Nuclear pore complexes(NPCs)regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells.Intrinsically disordered Phe-Gly nucleoporins(FG-Nups)line the central conduit of NPCs to impart a...Nuclear pore complexes(NPCs)regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells.Intrinsically disordered Phe-Gly nucleoporins(FG-Nups)line the central conduit of NPCs to impart a selective barrier where large proteins are excluded unless bound to a transport receptor(karyopherin;Kap).Here,we assess“Kap-centric”NPC models,which postulate that Kaps participate in establishing the selective barrier.We combine biomimetic nanopores,formed by tethering Nsp1 to the inner wall of a solid-state nanopore,with coarse-grained modeling to show that yeast Kap95 exhibits two populations in Nsp1-coated pores:one population that is transported across the pore in milliseconds,and a second population that is stably assembled within the FG mesh of the pore.Ionic current measurements show a conductance decrease for increasing Kap concentrations and noise data indicate an increase in rigidity of the FG-mesh.Modeling reveals an accumulation of Kap95 near the pore wall,yielding a conductance decrease.We find that Kaps only mildly affect the conformation of the Nsp1 mesh and that,even at high concentrations,Kaps only bind at most 8%of the FG-motifs in the nanopore,indicating that Kap95 occupancy is limited by steric constraints rather than by depletion of available FG-motifs.Our data provide an alternative explanation of the origin of bimodal NPC binding of Kaps,where a stable population of Kaps binds avidly to the NPC periphery,while fast transport proceeds via a central FG-rich channel through lower affinity interactions between Kaps and the cohesive domains of Nsp1.展开更多
Importin proteins were originally characterized for their central role in protein transport through the nuclear pores, the only intracellular entry to the nucleus. This vital function must be tightly regulated to cont...Importin proteins were originally characterized for their central role in protein transport through the nuclear pores, the only intracellular entry to the nucleus. This vital function must be tightly regulated to control access by transcription factors and other nuclear proteins to genomic DNA, to achieve appropriate modulation of cellular behaviors affecting cell fate. Importin-mediated nucleocytoplasmic transport relies on their specific recognition of cargoes, with each importin binding to distinct and overlapping protein subsets. Knowledge of importin function has expanded substantially in regard to three key developmental systems： embryonic stem cells, muscle cells and the germ line. In the decade since the potential for regulated nucleocytoplasmic transport to contribute to spermatogenesis was proposed, we and others have shown that the importins that ferry transcription factors into the nucleus perform additional roles, which control cell fate. This review presents key findings from studies of mammalian spermatogenesis that reveal potential new pathways by which male fertility and infertility arise. These studies of germline genesis illuminate new ways in which importin proteins govern cellular differentiation, includ ng v a d rect ng proteins to d st nct ntrace ular compartments and by determining cellular stress responses.展开更多
The nucleotide-binding and leucine-rich repeat(NLR)proteins comprise a major class of intracellular immune receptors that are capable of detecting pathogen-derived molecules and activating immunity and cell death in p...The nucleotide-binding and leucine-rich repeat(NLR)proteins comprise a major class of intracellular immune receptors that are capable of detecting pathogen-derived molecules and activating immunity and cell death in plants.The activity of some NLRs,particularly the Toll-like/interleukin-1 receptor(TIR)type,is highly correlated with their nucleocytoplasmic distribution.However,whether and how the nucleocytoplasmic homeostasis of NLRs is coordinated through a bidirectional nuclear shuttling mechanism remains unclear.Here,we identified a nuclear transport receptor,KA120,which is capable of affecting the nucleocytoplasmic distribution of an NLR protein and is essential in preventing its autoactivation.We showed that the ka120 mutant displays an autoimmune phenotype and NLR-induced transcriptome features.Through a targeted genetic screen using an artificial NLR microRNA library,we identified the TIR-NLR gene SNC1 as a genetic interactor of KA120.Loss-of-function snc1 mutations as well as compromising SNC1 protein activities all substantially suppressed ka120-induced autoimmune activation,and the enhanced SNC1 activity upon loss of KA120 functionappeared to occur at the protein level.Overexpression of KA120 efficiently repressed SNC1 activity and led to a nearly complete suppression of the autoimmune phenotype caused by the gain-of-function snc1-1 mutation or SNC1 overexpression in transgenic plants.Further florescence imaging analysis indicated that SNC1 undergoes altered nucleocytoplasmic distribution with significantly reduced nuclear signal when KA120 is constitutively expressed,supporting a role of KA120 in coordinating SNC1 nuclear abundance and activity.Consistently,compromising the SNC1 nuclear level by disrupting the nuclear pore complex could also partially rescue ka120-induced autoimmunity.Collectively,our study demonstrates that KA120 is essential to avoid autoimmune activation in the absence of pathogens and is required to constrain the nuclear activity of SNC1,possibly through coordinating SNC1 nucleocytoplasmic homeostasis as a potential mechanism.展开更多
Aim:Patients with glioblastomas demonstrate well‑documented immunological impairments including decreased numbers of mature dendritic cells(DCs).Recent data identified karyopherin a2(KPNA2),a nucleocytoplasmic shuttli...Aim:Patients with glioblastomas demonstrate well‑documented immunological impairments including decreased numbers of mature dendritic cells(DCs).Recent data identified karyopherin a2(KPNA2),a nucleocytoplasmic shuttling receptor,as diagnostic and prognostic biomarker for gliomas.The aim of this ongoing study is to correlate parameters of immunity and nucleocytoplasmic transport in glioblastoma patients.Methods:We preoperatively collected serum from 17 patients with glioblastomas and determined DC subsets(HLA DR+Lin-,CD34-,CD45+,CD123+,CD11+were analyzed)using a 6‑color flow cytometry panel.Expression levels of KPNA2 and nuclear accumulation of p53 were evaluated semi‑quantitatively by immunohistochemistry.O6‑methylguanine DNA methyltransferase(MGMT)and isocitrate dehydrogenase‑1(IDH‑1)status were assessed by pyrosequencing and immunohistochemistry,respectively.Results:Median expression levels for both KPNA2 and p53 were 5-10%.IDH‑1‑R132H mutation and MGMT promoter hypermethylation was detected in 3/16 and 1/9 patients,respectively.Mean counts of total mature DCs,myeloid DCs and plasmacytoid DCs were 9.6,2.1,3.4 cells/μL.A preliminary analysis suggests an association between low KPNA2 nuclear expression and increased numbers of mature DCs.However,this correlation did not reach statistical significance so far(P=0.077).Conclusion:Our preliminary data may indicate a role of KPNA2 in the impaired maturation of DCs observed in glioblastoma patients.展开更多
基金funded by NWO-I programme“Projectruimte”,Grant No.16PR3242-1.
文摘Nuclear pore complexes(NPCs)regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells.Intrinsically disordered Phe-Gly nucleoporins(FG-Nups)line the central conduit of NPCs to impart a selective barrier where large proteins are excluded unless bound to a transport receptor(karyopherin;Kap).Here,we assess“Kap-centric”NPC models,which postulate that Kaps participate in establishing the selective barrier.We combine biomimetic nanopores,formed by tethering Nsp1 to the inner wall of a solid-state nanopore,with coarse-grained modeling to show that yeast Kap95 exhibits two populations in Nsp1-coated pores:one population that is transported across the pore in milliseconds,and a second population that is stably assembled within the FG mesh of the pore.Ionic current measurements show a conductance decrease for increasing Kap concentrations and noise data indicate an increase in rigidity of the FG-mesh.Modeling reveals an accumulation of Kap95 near the pore wall,yielding a conductance decrease.We find that Kaps only mildly affect the conformation of the Nsp1 mesh and that,even at high concentrations,Kaps only bind at most 8%of the FG-motifs in the nanopore,indicating that Kap95 occupancy is limited by steric constraints rather than by depletion of available FG-motifs.Our data provide an alternative explanation of the origin of bimodal NPC binding of Kaps,where a stable population of Kaps binds avidly to the NPC periphery,while fast transport proceeds via a central FG-rich channel through lower affinity interactions between Kaps and the cohesive domains of Nsp1.
文摘Importin proteins were originally characterized for their central role in protein transport through the nuclear pores, the only intracellular entry to the nucleus. This vital function must be tightly regulated to control access by transcription factors and other nuclear proteins to genomic DNA, to achieve appropriate modulation of cellular behaviors affecting cell fate. Importin-mediated nucleocytoplasmic transport relies on their specific recognition of cargoes, with each importin binding to distinct and overlapping protein subsets. Knowledge of importin function has expanded substantially in regard to three key developmental systems： embryonic stem cells, muscle cells and the germ line. In the decade since the potential for regulated nucleocytoplasmic transport to contribute to spermatogenesis was proposed, we and others have shown that the importins that ferry transcription factors into the nucleus perform additional roles, which control cell fate. This review presents key findings from studies of mammalian spermatogenesis that reveal potential new pathways by which male fertility and infertility arise. These studies of germline genesis illuminate new ways in which importin proteins govern cellular differentiation, includ ng v a d rect ng proteins to d st nct ntrace ular compartments and by determining cellular stress responses.
基金X.Shen and X.Shi were supported by Tsinghua-Peking Joint Center tor Life SciencesThis project was supported by the USDA National Institute of Food and Agriculture(HATCH project CA-B-PLB-0243-H)+1 种基金the National Science Foundation(grant MCB-2049931)startup funds from Inno-vative Genomics Institute and University of California Berkeley.
文摘The nucleotide-binding and leucine-rich repeat(NLR)proteins comprise a major class of intracellular immune receptors that are capable of detecting pathogen-derived molecules and activating immunity and cell death in plants.The activity of some NLRs,particularly the Toll-like/interleukin-1 receptor(TIR)type,is highly correlated with their nucleocytoplasmic distribution.However,whether and how the nucleocytoplasmic homeostasis of NLRs is coordinated through a bidirectional nuclear shuttling mechanism remains unclear.Here,we identified a nuclear transport receptor,KA120,which is capable of affecting the nucleocytoplasmic distribution of an NLR protein and is essential in preventing its autoactivation.We showed that the ka120 mutant displays an autoimmune phenotype and NLR-induced transcriptome features.Through a targeted genetic screen using an artificial NLR microRNA library,we identified the TIR-NLR gene SNC1 as a genetic interactor of KA120.Loss-of-function snc1 mutations as well as compromising SNC1 protein activities all substantially suppressed ka120-induced autoimmune activation,and the enhanced SNC1 activity upon loss of KA120 functionappeared to occur at the protein level.Overexpression of KA120 efficiently repressed SNC1 activity and led to a nearly complete suppression of the autoimmune phenotype caused by the gain-of-function snc1-1 mutation or SNC1 overexpression in transgenic plants.Further florescence imaging analysis indicated that SNC1 undergoes altered nucleocytoplasmic distribution with significantly reduced nuclear signal when KA120 is constitutively expressed,supporting a role of KA120 in coordinating SNC1 nuclear abundance and activity.Consistently,compromising the SNC1 nuclear level by disrupting the nuclear pore complex could also partially rescue ka120-induced autoimmunity.Collectively,our study demonstrates that KA120 is essential to avoid autoimmune activation in the absence of pathogens and is required to constrain the nuclear activity of SNC1,possibly through coordinating SNC1 nucleocytoplasmic homeostasis as a potential mechanism.
文摘Aim:Patients with glioblastomas demonstrate well‑documented immunological impairments including decreased numbers of mature dendritic cells(DCs).Recent data identified karyopherin a2(KPNA2),a nucleocytoplasmic shuttling receptor,as diagnostic and prognostic biomarker for gliomas.The aim of this ongoing study is to correlate parameters of immunity and nucleocytoplasmic transport in glioblastoma patients.Methods:We preoperatively collected serum from 17 patients with glioblastomas and determined DC subsets(HLA DR+Lin-,CD34-,CD45+,CD123+,CD11+were analyzed)using a 6‑color flow cytometry panel.Expression levels of KPNA2 and nuclear accumulation of p53 were evaluated semi‑quantitatively by immunohistochemistry.O6‑methylguanine DNA methyltransferase(MGMT)and isocitrate dehydrogenase‑1(IDH‑1)status were assessed by pyrosequencing and immunohistochemistry,respectively.Results:Median expression levels for both KPNA2 and p53 were 5-10%.IDH‑1‑R132H mutation and MGMT promoter hypermethylation was detected in 3/16 and 1/9 patients,respectively.Mean counts of total mature DCs,myeloid DCs and plasmacytoid DCs were 9.6,2.1,3.4 cells/μL.A preliminary analysis suggests an association between low KPNA2 nuclear expression and increased numbers of mature DCs.However,this correlation did not reach statistical significance so far(P=0.077).Conclusion:Our preliminary data may indicate a role of KPNA2 in the impaired maturation of DCs observed in glioblastoma patients.
