The human myxovirus resistance 2(Mx2/Mx B)protein,a member of interferon(IFN)-inducible dynamin-like large GTPases,restricts a number of virus infections.Inhibition of these viruses occurs at poorly-defined steps afte...The human myxovirus resistance 2(Mx2/Mx B)protein,a member of interferon(IFN)-inducible dynamin-like large GTPases,restricts a number of virus infections.Inhibition of these viruses occurs at poorly-defined steps after viral entry and has a common requirement for Mx B oligomerization.However,the GTPase activity is essential for the anti-viral effects of Mx B against herpesviruses and HBV but not HIV-1.To understand the role of Mx B GTPase activity,including GTP binding and GTP hydrolysis,in restriction of HIV-1 infection,we genetically separated these two functions and evaluated their contributions to restriction.We found that both the GTP binding and hydrolysis function of Mx B involved in the restriction of HIV-1 replication.The GTPase activity of Mx B contributed to its nuclear location,interaction with nucleoporins(NUPs)and HIV-1 capsids.Furthermore,Mx B disrupted the association between NUPs and HIV-1 cores dependently upon its GTPase activity.The function of GTPase activity was therefore multi-faceted,led to fundamentally distinct mechanisms employed by wild-type Mx B and GTPase activity defective Mx B mutations to restrict HIV-1 replication.展开更多
AIM: To investigate the expression of nucleoporin 88 (Nup88) in hepatitis B virus (HBV) and C virus (HCV)-related liver diseases. METHODS: We generated a new monoclonal Nup88 antibody to investigate the Nup88 protein ...AIM: To investigate the expression of nucleoporin 88 (Nup88) in hepatitis B virus (HBV) and C virus (HCV)-related liver diseases. METHODS: We generated a new monoclonal Nup88 antibody to investigate the Nup88 protein expression by immunohistochemistry (IHC) in 294 paraffin-embedded liver specimens comprising all stages of hepatocellular carcinogenesis. In addition, in cell culture experiments HBV-positive (HepG2.2.15 and HB611) and HBV-negative (HepG2) hepatoma cell lines were tested for the Nup88 expression by Western-immunoblotting to test data obtained by IHC.RESULTS: Specific Nup88 expression was found in chronic HCV hepatitis and unspecific chronic hepatitis, whereas no or very weak Nup88 expression was detected in normal liver. The Nup88 expression was markedly reduced or missing in mild chronic HBV infection and inversely correlated with HBcAg expression. Irrespective of the HBV- or HCV-status, increasing Nup88 expression was observed in cirrhosis and dysplastic nodules, and Nup88 was highly expressed in hepatocellular carcinomas. The intensity of Nup88 expression significantly increased during carcinogenesis (P < 0.0001) and correlated with dedifferentiation (P < 0.0001). Interestingly, Nup88 protein expression was significantly downregulated in HBV-positive HepG2.2.15 (P < 0.002) and HB611 (P < 0.001) cell lines as compared to HBV-negative HepG2 cells. CONCLUSION: Based on our immunohistochemical data, HBV and HCV are unlikely to influence the expression of Nup88 in cirrhotic and neoplastic liver tissue, but point to an interaction of HBV with the nuclear pore in chronic hepatitis. The expression of Nup88 in nonneoplastic liver tissue might reflect enhanced metabolic activity of the liver tissue. Our data strongly indicate a dichotomous role for Nup88 in non-neoplastic and neoplastic conditions of the liver.展开更多
Nucleoporins(Nups)are important components of nuclear pore complexes(NPCs).NPCs control gene expression,cells proliferation and differentiation by mediating exchange of cellular signal molecules on both nuclear and cy...Nucleoporins(Nups)are important components of nuclear pore complexes(NPCs).NPCs control gene expression,cells proliferation and differentiation by mediating exchange of cellular signal molecules on both nuclear and cytoplasmic sides.Using subtractive screening,3'end fragment of Nup93 from the testis cDNA library of the rice field eel was obtained.Full-length cDNA of the gene was further cloned by degenerate PCR and 5'RACE methods.Sequence analysis indicated that the homology of the rice field eel Nup93 were 36.5%with yeast Nic96,94.6%and 90.5%with Nup93 of zebrafish and human,respectively.Phylogenetic analysis showed that the rice field eel Nup93 fits with Nup93 of the other fishes.Real-time PCR result showed that expression of Nup93 in gonads and kidney were much higher than in other tissues,and different expression quantities among gonads of three sexes were also observed,suggesting that Nup93 may involve in gonad development.展开更多
Super-resolution imaging techniques have broken the diffraction-limited resolution of light microscopy.However,acquiring three-dimensional(3D)super-resolution information about structures and dynamic processes in live...Super-resolution imaging techniques have broken the diffraction-limited resolution of light microscopy.However,acquiring three-dimensional(3D)super-resolution information about structures and dynamic processes in live cells at high speed remains challenging.Recently,the development of high-speed single-point edge-excitation subdiffraction(SPEED)microscopy,along with its 2D-to-3D transformation algorithm,provides a practical and effective approach to achieving 3D subdiffraction-limit information in subcellular structures and organelles with rotational symmetry.One of the major benefits of SPEED microscopy is that it does not rely on complex optical components and can be implemented on a standard,inverted epifluorescence microscope,simplifying the process of sample preparation and the expertise requirement.SPEED microscopy is specifically designed to obtain 2D spatial locations of individual immobile or moving fluorescent molecules inside submicrometer biological channels or cavities at high spatiotemporal resolution.The collected data are then subjected to postlocalization 2D-to-3D transformation to obtain 3D super-resolution structural and dynamic information.In recent years,SPEED microscopy has provided significant insights into nucleocytoplasmic transport across the nuclear pore complex(NPC)and cytoplasm-cilium trafficking through the ciliary transition zone.This Review focuses on the applications of SPEED microscopy in studying the structure and function of nuclear pores.展开更多
O-glycosylation of the nuclear pore complex(NPC)by O-linked N-acetylglucosamine(O-GlcNAc)is conserved within metazoans.Many nucleoporins(Nups)comprising the NPC are constitutively O-GlcNAcylated,but the functional rol...O-glycosylation of the nuclear pore complex(NPC)by O-linked N-acetylglucosamine(O-GlcNAc)is conserved within metazoans.Many nucleoporins(Nups)comprising the NPC are constitutively O-GlcNAcylated,but the functional role of this modification remains enigmatic.Weshowthat loss ofO-GlcNAc,induced by either inhibition ofO-GlcNAc transferase(OGT)or deletion of the gene encoding OGT,leads to decreased cellular levels of a number of natively O-GlcNAcylated Nups.Loss of O-GlcNAc enables increased ubiquitination of these Nups and their increased proteasomal degradation.The decreased half-life of these deglycosylated Nups manifests in their gradual loss from the NPC and a downstream malfunction of the nuclear pore selective permeability barrier in both dividing and post-mitotic cells.These findings define a critical role of O-GlcNAc modification of the NPC in maintaining its composition and the function of the selectivity filter.The results implicate NPC glycosylation as a regulator of NPC function and reveal the role of conserved glycosylation of the NPC among metazoans.展开更多
Flowering locus C(FLC)is a central transcriptional repressor that controls flowering time.However,how FLC is imported into the nucleus is unknown.Here,we report that Arabidopsis nucleoporins 62(NUP62),NUP58,and NUP54 ...Flowering locus C(FLC)is a central transcriptional repressor that controls flowering time.However,how FLC is imported into the nucleus is unknown.Here,we report that Arabidopsis nucleoporins 62(NUP62),NUP58,and NUP54 composed NUP62-subcomplex modulates FLC nuclear import during floral transition in an importinα-independent manner,via direct interaction.NUP62 recruits FLC to the cytoplasmic filaments and imports it into the nucleus through the NUP62-subcomplex composed central channel.Importinβsupersensitive to ABA and drought 2(SAD2),a carrier protein,is critical for FLC nuclear import and flower transition,which facilitates FLC import into the nucleus mainly through the NUP62-subcomplex.Proteomics,RNAseq,and cell biological analyses indicate that the NUP62-subcomplex mainly mediates the nuclear import of cargos with unconventional nuclear localization sequences(NLSs),such as FLC.Our findings illustrate the mechanisms of the NUP62-subcomplex and SAD2 on FLC nuclear import process and floral transition,and provide insights into the role of NUP62-subcomplex and SAD2 in protein nucleocytoplasmic transport in plants.展开更多
基金supported by the National Science Foundation of China(81271818 and 81471940 to YF,and 81471941,81871659 and 81828005 to WH)
文摘The human myxovirus resistance 2(Mx2/Mx B)protein,a member of interferon(IFN)-inducible dynamin-like large GTPases,restricts a number of virus infections.Inhibition of these viruses occurs at poorly-defined steps after viral entry and has a common requirement for Mx B oligomerization.However,the GTPase activity is essential for the anti-viral effects of Mx B against herpesviruses and HBV but not HIV-1.To understand the role of Mx B GTPase activity,including GTP binding and GTP hydrolysis,in restriction of HIV-1 infection,we genetically separated these two functions and evaluated their contributions to restriction.We found that both the GTP binding and hydrolysis function of Mx B involved in the restriction of HIV-1 replication.The GTPase activity of Mx B contributed to its nuclear location,interaction with nucleoporins(NUPs)and HIV-1 capsids.Furthermore,Mx B disrupted the association between NUPs and HIV-1 cores dependently upon its GTPase activity.The function of GTPase activity was therefore multi-faceted,led to fundamentally distinct mechanisms employed by wild-type Mx B and GTPase activity defective Mx B mutations to restrict HIV-1 replication.
文摘AIM: To investigate the expression of nucleoporin 88 (Nup88) in hepatitis B virus (HBV) and C virus (HCV)-related liver diseases. METHODS: We generated a new monoclonal Nup88 antibody to investigate the Nup88 protein expression by immunohistochemistry (IHC) in 294 paraffin-embedded liver specimens comprising all stages of hepatocellular carcinogenesis. In addition, in cell culture experiments HBV-positive (HepG2.2.15 and HB611) and HBV-negative (HepG2) hepatoma cell lines were tested for the Nup88 expression by Western-immunoblotting to test data obtained by IHC.RESULTS: Specific Nup88 expression was found in chronic HCV hepatitis and unspecific chronic hepatitis, whereas no or very weak Nup88 expression was detected in normal liver. The Nup88 expression was markedly reduced or missing in mild chronic HBV infection and inversely correlated with HBcAg expression. Irrespective of the HBV- or HCV-status, increasing Nup88 expression was observed in cirrhosis and dysplastic nodules, and Nup88 was highly expressed in hepatocellular carcinomas. The intensity of Nup88 expression significantly increased during carcinogenesis (P < 0.0001) and correlated with dedifferentiation (P < 0.0001). Interestingly, Nup88 protein expression was significantly downregulated in HBV-positive HepG2.2.15 (P < 0.002) and HB611 (P < 0.001) cell lines as compared to HBV-negative HepG2 cells. CONCLUSION: Based on our immunohistochemical data, HBV and HCV are unlikely to influence the expression of Nup88 in cirrhotic and neoplastic liver tissue, but point to an interaction of HBV with the nuclear pore in chronic hepatitis. The expression of Nup88 in nonneoplastic liver tissue might reflect enhanced metabolic activity of the liver tissue. Our data strongly indicate a dichotomous role for Nup88 in non-neoplastic and neoplastic conditions of the liver.
基金Supported by the National Natural Science Foundation of China,the National Key Basic Research Project(No.2004CB117400)the Program for New Century Excellent Talents in Universitythe Key Project of Chinese Ministry of Education.
文摘Nucleoporins(Nups)are important components of nuclear pore complexes(NPCs).NPCs control gene expression,cells proliferation and differentiation by mediating exchange of cellular signal molecules on both nuclear and cytoplasmic sides.Using subtractive screening,3'end fragment of Nup93 from the testis cDNA library of the rice field eel was obtained.Full-length cDNA of the gene was further cloned by degenerate PCR and 5'RACE methods.Sequence analysis indicated that the homology of the rice field eel Nup93 were 36.5%with yeast Nic96,94.6%and 90.5%with Nup93 of zebrafish and human,respectively.Phylogenetic analysis showed that the rice field eel Nup93 fits with Nup93 of the other fishes.Real-time PCR result showed that expression of Nup93 in gonads and kidney were much higher than in other tissues,and different expression quantities among gonads of three sexes were also observed,suggesting that Nup93 may involve in gonad development.
基金supported by grants from the US NIH(NIH GM097037,GM116204 and GM122552 to W.Y.).
文摘Super-resolution imaging techniques have broken the diffraction-limited resolution of light microscopy.However,acquiring three-dimensional(3D)super-resolution information about structures and dynamic processes in live cells at high speed remains challenging.Recently,the development of high-speed single-point edge-excitation subdiffraction(SPEED)microscopy,along with its 2D-to-3D transformation algorithm,provides a practical and effective approach to achieving 3D subdiffraction-limit information in subcellular structures and organelles with rotational symmetry.One of the major benefits of SPEED microscopy is that it does not rely on complex optical components and can be implemented on a standard,inverted epifluorescence microscope,simplifying the process of sample preparation and the expertise requirement.SPEED microscopy is specifically designed to obtain 2D spatial locations of individual immobile or moving fluorescent molecules inside submicrometer biological channels or cavities at high spatiotemporal resolution.The collected data are then subjected to postlocalization 2D-to-3D transformation to obtain 3D super-resolution structural and dynamic information.In recent years,SPEED microscopy has provided significant insights into nucleocytoplasmic transport across the nuclear pore complex(NPC)and cytoplasm-cilium trafficking through the ciliary transition zone.This Review focuses on the applications of SPEED microscopy in studying the structure and function of nuclear pores.
基金supported by a Discovery Grant(grant no.RGPIN/298406-2010)fromthe Natural Sciences and Engineering Research(NSERC),and the Canadian Institutes of Health Research(CIHR)(grant no.MOP-123341).Y.Z.thanks the CIHR for support through a postdoctoral fellowship.D.J.V.acknowledges the kind support of the Canada Research Chairs Program for a Tier I Canada Research Chair in Chemical Glycobiology and NSERC for support as an E.W.R.Steacie Memorial Fellow.N.Z.acknowledges the support from the National Heart Lung and Blood Institute(P01HL107153).
文摘O-glycosylation of the nuclear pore complex(NPC)by O-linked N-acetylglucosamine(O-GlcNAc)is conserved within metazoans.Many nucleoporins(Nups)comprising the NPC are constitutively O-GlcNAcylated,but the functional role of this modification remains enigmatic.Weshowthat loss ofO-GlcNAc,induced by either inhibition ofO-GlcNAc transferase(OGT)or deletion of the gene encoding OGT,leads to decreased cellular levels of a number of natively O-GlcNAcylated Nups.Loss of O-GlcNAc enables increased ubiquitination of these Nups and their increased proteasomal degradation.The decreased half-life of these deglycosylated Nups manifests in their gradual loss from the NPC and a downstream malfunction of the nuclear pore selective permeability barrier in both dividing and post-mitotic cells.These findings define a critical role of O-GlcNAc modification of the NPC in maintaining its composition and the function of the selectivity filter.The results implicate NPC glycosylation as a regulator of NPC function and reveal the role of conserved glycosylation of the NPC among metazoans.
基金supported by grants from the National Natural Science Foundation(31970730 and 32170721)to H.Hthe Baichuan felowship from College of Life Science and Technology,Huazhong Agricultural University to D.X。
文摘Flowering locus C(FLC)is a central transcriptional repressor that controls flowering time.However,how FLC is imported into the nucleus is unknown.Here,we report that Arabidopsis nucleoporins 62(NUP62),NUP58,and NUP54 composed NUP62-subcomplex modulates FLC nuclear import during floral transition in an importinα-independent manner,via direct interaction.NUP62 recruits FLC to the cytoplasmic filaments and imports it into the nucleus through the NUP62-subcomplex composed central channel.Importinβsupersensitive to ABA and drought 2(SAD2),a carrier protein,is critical for FLC nuclear import and flower transition,which facilitates FLC import into the nucleus mainly through the NUP62-subcomplex.Proteomics,RNAseq,and cell biological analyses indicate that the NUP62-subcomplex mainly mediates the nuclear import of cargos with unconventional nuclear localization sequences(NLSs),such as FLC.Our findings illustrate the mechanisms of the NUP62-subcomplex and SAD2 on FLC nuclear import process and floral transition,and provide insights into the role of NUP62-subcomplex and SAD2 in protein nucleocytoplasmic transport in plants.
