of important biological processes,including tumorigenesis and cancer cell migration.However,the regulatory mechanism of TNFAIP1 degradation re...Tumor necrosis factor alpha-induced protein 1(TNFAIP1)modulates a plethora of important biological processes,including tumorigenesis and cancer cell migration.However,the regulatory mechanism of TNFAIP1 degradation remains largely elusive.In the present study,with a label-free quantitative proteomic approach,TNFAIP1 was identified as a novel ubiquitin target of the Cullin-RING E3 ubiquitin ligase(CRL)complex.More importantly,Cul3-ROC1(CRL3),a subfamily of CRLs,was identified to specifically interact with TNFAIP1 and promote its polyubiquitination and degradation.Mechanistically,BTBD9,a specific adaptor component of CRL3 complex,was further defined to bind and promote the ubiquitination and degradation of TNFAIP1 in cells.As such,downregulation of BTBD9 promoted lung cancer cell migration by upregulating the expression of TNFAIP1,whereas TNFAIP1 deletion abrogated this effect.Finally,bioinformatics and clinical sample analyses revealed that BTBD9 was downregulated while TNFAIP1 was overexpressed in human lung cancer,which was associated with poor overall survival of patients.Taken together,these findings reveal a previously unrecognized mechanism by which the CRL3^(BTBD9) ubiquitin ligase controls TNFAIP1 degradation to regulate cancer cell migration.展开更多
为了探究鸡肿瘤坏死因子α诱导蛋白8样1(TNF alpha induced protein 8 like 1,TNFAIP8L1)基因序列特征,以及叶酸(FA)和甲氨蝶呤(MTX)对TNFAIP8L1基因在鸡胚肝脏组织不同发育阶段表达的影响,试验将孵化至第7天的240枚琅琊鸡种蛋分为生理...为了探究鸡肿瘤坏死因子α诱导蛋白8样1(TNF alpha induced protein 8 like 1,TNFAIP8L1)基因序列特征,以及叶酸(FA)和甲氨蝶呤(MTX)对TNFAIP8L1基因在鸡胚肝脏组织不同发育阶段表达的影响,试验将孵化至第7天的240枚琅琊鸡种蛋分为生理盐水组(注射0.1 mL生理盐水)、FA组[注射0.1 mL FA溶液(90μg FA溶于0.1 mL生理盐水中)]、MTX组[注射0.1 mL MTX溶液(5μg MTX溶于0.1 mL生理盐水中)]、FA和MTX组[注射0.1 mL FA+MTX混合液(90μg FA+5μg MTX溶于0.1 mL生理盐水中)],每组4个重复,每个重复15枚,利用PCR技术克隆鸡TNFAIP8L1基因开放阅读框(ORF),并对其进行生物信息学分析,同时利用荧光定量PCR方法分析FA和MTX对TNFAIP8L1基因在鸡胚孵化至第10天(10E)、第13天(13E)、第16天(16E)、第19天(19E)和出壳第1天(1D)肝脏中的表达差异。结果表明:克隆获得琅琊鸡TNFAIP8L1基因序列长726 bp,其中ORF区序列长561 bp,编码186个氨基酸。氨基酸多序列比对显示琅琊鸡与绿头鸭的氨基酸序列一致性最高(为97.85%),与马的一致性最低(为64.52%)。鸡TNFAIP8L1蛋白是一种亲水稳定蛋白;二级结构预测以α-螺旋(为72.04%)和无规则卷曲(为23.66%)为主要结构,三级结构与5jxd.1模板相似,其序列同源性为62.37%。10E和16E时,FA组和MTX组TNFAIP8L1基因相对表达量均显著低于生理盐水组(P<0.05);19E和1D时,FA组和MTX组TNFAIP8L1基因相对表达量与生理盐水组差异不显著(P>0.05)。说明在鸡胚发育前期FA和MTX可能对TNFAIP8L1基因表达有抑制作用。展开更多
基金The Chinese Minister of Science and Technology grant(2016YFA0501800)the National Natural Science Foundation of China(grant Nos.81625018,81820108022,81772470,81572340,81602072,81772459)+3 种基金the Innovation Program of Shanghai Municipal Education Commission(2019-01-07-00-10-E00056)the Program of Shanghai Academic/Technology Research Leader(18XD1403800)the National Thirteenth Five-Year Science and Technology Major Special Project for New Drug and Development(2017ZX09304001)the“Shuguang Program”supported by Shanghai Education Development Foundation(14SG07)supported this work.
文摘Tumor necrosis factor alpha-induced protein 1(TNFAIP1)modulates a plethora of important biological processes,including tumorigenesis and cancer cell migration.However,the regulatory mechanism of TNFAIP1 degradation remains largely elusive.In the present study,with a label-free quantitative proteomic approach,TNFAIP1 was identified as a novel ubiquitin target of the Cullin-RING E3 ubiquitin ligase(CRL)complex.More importantly,Cul3-ROC1(CRL3),a subfamily of CRLs,was identified to specifically interact with TNFAIP1 and promote its polyubiquitination and degradation.Mechanistically,BTBD9,a specific adaptor component of CRL3 complex,was further defined to bind and promote the ubiquitination and degradation of TNFAIP1 in cells.As such,downregulation of BTBD9 promoted lung cancer cell migration by upregulating the expression of TNFAIP1,whereas TNFAIP1 deletion abrogated this effect.Finally,bioinformatics and clinical sample analyses revealed that BTBD9 was downregulated while TNFAIP1 was overexpressed in human lung cancer,which was associated with poor overall survival of patients.Taken together,these findings reveal a previously unrecognized mechanism by which the CRL3^(BTBD9) ubiquitin ligase controls TNFAIP1 degradation to regulate cancer cell migration.
文摘为了探究鸡肿瘤坏死因子α诱导蛋白8样1(TNF alpha induced protein 8 like 1,TNFAIP8L1)基因序列特征,以及叶酸(FA)和甲氨蝶呤(MTX)对TNFAIP8L1基因在鸡胚肝脏组织不同发育阶段表达的影响,试验将孵化至第7天的240枚琅琊鸡种蛋分为生理盐水组(注射0.1 mL生理盐水)、FA组[注射0.1 mL FA溶液(90μg FA溶于0.1 mL生理盐水中)]、MTX组[注射0.1 mL MTX溶液(5μg MTX溶于0.1 mL生理盐水中)]、FA和MTX组[注射0.1 mL FA+MTX混合液(90μg FA+5μg MTX溶于0.1 mL生理盐水中)],每组4个重复,每个重复15枚,利用PCR技术克隆鸡TNFAIP8L1基因开放阅读框(ORF),并对其进行生物信息学分析,同时利用荧光定量PCR方法分析FA和MTX对TNFAIP8L1基因在鸡胚孵化至第10天(10E)、第13天(13E)、第16天(16E)、第19天(19E)和出壳第1天(1D)肝脏中的表达差异。结果表明:克隆获得琅琊鸡TNFAIP8L1基因序列长726 bp,其中ORF区序列长561 bp,编码186个氨基酸。氨基酸多序列比对显示琅琊鸡与绿头鸭的氨基酸序列一致性最高(为97.85%),与马的一致性最低(为64.52%)。鸡TNFAIP8L1蛋白是一种亲水稳定蛋白;二级结构预测以α-螺旋(为72.04%)和无规则卷曲(为23.66%)为主要结构,三级结构与5jxd.1模板相似,其序列同源性为62.37%。10E和16E时,FA组和MTX组TNFAIP8L1基因相对表达量均显著低于生理盐水组(P<0.05);19E和1D时,FA组和MTX组TNFAIP8L1基因相对表达量与生理盐水组差异不显著(P>0.05)。说明在鸡胚发育前期FA和MTX可能对TNFAIP8L1基因表达有抑制作用。
