目的采用酵母双杂交体系寻找与脆性X智力低下蛋白(Fragile X men-tal retadation protein,FMRP)相互作用的蛋白,探讨FMRP的生物医学功能。方法以编码FMRP_(Pro327-Thr518)肽段的cDNA序列与pBTM116质粒DNA结合结构域重组作为"钓饵&q...目的采用酵母双杂交体系寻找与脆性X智力低下蛋白(Fragile X men-tal retadation protein,FMRP)相互作用的蛋白,探讨FMRP的生物医学功能。方法以编码FMRP_(Pro327-Thr518)肽段的cDNA序列与pBTM116质粒DNA结合结构域重组作为"钓饵",从小鼠胚胎cDNA文库中筛选与之相互作用蛋白的cDNA。结果筛选出13个与FMRP_(Pro327-Thr518)肽段特异性地相互作用的克隆,其中12个为小鼠泛素转运酶9(UBC9),1个是在GenBank中无高同源序列的未知cDNA序列。结论小鼠UBC9与人UBC9的氨基酸序列完全相同,且FMRP与UBC9蛋白表达的时空特征相似,因此认为人UBC9是与FMRP相互作用的蛋白,其相互作用的生物学意义有待进一步研究。本工作表明酵母双杂交体系是研究蛋白质相互作用的有效方法。展开更多
Fragiie X syndrome is the most common form of inherited mental retardation disease, resulting from absent of expression of its disease gene FMR1. To study the function of the fragiie X mental retardation protein (FMRP...Fragiie X syndrome is the most common form of inherited mental retardation disease, resulting from absent of expression of its disease gene FMR1. To study the function of the fragiie X mental retardation protein (FMRP) through protein/protein interaction, a mouse embryo cDNA library was screened by the yeast two-hybrid system. A clone was found to interact specifically with FMRP. The cDNA of this clone ( Genbank accession number af102875 ) encoded a protein highly homologous to human G/T mismatch-specific DNA thymine glycosylase ( hTDG ). Interactions between various alternatively spliced FMRP isoforms and a series of mTDG deletion proteins were further studied in the yeast two-hybrid system and their interaction amino acid regions were determined. interaction between FMRP and TDG existed inside exon 13 of FMRP ( amino acid residue 397-425 ) and around amino acid residue 122-346 of TDG. These results will be helpful to the study of the biological role of FMRP.展开更多
The type 2 DNA topoisomerases(Top2)are conserved enzymes and biomarkers for cell proliferation.The catalytic activities of the human isoform Top2a are essential for the regulation of DNA topology during DNA replicatio...The type 2 DNA topoisomerases(Top2)are conserved enzymes and biomarkers for cell proliferation.The catalytic activities of the human isoform Top2a are essential for the regulation of DNA topology during DNA replication,transcription,and chromosome segregation.Top2a is a prominent target for anti-cancer drugs and is highly regulated by post-translational modifications(PTM).Despite an increasing number of proteomic studies,the extent of PTM in cancer cells and its importance in drug response remains largely uncharacterized.In this review,we highlight the different modifications affecting the human Top2a in healthy and cancer cells,taking advantage of the structure-function information accumulated in the past decades.We also overview the regulation of Top2a by PTM,the level of PTM in cancer cells,and the resistance to therapeutic compounds targeting the Top2 enzyme.Altogether,this review underlines the importance of future studies addressing more systematically the interplay between PTM and Top2 drug resistance.展开更多
Heterogeneity of biological samples is usually considered a major obstacle for three-dimensional (3D) structure determination of macromolecular complexes. Heterogeneity may occur at the level of composition or conform...Heterogeneity of biological samples is usually considered a major obstacle for three-dimensional (3D) structure determination of macromolecular complexes. Heterogeneity may occur at the level of composition or conformational variability of complexes and affects most 3D structure determination methods that rely on signal averaging. Here, an approach is described that allows sorting structural states based on a 3D statistical approach, the 3D sampling and classification (3D-SC) of 3D structures derived from single particles imaged by cryo electron microscopy (cryo-EM). The method is based on jackknifing & bootstrapping of 3D sub-ensembles and 3D multivariate statistical analysis followed by 3D classification. The robustness of the statistical sorting procedure is corroborated using model data from an RNA polymerase structure and experimental data from a ribosome complex. It allows resolving multiple states within heterogeneous complexes that thus become amendable for a structural analysis despite of their highly flexible nature. The method has important implications for high-resolution structural studies and allows describing structure ensembles to provide insights into the dynamics of multi-component macromolecular assemblies.展开更多
Dentin is a mineralized tissue with a chemical composition similar to bone but with a higher mineralized density and rigidity.It constitutes the central structure of the tooth between the internal pulp and external en...Dentin is a mineralized tissue with a chemical composition similar to bone but with a higher mineralized density and rigidity.It constitutes the central structure of the tooth between the internal pulp and external enamel toward the oral cavity or cementum toward the underlying roots.Inherited dentin defects occur in a variety of rare genetic diseases.They can manifest as“isolated”occurrences such as in dentinogenesis imperfecta(DI)or dentin dysplasia(DD)or can be associated with other symptoms in diseases such as osteogenesis imperfecta,Goldblatt syndrome,microcephalic osteodysplastic primordial dwarfism type Ⅱ,among others.展开更多
Class switch recombination(CSR)occurs at the IgH locus and replaces the immunoglobulin(Ig)isotype expressed from IgM to IgG,IgE or IgA,endowing the B cell receptor with novel effector functions.CSR is triggered by act...Class switch recombination(CSR)occurs at the IgH locus and replaces the immunoglobulin(Ig)isotype expressed from IgM to IgG,IgE or IgA,endowing the B cell receptor with novel effector functions.CSR is triggered by activation-induced cytidine deaminase(AID),1 an enzyme that deaminates cytosines to uracils in single-stranded DNA exposed by transcription.The distinct antibody isotypes are encoded in the IgH locus in individual transcription units composed of a cytokine-inducible promoter,an intronic exon,and a switch region(Sx),followed by the exons encoding the constant region(Cx)(Fig.S1a).During CSR,the choice of recombination to a particular isotype is determined by the stimulation-dependent activation of specific promoters,triggering the generation of noncoding germline transcripts(GLTs).2 Thus far,the transcriptional regulation of the IgH locus is known to be controlled by the Eμenhancer,located downstream of the variable region and upstream of the donor switch region(Sμ),and the 3′regulatory region(3′RR)super-enhancer located downstream of Cα.展开更多
Retinoic-acid-related Orphan Receptors (RORs) regulate the maintenance of the circadian rhythm and immune response among others and are involved in increasing numbers of pathologies including autoimmune diseases, canc...Retinoic-acid-related Orphan Receptors (RORs) regulate the maintenance of the circadian rhythm and immune response among others and are involved in increasing numbers of pathologies including autoimmune diseases, cancer and neurological disorders leading to huge interest in the development of ROR ligands. Here, we describe the synthesis of six novel Gemini cholesterol analogues and the binding mode of one of them to RORγ.展开更多
文摘目的采用酵母双杂交体系寻找与脆性X智力低下蛋白(Fragile X men-tal retadation protein,FMRP)相互作用的蛋白,探讨FMRP的生物医学功能。方法以编码FMRP_(Pro327-Thr518)肽段的cDNA序列与pBTM116质粒DNA结合结构域重组作为"钓饵",从小鼠胚胎cDNA文库中筛选与之相互作用蛋白的cDNA。结果筛选出13个与FMRP_(Pro327-Thr518)肽段特异性地相互作用的克隆,其中12个为小鼠泛素转运酶9(UBC9),1个是在GenBank中无高同源序列的未知cDNA序列。结论小鼠UBC9与人UBC9的氨基酸序列完全相同,且FMRP与UBC9蛋白表达的时空特征相似,因此认为人UBC9是与FMRP相互作用的蛋白,其相互作用的生物学意义有待进一步研究。本工作表明酵母双杂交体系是研究蛋白质相互作用的有效方法。
文摘Fragiie X syndrome is the most common form of inherited mental retardation disease, resulting from absent of expression of its disease gene FMR1. To study the function of the fragiie X mental retardation protein (FMRP) through protein/protein interaction, a mouse embryo cDNA library was screened by the yeast two-hybrid system. A clone was found to interact specifically with FMRP. The cDNA of this clone ( Genbank accession number af102875 ) encoded a protein highly homologous to human G/T mismatch-specific DNA thymine glycosylase ( hTDG ). Interactions between various alternatively spliced FMRP isoforms and a series of mTDG deletion proteins were further studied in the yeast two-hybrid system and their interaction amino acid regions were determined. interaction between FMRP and TDG existed inside exon 13 of FMRP ( amino acid residue 397-425 ) and around amino acid residue 122-346 of TDG. These results will be helpful to the study of the biological role of FMRP.
基金This work was supported by the Fondation ARC and the grant ANR-10-LABX-0030-INRT(managed by the Agence Nationale de la Recherche under the frame programme Investissements d’Avenir ANR-10-IDEX-0002-02)The authors acknowledge the support and the use of resources of the French Infrastructure for Integrated Structural Biology FRISBI ANR-10-INBS-05 and of Instruct-ERIC.
文摘The type 2 DNA topoisomerases(Top2)are conserved enzymes and biomarkers for cell proliferation.The catalytic activities of the human isoform Top2a are essential for the regulation of DNA topology during DNA replication,transcription,and chromosome segregation.Top2a is a prominent target for anti-cancer drugs and is highly regulated by post-translational modifications(PTM).Despite an increasing number of proteomic studies,the extent of PTM in cancer cells and its importance in drug response remains largely uncharacterized.In this review,we highlight the different modifications affecting the human Top2a in healthy and cancer cells,taking advantage of the structure-function information accumulated in the past decades.We also overview the regulation of Top2a by PTM,the level of PTM in cancer cells,and the resistance to therapeutic compounds targeting the Top2 enzyme.Altogether,this review underlines the importance of future studies addressing more systematically the interplay between PTM and Top2 drug resistance.
文摘Heterogeneity of biological samples is usually considered a major obstacle for three-dimensional (3D) structure determination of macromolecular complexes. Heterogeneity may occur at the level of composition or conformational variability of complexes and affects most 3D structure determination methods that rely on signal averaging. Here, an approach is described that allows sorting structural states based on a 3D statistical approach, the 3D sampling and classification (3D-SC) of 3D structures derived from single particles imaged by cryo electron microscopy (cryo-EM). The method is based on jackknifing & bootstrapping of 3D sub-ensembles and 3D multivariate statistical analysis followed by 3D classification. The robustness of the statistical sorting procedure is corroborated using model data from an RNA polymerase structure and experimental data from a ribosome complex. It allows resolving multiple states within heterogeneous complexes that thus become amendable for a structural analysis despite of their highly flexible nature. The method has important implications for high-resolution structural studies and allows describing structure ensembles to provide insights into the dynamics of multi-component macromolecular assemblies.
基金the actions of the projects Offensives Sciences INTERREG IV A27 and“RARENET:No.1.7,a trinational network for education,research and management of complex and rare disorders in the Upper Rhine”co-financed by the European Regional Development Fund(ERDF)of the European Union in the framework of the INTERREG V Upper Rhine program as well as to the ERN(European reference network)CRANIO initiative.ABZ is a USIAS 2015 Fellow of the Institute of Advanced Studies(Institut d’Etudes Avancees)de l’Universite´de Strasbourg,France.It was also supported by the grant ANR10-LABX-0030-INRT,a French State fund managed by the Agence Nationale de la Recherche under the frame programme Investissements d’Avenir labelled ANR-10-IDEX0002-02.the Projet E-GENODENT financed by the Fonds d’Intervention Re´gionale(FIR)of the Agence Re´gionale de Sante´Grand Est(2019-2022)+1 种基金the“Impulsion Recherche”financial support of the“Filie`re de Sante´Maladies Rares TETECOU”2021 and 2022 and acknowledge the Fondation Force for supporting the DIAGNODENT project(2023-2026)the study design,data collection and analysis,decision to publish,or preparation of the manuscript.
文摘Dentin is a mineralized tissue with a chemical composition similar to bone but with a higher mineralized density and rigidity.It constitutes the central structure of the tooth between the internal pulp and external enamel toward the oral cavity or cementum toward the underlying roots.Inherited dentin defects occur in a variety of rare genetic diseases.They can manifest as“isolated”occurrences such as in dentinogenesis imperfecta(DI)or dentin dysplasia(DD)or can be associated with other symptoms in diseases such as osteogenesis imperfecta,Goldblatt syndrome,microcephalic osteodysplastic primordial dwarfism type Ⅱ,among others.
基金supported by the IGBMC’s International PhD Program LABEX fellowship and by the Fondation Recherche Médicalesupported by the grant ANR-10-LABX-0030-INRT,a French State fund managed by the Agence Nationale de la Recherche under the program Investissements d’Avenir labeled ANR-10-IDEX-0002-02.
文摘Class switch recombination(CSR)occurs at the IgH locus and replaces the immunoglobulin(Ig)isotype expressed from IgM to IgG,IgE or IgA,endowing the B cell receptor with novel effector functions.CSR is triggered by activation-induced cytidine deaminase(AID),1 an enzyme that deaminates cytosines to uracils in single-stranded DNA exposed by transcription.The distinct antibody isotypes are encoded in the IgH locus in individual transcription units composed of a cytokine-inducible promoter,an intronic exon,and a switch region(Sx),followed by the exons encoding the constant region(Cx)(Fig.S1a).During CSR,the choice of recombination to a particular isotype is determined by the stimulation-dependent activation of specific promoters,triggering the generation of noncoding germline transcripts(GLTs).2 Thus far,the transcriptional regulation of the IgH locus is known to be controlled by the Eμenhancer,located downstream of the variable region and upstream of the donor switch region(Sμ),and the 3′regulatory region(3′RR)super-enhancer located downstream of Cα.
基金supported financially by the Xunta de Galicia(ED431C2017/70).
文摘Retinoic-acid-related Orphan Receptors (RORs) regulate the maintenance of the circadian rhythm and immune response among others and are involved in increasing numbers of pathologies including autoimmune diseases, cancer and neurological disorders leading to huge interest in the development of ROR ligands. Here, we describe the synthesis of six novel Gemini cholesterol analogues and the binding mode of one of them to RORγ.
