Ras proteins,which are important for cellular signaling and cancer development,are activated by guanine nucleotide exchange factors,among which son-of-sevenless(Sos)plays a pivotal role.The catalytic activity of Sos i...Ras proteins,which are important for cellular signaling and cancer development,are activated by guanine nucleotide exchange factors,among which son-of-sevenless(Sos)plays a pivotal role.The catalytic activity of Sos is regulated by its interaction with Ras·GTP at the allosteric site of Sos.Although much research has been conducted,the structural details of the dynamic interactions between Ras·GTP and Sos at the allosteric site in solution are not completely understood.In this study,paramagnetic relaxation enhancement(PRE)was used to examine the binding of HRas·GTP to the allosteric site of Soscat.A site-specific paramagnetic probe was attached to Soscat,revealing crucial interface information,which was then used to build structural models of the HRas-Soscat complex via HADDOCK.Our results indicate that the dominant conformation of HRas·GTP bound to Soscat at the allosteric site is highly consistent with the established crystal structure while also identifying a transient conformation with a novel interface between HRas and Sos unseen in previous crystal structures.展开更多
Rat sarcoma virus oncogene(RAS)proteins are of crucial oncogenic proteins and are involved in several essential intracellular processes.The RAS protein has an intrinsic metal binding site for Mg^(2+),which is importan...Rat sarcoma virus oncogene(RAS)proteins are of crucial oncogenic proteins and are involved in several essential intracellular processes.The RAS protein has an intrinsic metal binding site for Mg^(2+),which is important for the conformational stability of the active site.Recently,it was reported that a second metal ion binding site,located further from the active site in HRAS(Harvey RAS homolog),binds Ca^(2+) with millimolar affinity.As one of the most abundant metal ions in cells,Mn^(2+) is a potential candidate for the second metal ion binding site in RAS proteins.Here,we examined the interaction of Mn^(2+) with HRAS and KRAS(Kirsten RAS homolog)using high resolution NMR spectroscopy.The NMR data showed that both the second metal ion binding site and the switch I and II regions bind Mn^(2+) in the RAS proteins.Furthermore,our paramagnetic NMR results disclosed the conformational differences in helix a3 and the following loop between HRAS and KRAS,accompanied by the association with metal ion binding.These results provide new insights into the interaction of RAS proteins and Mn^(2+) in the respective biological processes in cells.展开更多
Background:The precise insertion of large DNA fragments(>3–5 kb)remains one of the key obstacles in establishment of genetically modified murine models.Methods:A 21 kb large DNA fragment containing three tandemly ...Background:The precise insertion of large DNA fragments(>3–5 kb)remains one of the key obstacles in establishment of genetically modified murine models.Methods:A 21 kb large DNA fragment containing three tandemly linked copies of the human HRAS gene was inserted into the genome of C57BL/6J mouse,generating a mouse model designated as KI.C57-ras(or named NF-h HRAS).Whole-genome sequencing and Sanger sequencing were utilized to it confirm precise insertion and copy number.The stability of transgene expression among different generations was verified from multiple aspects using by digital PCR,western blot and DNA sequencing.To assess tumor susceptibility in the mouse model,N-Nitroso-N-methylurea(MNU)was administered at a dosage of 75 mg/kg.Histopathological examinations were conducted using hematoxylin and eosin(H&E)staining.Results:The HRAS DNA fragment was inserted into mouse chromosome 15E1 site,locating between 80623202 bp and 80625020 bp.NF-h HRAS mice exhibited stable inheritance and displayed consistent phenotypes across individuals.Moreover,this mouse model exhibited a high susceptibility to carcinogens.Upon administration of MNU the earliest mortality onset was earlier than that of wild-type littermates(day 65 vs.day 78 for male and day 56 vs.day 84 for female).Notably,100%of the NF-h HRAS transgenic mice developed tumors,with approximately 84%of male NF-h HRAS mice exhibiting specific tumor types,such as squamous cell carcinoma or squamous cell papilloma,which was consistent with the previously reported carcinogenic rasH2 mouse model.The types of tumors and the target organs exhibited diversity in NFh HRAS mice,while the spontaneous tumor incidence remained low(1/50).Conclusions:The NF-h HRAS mice demonstrated excellent genetic stability,a reproducible phenotype,and high susceptibility to carcinogens,indicating their potential utility in non-clinical safety evaluations of drugs as per the S1B guidelines issued by the ICH(The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use).展开更多
Hepatocellular carcinoma(HCC),a prevalent solid carcinoma of significant concern,is an aggressive and often fatal disease with increasing global incidence rates and poor therapeutic outcomes.The etiology and pathologi...Hepatocellular carcinoma(HCC),a prevalent solid carcinoma of significant concern,is an aggressive and often fatal disease with increasing global incidence rates and poor therapeutic outcomes.The etiology and pathological progression of non-alcoholic steatohepatitis(NASH)-related HCC is multifactorial and multistage.However,no single animal model can accurately mimic the full NASH-related HCC pathological progression,posing considerable challenges to transition and mechanistic studies.Herein,a novel conditional inducible wild-type human HRAS overexpressed mouse model(HRAS-HCC)was established,demonstrating 100%morbidity and mortality within approximately one month under normal dietary and lifestyle conditions.Advanced symptoms of HCC such as ascites,thrombus,internal hemorrhage,jaundice,and lung metastasis were successfully replicated in mice.In-depth pathological features of NASH-related HCC were demonstrated by pathological staining,biochemical analyses,and typical marker gene detections.Combined murine anti-PD-1 and sorafenib treatment effectively prolonged mouse survival,further confirming the accuracy and reliability of the model.Based on protein-protein interaction(PPI)network and RNA sequencing analyses,we speculated that overexpression of HRAS may initiate the THBS1-COL4A3 axis to induce NASH with severe fibrosis,with subsequent progression to HCC.Collectively,our study successfully duplicated natural sequential progression in a single murine model over a very short period,providing an accurate and reliable preclinical tool for therapeutic evaluations targeting the NASH to HCC continuum.展开更多
目的肝细胞癌(hepatocellular carcinoma,HCC)的发生具有显著的性别差异,但其分子机制尚不清楚。本研究通过检测Hras12V转基因小鼠雌雄肝肿瘤组织的蛋白质组学表达差异,探讨肝肿瘤发生的性别差异机制。方法采用双向荧光差异凝胶电泳(two...目的肝细胞癌(hepatocellular carcinoma,HCC)的发生具有显著的性别差异,但其分子机制尚不清楚。本研究通过检测Hras12V转基因小鼠雌雄肝肿瘤组织的蛋白质组学表达差异,探讨肝肿瘤发生的性别差异机制。方法采用双向荧光差异凝胶电泳(two dimension difference gel electrophoresis,2D-DIGE)和基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS)技术检测Hras12V雌雄小鼠肝肿瘤组织的蛋白质组学差异表达谱,分离并鉴定差异表达蛋白,应用蛋白质印迹法验证2D-DIGE结果,并对差异表达蛋白进行生物信息学分析,包括蛋白功能注释、分类(gene ontology,GO)分析、京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)通路分析。结果经双向荧光差异凝胶电泳-图像分析得到1 313个蛋白点,其中差异倍数≥1.5(P<0.05)的蛋白点553个。从中选择94个差异蛋白点进行MALDI-TOF-MS鉴定,得到56种差异性表达的蛋白质。其中,与雌鼠相比,雄鼠肝肿瘤组织中高表达的蛋白有43种,低表达的蛋白有13种。选取4个差异蛋白FABP1、Albumin、Prdx6和CALR进行了蛋白质印迹法验证,其中FABP1在雄鼠肝癌中显著上调,Albumin和Prdx6在雄鼠肝癌中显著下调,CALR在雌雄小鼠肝癌中差异无统计学意义,结果与质谱数据基本一致,证明了2D-DIGE结果的可靠性。对雌雄鼠肝肿瘤组织中的差异蛋白进行GO分析,细胞组分分析显示,差异蛋白主要分布在细胞质、细胞溶质、线粒体和细胞核中;分子功能分析显示,差异蛋白主要承担poly(A)RNA结合、ATP结合和酶结合等功能;生物过程分析显示,差异蛋白主要参与运输、脂代谢过程和凋亡过程的负调控等。KEGG通路分析结果表明,差异蛋白涉及9条信号通路,主要通路包括代谢途径、内质网加工过程和细胞色素P450的异物代谢等通路。结论 Hras12V转基因小鼠雌雄肝肿瘤组织的差异性蛋白表达的检测,为研究性别差异性肝癌发生分子机制提供了基础生物学信息和有益的线索。展开更多
基金supported by the Fundamental Research Funds for the Central Universities(WK9100000046).
文摘Ras proteins,which are important for cellular signaling and cancer development,are activated by guanine nucleotide exchange factors,among which son-of-sevenless(Sos)plays a pivotal role.The catalytic activity of Sos is regulated by its interaction with Ras·GTP at the allosteric site of Sos.Although much research has been conducted,the structural details of the dynamic interactions between Ras·GTP and Sos at the allosteric site in solution are not completely understood.In this study,paramagnetic relaxation enhancement(PRE)was used to examine the binding of HRas·GTP to the allosteric site of Soscat.A site-specific paramagnetic probe was attached to Soscat,revealing crucial interface information,which was then used to build structural models of the HRas-Soscat complex via HADDOCK.Our results indicate that the dominant conformation of HRas·GTP bound to Soscat at the allosteric site is highly consistent with the established crystal structure while also identifying a transient conformation with a novel interface between HRas and Sos unseen in previous crystal structures.
基金supported by the Ministry of Science and Technology of China(2021YFA1600304)the National Natural Science Foundation of China(22161142018,21991081,22174074 and 22374126).
文摘Rat sarcoma virus oncogene(RAS)proteins are of crucial oncogenic proteins and are involved in several essential intracellular processes.The RAS protein has an intrinsic metal binding site for Mg^(2+),which is important for the conformational stability of the active site.Recently,it was reported that a second metal ion binding site,located further from the active site in HRAS(Harvey RAS homolog),binds Ca^(2+) with millimolar affinity.As one of the most abundant metal ions in cells,Mn^(2+) is a potential candidate for the second metal ion binding site in RAS proteins.Here,we examined the interaction of Mn^(2+) with HRAS and KRAS(Kirsten RAS homolog)using high resolution NMR spectroscopy.The NMR data showed that both the second metal ion binding site and the switch I and II regions bind Mn^(2+) in the RAS proteins.Furthermore,our paramagnetic NMR results disclosed the conformational differences in helix a3 and the following loop between HRAS and KRAS,accompanied by the association with metal ion binding.These results provide new insights into the interaction of RAS proteins and Mn^(2+) in the respective biological processes in cells.
基金National Key R&D Program of China,Grant/Award Number:2023YFC3402000National Institutes for Food and Drug Control,State Key Laboratory of Drug Regulatory Science,Grant/Award Number:2023SKLDRS0124。
文摘Background:The precise insertion of large DNA fragments(>3–5 kb)remains one of the key obstacles in establishment of genetically modified murine models.Methods:A 21 kb large DNA fragment containing three tandemly linked copies of the human HRAS gene was inserted into the genome of C57BL/6J mouse,generating a mouse model designated as KI.C57-ras(or named NF-h HRAS).Whole-genome sequencing and Sanger sequencing were utilized to it confirm precise insertion and copy number.The stability of transgene expression among different generations was verified from multiple aspects using by digital PCR,western blot and DNA sequencing.To assess tumor susceptibility in the mouse model,N-Nitroso-N-methylurea(MNU)was administered at a dosage of 75 mg/kg.Histopathological examinations were conducted using hematoxylin and eosin(H&E)staining.Results:The HRAS DNA fragment was inserted into mouse chromosome 15E1 site,locating between 80623202 bp and 80625020 bp.NF-h HRAS mice exhibited stable inheritance and displayed consistent phenotypes across individuals.Moreover,this mouse model exhibited a high susceptibility to carcinogens.Upon administration of MNU the earliest mortality onset was earlier than that of wild-type littermates(day 65 vs.day 78 for male and day 56 vs.day 84 for female).Notably,100%of the NF-h HRAS transgenic mice developed tumors,with approximately 84%of male NF-h HRAS mice exhibiting specific tumor types,such as squamous cell carcinoma or squamous cell papilloma,which was consistent with the previously reported carcinogenic rasH2 mouse model.The types of tumors and the target organs exhibited diversity in NFh HRAS mice,while the spontaneous tumor incidence remained low(1/50).Conclusions:The NF-h HRAS mice demonstrated excellent genetic stability,a reproducible phenotype,and high susceptibility to carcinogens,indicating their potential utility in non-clinical safety evaluations of drugs as per the S1B guidelines issued by the ICH(The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use).
基金supported by the National Institutes for Food and Drug Control,State Key Laboratory of Drug Regulatory Science。
文摘Hepatocellular carcinoma(HCC),a prevalent solid carcinoma of significant concern,is an aggressive and often fatal disease with increasing global incidence rates and poor therapeutic outcomes.The etiology and pathological progression of non-alcoholic steatohepatitis(NASH)-related HCC is multifactorial and multistage.However,no single animal model can accurately mimic the full NASH-related HCC pathological progression,posing considerable challenges to transition and mechanistic studies.Herein,a novel conditional inducible wild-type human HRAS overexpressed mouse model(HRAS-HCC)was established,demonstrating 100%morbidity and mortality within approximately one month under normal dietary and lifestyle conditions.Advanced symptoms of HCC such as ascites,thrombus,internal hemorrhage,jaundice,and lung metastasis were successfully replicated in mice.In-depth pathological features of NASH-related HCC were demonstrated by pathological staining,biochemical analyses,and typical marker gene detections.Combined murine anti-PD-1 and sorafenib treatment effectively prolonged mouse survival,further confirming the accuracy and reliability of the model.Based on protein-protein interaction(PPI)network and RNA sequencing analyses,we speculated that overexpression of HRAS may initiate the THBS1-COL4A3 axis to induce NASH with severe fibrosis,with subsequent progression to HCC.Collectively,our study successfully duplicated natural sequential progression in a single murine model over a very short period,providing an accurate and reliable preclinical tool for therapeutic evaluations targeting the NASH to HCC continuum.
文摘目的肝细胞癌(hepatocellular carcinoma,HCC)的发生具有显著的性别差异,但其分子机制尚不清楚。本研究通过检测Hras12V转基因小鼠雌雄肝肿瘤组织的蛋白质组学表达差异,探讨肝肿瘤发生的性别差异机制。方法采用双向荧光差异凝胶电泳(two dimension difference gel electrophoresis,2D-DIGE)和基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS)技术检测Hras12V雌雄小鼠肝肿瘤组织的蛋白质组学差异表达谱,分离并鉴定差异表达蛋白,应用蛋白质印迹法验证2D-DIGE结果,并对差异表达蛋白进行生物信息学分析,包括蛋白功能注释、分类(gene ontology,GO)分析、京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes,KEGG)通路分析。结果经双向荧光差异凝胶电泳-图像分析得到1 313个蛋白点,其中差异倍数≥1.5(P<0.05)的蛋白点553个。从中选择94个差异蛋白点进行MALDI-TOF-MS鉴定,得到56种差异性表达的蛋白质。其中,与雌鼠相比,雄鼠肝肿瘤组织中高表达的蛋白有43种,低表达的蛋白有13种。选取4个差异蛋白FABP1、Albumin、Prdx6和CALR进行了蛋白质印迹法验证,其中FABP1在雄鼠肝癌中显著上调,Albumin和Prdx6在雄鼠肝癌中显著下调,CALR在雌雄小鼠肝癌中差异无统计学意义,结果与质谱数据基本一致,证明了2D-DIGE结果的可靠性。对雌雄鼠肝肿瘤组织中的差异蛋白进行GO分析,细胞组分分析显示,差异蛋白主要分布在细胞质、细胞溶质、线粒体和细胞核中;分子功能分析显示,差异蛋白主要承担poly(A)RNA结合、ATP结合和酶结合等功能;生物过程分析显示,差异蛋白主要参与运输、脂代谢过程和凋亡过程的负调控等。KEGG通路分析结果表明,差异蛋白涉及9条信号通路,主要通路包括代谢途径、内质网加工过程和细胞色素P450的异物代谢等通路。结论 Hras12V转基因小鼠雌雄肝肿瘤组织的差异性蛋白表达的检测,为研究性别差异性肝癌发生分子机制提供了基础生物学信息和有益的线索。
