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
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).
