目的克隆细粒棘球绦虫DNA损伤修复蛋白Rad50(DNA damagerepair protein Rad50 of Echinococcus granulosus,EgRad50)基因全长序列,并进行生物信息学分析。方法利用PCR技术克隆EgRad50基因全长序列,并通过NCBI蛋白数据库获取EgRad50的氨...目的克隆细粒棘球绦虫DNA损伤修复蛋白Rad50(DNA damagerepair protein Rad50 of Echinococcus granulosus,EgRad50)基因全长序列,并进行生物信息学分析。方法利用PCR技术克隆EgRad50基因全长序列,并通过NCBI蛋白数据库获取EgRad50的氨基酸序列,利用ProtParam、ProtScale、SignalP 5.0、TMHMM和ProtComp 9.0预测EgRad50蛋白的理化性质、MotifScan和NetPhos 3.1预测翻译后修饰位点、DNAStar和IEDB预测抗原表位、CDD预测结构域、SOPMA预测二级结构、Swiss-Model和VMD构建三级结构、DNAMAN进行多序列分析、MEGA构建系统进化树。结果克隆获得EgRad50基因全长序列共4073 bp,生物信息学预测EgRad50蛋白具有亲水性,分子质量为156.45 ku,无信号肽裂解位点,无跨膜区域,定位于细胞核和细胞质。二级结构以α螺旋为主,约占77.41%。EgRad50蛋白包括ATP酶相关(ATPases associated,AAA)特征结构域、DNA双链断裂修复ATP酶结构域和ATP结合盒(ATP-binding cassette,ABC)结构域。多序列比对发现该蛋白与多房棘球绦虫同源性为92.70%,与其他物种同源性较低。系统发育树表明EgRad50与智人、小鼠等哺乳动物Rad50亲缘关系较远。结论成功克隆并鉴定EgRad50基因,EgRad50蛋白主要参与DNA修复、细胞进程以及端粒维持等生物过程,是DNA损伤修复途径的重要蛋白。展开更多
Dear Editor,ATP-binding cassette(ABC)-ATPase(RAD50),together with meiotic recombination 11 homolog 1(MRE11)subunits,to form MRE11-RAD50 complex,plays important roles in recognition of double-stranded DNA(dsDNA)and ini...Dear Editor,ATP-binding cassette(ABC)-ATPase(RAD50),together with meiotic recombination 11 homolog 1(MRE11)subunits,to form MRE11-RAD50 complex,plays important roles in recognition of double-stranded DNA(dsDNA)and initiation of consequent inflammatory cascade1.Acute lung injury(ALI)and acute respiratory destress syndrome(ARDS)are systemic uncontrolled inflammation and life-threatening.However,the function of the DNA sensor in ALI/ARDS remains poorly defined.Here we investigated functions of RAD50 using mouse primary macrophages and conditionally RAD50 knockout mice in vitro and in a lipopolysaccharide(LPS)-induced lung injury model.展开更多
As prostate cancer(PCa)is one of the most commonly diagnosed cancer worldwide,identifying potential prognostic bio-markers is crucial.In this study,the survival information,gene expression,and protein expression data ...As prostate cancer(PCa)is one of the most commonly diagnosed cancer worldwide,identifying potential prognostic bio-markers is crucial.In this study,the survival information,gene expression,and protein expression data of 344 PCa cases were collected from the Cancer Proteome Atlas(TCPA)and the Cancer Genome Atlas(TCGA)to investigate the potential prognostic biomarkers.The integrated prognosis-related proteins(IPRPs)model was constructed based on the risk score of each patients using machine-learning algorithm.IPRPs model suggested that Elevated RAD50 expression(p=0.016)and down-regulated SMAD4 expression(p=0.017)were significantly correlated with unfavorable outcomes for PCa patients.Immunohistochemical(IHC)staining and western blot(WB)analysis revealed significant differential expression of SMAD4 and RAD50 protein between tumor and normal tissues in validation cohort.According to the overall IHC score,patients with low SMAD4(p<0.0001)expression and high RAD50 expression(p=0.0001)were significantly correlated with poor outcomes.Besides,expression of SMAD4 showed significantly negative correlation with most immune checkpoint molecules,and the low SMAD4 expression group exhibited significantly high levels of LAG3(p<0.05),TGFβ(p<0.001),and PD-L1(p<0.05)compared with the high SMAD4 expression group in the validation cohort.Patients with low SMAD4 expression had significantly higher infiltration of memory B cells(p=0.002),CD8+T cells(p<0.001),regulatory T cells(p=0.006),M2-type macrophages(p<0.001),and significantly lower infiltration of naïve B cells(p=0.002),plasma cells(p<0.001),resting memory CD4+T cells(p<0.001)and eosinophils(p=0.045).Candidate proteins were mainly involved in antigen processing and presentation,stem cell differentiation,and type I interferon pathways.展开更多
基金The authors are grateful for financial support from the National Natural Science Foundation of China(81870007,81920108001,81800024,81900025,81870023,81700025)the Zhejiang Provincial Natural Science Foundation(LD19H160001)the Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents(2016-63).
文摘Dear Editor,ATP-binding cassette(ABC)-ATPase(RAD50),together with meiotic recombination 11 homolog 1(MRE11)subunits,to form MRE11-RAD50 complex,plays important roles in recognition of double-stranded DNA(dsDNA)and initiation of consequent inflammatory cascade1.Acute lung injury(ALI)and acute respiratory destress syndrome(ARDS)are systemic uncontrolled inflammation and life-threatening.However,the function of the DNA sensor in ALI/ARDS remains poorly defined.Here we investigated functions of RAD50 using mouse primary macrophages and conditionally RAD50 knockout mice in vitro and in a lipopolysaccharide(LPS)-induced lung injury model.
基金supported by National Key Research and Development Project(No.2019YFC1316000)National Natural Science Foundation of China(No.81772706 and No.81802525).
文摘As prostate cancer(PCa)is one of the most commonly diagnosed cancer worldwide,identifying potential prognostic bio-markers is crucial.In this study,the survival information,gene expression,and protein expression data of 344 PCa cases were collected from the Cancer Proteome Atlas(TCPA)and the Cancer Genome Atlas(TCGA)to investigate the potential prognostic biomarkers.The integrated prognosis-related proteins(IPRPs)model was constructed based on the risk score of each patients using machine-learning algorithm.IPRPs model suggested that Elevated RAD50 expression(p=0.016)and down-regulated SMAD4 expression(p=0.017)were significantly correlated with unfavorable outcomes for PCa patients.Immunohistochemical(IHC)staining and western blot(WB)analysis revealed significant differential expression of SMAD4 and RAD50 protein between tumor and normal tissues in validation cohort.According to the overall IHC score,patients with low SMAD4(p<0.0001)expression and high RAD50 expression(p=0.0001)were significantly correlated with poor outcomes.Besides,expression of SMAD4 showed significantly negative correlation with most immune checkpoint molecules,and the low SMAD4 expression group exhibited significantly high levels of LAG3(p<0.05),TGFβ(p<0.001),and PD-L1(p<0.05)compared with the high SMAD4 expression group in the validation cohort.Patients with low SMAD4 expression had significantly higher infiltration of memory B cells(p=0.002),CD8+T cells(p<0.001),regulatory T cells(p=0.006),M2-type macrophages(p<0.001),and significantly lower infiltration of naïve B cells(p=0.002),plasma cells(p<0.001),resting memory CD4+T cells(p<0.001)and eosinophils(p=0.045).Candidate proteins were mainly involved in antigen processing and presentation,stem cell differentiation,and type I interferon pathways.
