【目的】探究环指蛋白20(ring finger protein 20,RNF20)对小鼠性腺脂肪组织(gonadal white adipose tissues,gWAT)及3T3-L1脂肪细胞中支链氨基酸(branched-chain amino acids,BCAAs)代谢的影响及调控机制。【方法】以雄性脂肪特异性Rn...【目的】探究环指蛋白20(ring finger protein 20,RNF20)对小鼠性腺脂肪组织(gonadal white adipose tissues,gWAT)及3T3-L1脂肪细胞中支链氨基酸(branched-chain amino acids,BCAAs)代谢的影响及调控机制。【方法】以雄性脂肪特异性Rnf20基因敲除小鼠(Rnf20^(flox/flox);adiponectin-Cre^(+),ASKO)及同窝野生型小鼠(Rnf20^(flox/flox);adiponectin-Cre^(-),WT)为研究对象,通过靶向代谢组学检测小鼠gWAT和血清中BCAAs含量,结合实时荧光定量PCR分析gWAT、肝脏及腓肠肌中BCAAs分解代谢基因(Bcat2、Bckdha等)的表达情况。利用siRNA干扰3T3-L1前体脂肪细胞内源性Rnf20基因表达,并诱导细胞成脂分化,通过油红O染色评估siRNF20(干扰组)和siNC(对照组)细胞的成脂分化效率,检测成脂分化标志基因、脂肪水解基因及BCAAs分解代谢基因的表达情况以及脂肪细胞培养基中BCAAs的含量。【结果】与WT小鼠相比,ASKO小鼠gWAT中BCAAs(亮氨酸、异亮氨酸、缬氨酸)含量均极显著升高(P<0.01),且BCAAs分解代谢相关基因Bcat2、Acad5、Ehhand、Hibch表达量显著或极显著降低(P<0.05;P<0.01);而血清中BCAAs含量及肝脏、腓肠肌中BCAAs分解代谢相关基因表达量均无显著变化(P>0.05)。与siNC组相比,体外敲除Rnf20基因可抑制3T3-L1细胞成脂分化,其中成脂标志基因Pparγ表达量显著降低(P<0.05),脂肪水解基因Adrb3表达量极显著升高(P<0.01);成熟脂肪细胞中BCAAs分解代谢关键基因Bcat2、Bckdha表达量随Rnf20基因敲除而降低,且siRNF20组培养基中BCAAs含量极显著升高(P<0.01)。【结论】脂肪细胞Rnf20基因通过转录调控BCAAs分解代谢关键基因(Bcat2、Bckdha),维持细胞内BCAAs稳态;敲除Rnf20基因可抑制BCAAs分解代谢,导致BCAAs在脂肪组织内累积。研究结果为深入探究Rnf20基因功能提供了新的方向,为大动物脂肪沉积性状研究提供了新的基因素材。展开更多
Objective:Ring finger protein 145(RNF145),an E3 ubiquitin ligase,is significantly upregulated in hepatocellular carcinoma(HCC).However,its role in HCC remains unknown.The study aimed to investigate the functions and u...Objective:Ring finger protein 145(RNF145),an E3 ubiquitin ligase,is significantly upregulated in hepatocellular carcinoma(HCC).However,its role in HCC remains unknown.The study aimed to investigate the functions and underlying mechanisms of RNF145 in HCC.Methods:The role of RNF145 in HCC was investigated using data from The Cancer Genome Atlas(TCGA)and in vitro experimental assays.Its oncogenic functions were assessed using the transwell migration assay and the wound-healing assay.The molecular mechanism was explored through protein immunoprecipitation and western blot analyses.Data from public databases were analyzed to correlate RNF145 expression with clinicopathological features.Univariate and multivariate Cox analyses established RNF145 as an independent prognostic factor.Subsequently,a prognostic nomogram was constructed.Results:RNF145 was upregulated in HCC.The expression level of RNF145 in HCC showed significant correlations with histological grade,pathological stage,and vascular invasion.Functionally,knockdown of RNF145 effectively abolished the migratory and invasive capacities of HCC cells.This pro-metastatic effect is mediated through the RNF145-driven ubiquitination and subsequent degradation of protocadherin 9(PCDH9).Conclusion:Our findings confirm the significant upregulation of RNF145 in HCC and promote metastasis by facilitating PCDH9 ubiquitination and degradation,highlighting its role as a prognostic biomarker and a potential therapeutic target.展开更多
RING protein 213(RNF213),the susceptibility gene for Moyamoya disease(MMD),possesses two active AAA+ATPase(ATPases Associated with diverse cellular Activities)modules,a RING,and RNF213-ZNFX1 finger(RZ finger)domains.S...RING protein 213(RNF213),the susceptibility gene for Moyamoya disease(MMD),possesses two active AAA+ATPase(ATPases Associated with diverse cellular Activities)modules,a RING,and RNF213-ZNFX1 finger(RZ finger)domains.Several RNF213 variants have been reported in MMD patients,including the p.R4810K variant(rs112735431),which is a founder polymorphism associated with MMDin EastAsia.To elucidate the function of RNF213 and its variant,we investigated the localization of RNF213 and the R4810K variant in this study.RNF213 induced circular hole structures near the nucleus,similar to lipid droplets(LDs),in U-2 OS cells.The holes decorated with tagged RNF213 protein were colocalized with mCherry-RAB5A and mCherry-peroxisome,but not mCherry-RAB9A and other organelles.RNF213 decorated the holes,and the structures were growing and changing to cylindrical objects,but not the R4810K variant efficiently.Furthermore,both AAA+ATPase modules and the ubiquitin ligase domain of RNF213 were required for the formation of the hole structures and cylindrical objects.These findings suggest that RNF213 activity may be involved in the formation of organelle-like structures,and this structure formation by RNF213 may be responsible for vascular functions.展开更多
目的探讨环指蛋白2(ring finger protein 2,RNF2)在胃腺癌组织中的表达及临床意义。方法采用免疫组化EnVision法检测80例胃腺癌及癌旁组织中RNF2蛋白表达水平,分析RNF2蛋白表达与胃腺癌临床病理特征及预后的关系。RT-qPCR法分析RNF2 mRN...目的探讨环指蛋白2(ring finger protein 2,RNF2)在胃腺癌组织中的表达及临床意义。方法采用免疫组化EnVision法检测80例胃腺癌及癌旁组织中RNF2蛋白表达水平,分析RNF2蛋白表达与胃腺癌临床病理特征及预后的关系。RT-qPCR法分析RNF2 mRNA在11例胃腺癌和癌旁组织中的表达水平。采用Spearman相关性分析RNF2与E-Cadherin蛋白表达的相关性。结果RNF2蛋白在胃腺癌组织(76.3%)中的表达明显高于癌旁组织(25.0%),差异有统计学意义(P﹤0.05)。RNF2表达与胃腺癌肿瘤大小、浸润深度、淋巴结转移、TNM分期及预后相关(P均﹤0.05),与患者性别、年龄、肿瘤分化程度、Lauren分型及神经侵犯无关(P﹥0.05)。RT-qPCR检测结果显示,RNF2 mRNA在胃腺癌组织(11.92±3.48)中的水平显著高于癌旁组织(11.11±2.38)(P﹤0.05)。相关性分析显示,RNF2与E-Cadherin在胃腺癌组织中的表达呈负相关(r_(s)=-0.278,P﹤0.05)。结论RNF2在胃腺癌的发生、发展中具有促进作用,表明RNF2可能成为胃腺癌治疗的潜在靶点。展开更多
为分析E3泛素连接酶环指蛋白125(ring finger protein 125,RNF125)在肝癌中的表达及临床预后意义,并探究RNF125对肝癌侵袭和转移的影响及其作用机制,通过ENCORI数据库和临床组织样本分析RNF125在肝癌和癌旁组织中的表达量,使用ENCORI数...为分析E3泛素连接酶环指蛋白125(ring finger protein 125,RNF125)在肝癌中的表达及临床预后意义,并探究RNF125对肝癌侵袭和转移的影响及其作用机制,通过ENCORI数据库和临床组织样本分析RNF125在肝癌和癌旁组织中的表达量,使用ENCORI数据库分析RNF125与肝癌预后的关系。构建敲低和过表达RNF125的肝癌细胞系,通过Transwell试验和细胞划痕试验检验RNF125表达水平对肝癌细胞迁移、侵袭能力的影响。通过动物试验验证调控RNF125对体内肿瘤生长情况及对肿瘤免疫微环境的影响,质谱分析寻找RNF125的靶蛋白,qPCR、蛋白质免疫印迹技术、泛素化试验验证RNF125与MYH9之间的关系,通过蛋白质免疫印迹和细胞免疫荧光技术确定调控RNF125与MYH9对EMT标志蛋白的影响。结果表明:RNF125在肝癌组织中表达较低,RNF125的高表达提示良好的预后。RNF125的表达水平与肝癌细胞迁移和侵袭能力呈负相关。过表达RNF125使肿瘤生长延迟并增加免疫细胞的浸润水平。RNF125特异性结合MYH9并促进其泛素化降解。RNF125与上皮细胞标志物的表达水平呈正相关,与间充质细胞标志物呈负相关。过表达MYH9可逆转RNF125对EMT标志蛋白的影响。综上,RNF125在肝癌中表达下调,RNF125的高表达与肝癌患者的良好预后相关,RNF125通过泛素化MYH9来抑制肝癌的EMT过程,从而抑制肝癌的迁移和侵袭。展开更多
口蹄疫病毒(FMDV)编码四种结构蛋白,其中VP1蛋白是病毒表面的主要衣壳蛋白,在病毒入侵和抗原性方面发挥关键作用,决定病毒的致病力和组织嗜性。2025年1月17日,中国农业科学院兰州兽医研究所郑海学研究员带领的科研团队在PLoS Pathogens...口蹄疫病毒(FMDV)编码四种结构蛋白,其中VP1蛋白是病毒表面的主要衣壳蛋白,在病毒入侵和抗原性方面发挥关键作用,决定病毒的致病力和组织嗜性。2025年1月17日,中国农业科学院兰州兽医研究所郑海学研究员带领的科研团队在PLoS Pathogens期刊上发表了题为“RING finger protein 5 is a key anti-FMDV host factor through inhibition of virion assembly”的研究论文,该研究揭示了环指蛋白5(RNF5)依赖其E3泛素酶活性介导FMDV VP1蛋白降解并限制病毒组装的功能机制,为广谱抗小RNA病毒药物的研发提供了理论依据,为抗病育种提供新思路。展开更多
Ring finger protein 122(RNF122),an E3 ubiquitin ligase,orchestrates antiviral immune responses in mammals by targeting retinoic acid-inducible gene 1 and melanoma differentiation-associated gene 5 for ubiquitination.H...Ring finger protein 122(RNF122),an E3 ubiquitin ligase,orchestrates antiviral immune responses in mammals by targeting retinoic acid-inducible gene 1 and melanoma differentiation-associated gene 5 for ubiquitination.However,its functional relevance in teleosts has yet to be clearly defined,particularly regarding the identification of substrate-specific regulatory sites.This study characterized RNF122 from mandarin fish(Siniperca chuatsi),termed scRNF122,and investigated its regulatory impact on stimulator of interferon genes(STING)-mediated antiviral signaling.Results showed that scRNF122 expression was up-regulated in response to mandarin fish ranavirus(MRV)infection,and its overexpression suppressed scSTING-mediated interferon(IFN)production and enhanced MRV replication.Co-immunoprecipitation confirmed a direct interaction between scRNF122 and scSTING.Functional assays demonstrated that scRNF122 facilitated scSTING degradation through the ubiquitin-proteasome pathway,a process impeded by MG132 treatment.Ubiquitination analyses of various scSTING mutants revealed that scRNF122 catalyzed scSTING ubiquitination at K95,K117,and K155 residues.Moreover,scRNF122 significantly impaired scSTING-dependent antiviral responses by engaging negative regulatory elements within the signaling cascade.Overall,scRNF122 was identified as a negative modulator of STING-mediated IFN signaling in mandarin fish,diminishing STING-dependent antiviral activity and promoting its degradation via the ubiquitin-proteasome pathway at lysine residues K95,K117,and K155.These findings provide mechanistic insight into the post-translational control of STING in teleosts and establish a foundation for future investigations into antiviral immune regulation.展开更多
文摘【目的】探究环指蛋白20(ring finger protein 20,RNF20)对小鼠性腺脂肪组织(gonadal white adipose tissues,gWAT)及3T3-L1脂肪细胞中支链氨基酸(branched-chain amino acids,BCAAs)代谢的影响及调控机制。【方法】以雄性脂肪特异性Rnf20基因敲除小鼠(Rnf20^(flox/flox);adiponectin-Cre^(+),ASKO)及同窝野生型小鼠(Rnf20^(flox/flox);adiponectin-Cre^(-),WT)为研究对象,通过靶向代谢组学检测小鼠gWAT和血清中BCAAs含量,结合实时荧光定量PCR分析gWAT、肝脏及腓肠肌中BCAAs分解代谢基因(Bcat2、Bckdha等)的表达情况。利用siRNA干扰3T3-L1前体脂肪细胞内源性Rnf20基因表达,并诱导细胞成脂分化,通过油红O染色评估siRNF20(干扰组)和siNC(对照组)细胞的成脂分化效率,检测成脂分化标志基因、脂肪水解基因及BCAAs分解代谢基因的表达情况以及脂肪细胞培养基中BCAAs的含量。【结果】与WT小鼠相比,ASKO小鼠gWAT中BCAAs(亮氨酸、异亮氨酸、缬氨酸)含量均极显著升高(P<0.01),且BCAAs分解代谢相关基因Bcat2、Acad5、Ehhand、Hibch表达量显著或极显著降低(P<0.05;P<0.01);而血清中BCAAs含量及肝脏、腓肠肌中BCAAs分解代谢相关基因表达量均无显著变化(P>0.05)。与siNC组相比,体外敲除Rnf20基因可抑制3T3-L1细胞成脂分化,其中成脂标志基因Pparγ表达量显著降低(P<0.05),脂肪水解基因Adrb3表达量极显著升高(P<0.01);成熟脂肪细胞中BCAAs分解代谢关键基因Bcat2、Bckdha表达量随Rnf20基因敲除而降低,且siRNF20组培养基中BCAAs含量极显著升高(P<0.01)。【结论】脂肪细胞Rnf20基因通过转录调控BCAAs分解代谢关键基因(Bcat2、Bckdha),维持细胞内BCAAs稳态;敲除Rnf20基因可抑制BCAAs分解代谢,导致BCAAs在脂肪组织内累积。研究结果为深入探究Rnf20基因功能提供了新的方向,为大动物脂肪沉积性状研究提供了新的基因素材。
基金supported by Guangdong Basic and Applied Basic Research Foundation(2024A1515012800)Chen Xiao-Ping Foundation for the Development of Science and Technology of Hubei Province(CXPJJH125001-2540).
文摘Objective:Ring finger protein 145(RNF145),an E3 ubiquitin ligase,is significantly upregulated in hepatocellular carcinoma(HCC).However,its role in HCC remains unknown.The study aimed to investigate the functions and underlying mechanisms of RNF145 in HCC.Methods:The role of RNF145 in HCC was investigated using data from The Cancer Genome Atlas(TCGA)and in vitro experimental assays.Its oncogenic functions were assessed using the transwell migration assay and the wound-healing assay.The molecular mechanism was explored through protein immunoprecipitation and western blot analyses.Data from public databases were analyzed to correlate RNF145 expression with clinicopathological features.Univariate and multivariate Cox analyses established RNF145 as an independent prognostic factor.Subsequently,a prognostic nomogram was constructed.Results:RNF145 was upregulated in HCC.The expression level of RNF145 in HCC showed significant correlations with histological grade,pathological stage,and vascular invasion.Functionally,knockdown of RNF145 effectively abolished the migratory and invasive capacities of HCC cells.This pro-metastatic effect is mediated through the RNF145-driven ubiquitination and subsequent degradation of protocadherin 9(PCDH9).Conclusion:Our findings confirm the significant upregulation of RNF145 in HCC and promote metastasis by facilitating PCDH9 ubiquitination and degradation,highlighting its role as a prognostic biomarker and a potential therapeutic target.
基金supported by grants(#15611607)from the Ministry of Education,Culture,Sports,Science and Technology of Japan to T.H.
文摘RING protein 213(RNF213),the susceptibility gene for Moyamoya disease(MMD),possesses two active AAA+ATPase(ATPases Associated with diverse cellular Activities)modules,a RING,and RNF213-ZNFX1 finger(RZ finger)domains.Several RNF213 variants have been reported in MMD patients,including the p.R4810K variant(rs112735431),which is a founder polymorphism associated with MMDin EastAsia.To elucidate the function of RNF213 and its variant,we investigated the localization of RNF213 and the R4810K variant in this study.RNF213 induced circular hole structures near the nucleus,similar to lipid droplets(LDs),in U-2 OS cells.The holes decorated with tagged RNF213 protein were colocalized with mCherry-RAB5A and mCherry-peroxisome,but not mCherry-RAB9A and other organelles.RNF213 decorated the holes,and the structures were growing and changing to cylindrical objects,but not the R4810K variant efficiently.Furthermore,both AAA+ATPase modules and the ubiquitin ligase domain of RNF213 were required for the formation of the hole structures and cylindrical objects.These findings suggest that RNF213 activity may be involved in the formation of organelle-like structures,and this structure formation by RNF213 may be responsible for vascular functions.
文摘为分析E3泛素连接酶环指蛋白125(ring finger protein 125,RNF125)在肝癌中的表达及临床预后意义,并探究RNF125对肝癌侵袭和转移的影响及其作用机制,通过ENCORI数据库和临床组织样本分析RNF125在肝癌和癌旁组织中的表达量,使用ENCORI数据库分析RNF125与肝癌预后的关系。构建敲低和过表达RNF125的肝癌细胞系,通过Transwell试验和细胞划痕试验检验RNF125表达水平对肝癌细胞迁移、侵袭能力的影响。通过动物试验验证调控RNF125对体内肿瘤生长情况及对肿瘤免疫微环境的影响,质谱分析寻找RNF125的靶蛋白,qPCR、蛋白质免疫印迹技术、泛素化试验验证RNF125与MYH9之间的关系,通过蛋白质免疫印迹和细胞免疫荧光技术确定调控RNF125与MYH9对EMT标志蛋白的影响。结果表明:RNF125在肝癌组织中表达较低,RNF125的高表达提示良好的预后。RNF125的表达水平与肝癌细胞迁移和侵袭能力呈负相关。过表达RNF125使肿瘤生长延迟并增加免疫细胞的浸润水平。RNF125特异性结合MYH9并促进其泛素化降解。RNF125与上皮细胞标志物的表达水平呈正相关,与间充质细胞标志物呈负相关。过表达MYH9可逆转RNF125对EMT标志蛋白的影响。综上,RNF125在肝癌中表达下调,RNF125的高表达与肝癌患者的良好预后相关,RNF125通过泛素化MYH9来抑制肝癌的EMT过程,从而抑制肝癌的迁移和侵袭。
文摘口蹄疫病毒(FMDV)编码四种结构蛋白,其中VP1蛋白是病毒表面的主要衣壳蛋白,在病毒入侵和抗原性方面发挥关键作用,决定病毒的致病力和组织嗜性。2025年1月17日,中国农业科学院兰州兽医研究所郑海学研究员带领的科研团队在PLoS Pathogens期刊上发表了题为“RING finger protein 5 is a key anti-FMDV host factor through inhibition of virion assembly”的研究论文,该研究揭示了环指蛋白5(RNF5)依赖其E3泛素酶活性介导FMDV VP1蛋白降解并限制病毒组装的功能机制,为广谱抗小RNA病毒药物的研发提供了理论依据,为抗病育种提供新思路。
基金supported by the National Key Research and Development Program of China(2022YFE0203900,2024YFD2401101)China Agriculture Research System(CARS-46)+1 种基金National Natural Science Foundation of China(32473201)Guangdong S&T Program(2022B1111030001,2024B1212040007)。
文摘Ring finger protein 122(RNF122),an E3 ubiquitin ligase,orchestrates antiviral immune responses in mammals by targeting retinoic acid-inducible gene 1 and melanoma differentiation-associated gene 5 for ubiquitination.However,its functional relevance in teleosts has yet to be clearly defined,particularly regarding the identification of substrate-specific regulatory sites.This study characterized RNF122 from mandarin fish(Siniperca chuatsi),termed scRNF122,and investigated its regulatory impact on stimulator of interferon genes(STING)-mediated antiviral signaling.Results showed that scRNF122 expression was up-regulated in response to mandarin fish ranavirus(MRV)infection,and its overexpression suppressed scSTING-mediated interferon(IFN)production and enhanced MRV replication.Co-immunoprecipitation confirmed a direct interaction between scRNF122 and scSTING.Functional assays demonstrated that scRNF122 facilitated scSTING degradation through the ubiquitin-proteasome pathway,a process impeded by MG132 treatment.Ubiquitination analyses of various scSTING mutants revealed that scRNF122 catalyzed scSTING ubiquitination at K95,K117,and K155 residues.Moreover,scRNF122 significantly impaired scSTING-dependent antiviral responses by engaging negative regulatory elements within the signaling cascade.Overall,scRNF122 was identified as a negative modulator of STING-mediated IFN signaling in mandarin fish,diminishing STING-dependent antiviral activity and promoting its degradation via the ubiquitin-proteasome pathway at lysine residues K95,K117,and K155.These findings provide mechanistic insight into the post-translational control of STING in teleosts and establish a foundation for future investigations into antiviral immune regulation.
