N-Methyl-D-aspartate receptors（NMDARs） play a central role in various physiological and pathological processes in the central nervous system.And they are commonly composed of four subunits,two GluN1 subunits and two...N-Methyl-D-aspartate receptors（NMDARs） play a central role in various physiological and pathological processes in the central nervous system.And they are commonly composed of four subunits,two GluN1 subunits and two GluN2 or GluN3 subunits.The different subunit compositions make NMDARs a heterogeneous population with distinct electrophysiological and pharmacological properties and thus with different abilities to conduct neuronal activities.The subunit composition,assembly process,and final structure of assembled NMDARs have been studied for years but no consensus has been achieved.In this study,we investigated the role of the amino terminal domain（ATD） of GluN2A in regulating NMDAR assembly.The ATD of GluN2A was first expressed in heterogeneous cells and the homodimer formation was investigated by fluorescent resonance energy transfer and non-reducing SDS- PAGE electrophoresis.Each of the three cysteine residues located in the ATD was mutated into alanine,and the homodimerization of the ATD or GluN2A,as well as the heteromeric assembly of NMDARs was assessed by non-reducing SDS- PAGE electrophoresis,co-immunoprecipitation and immunocytochemistry.We found that two cysteine residues,C87 and C320,in the ATD of the GluN2A subunit were required for the formation of disulfide bonds and GluN2A ATD homodimers.Furthermore,the disruption of GluN2A ATD domain dimerization had no influence on the assembly and surface expression of NMDARs.These results suggest that the two ATD domains of GluN2A are structurally adjacent in fully-assembled NMDARs.However,unlike GluN1,the homomerization of the ATD domain of GluN2A is not required for the assembly of NMDARs,implying that GluN2A and GluN1 play unequal roles in NMDAR assembly.展开更多
G protein-coupled receptors(GPCRs),crucial for diverse physiological responses,have traditionally been investigated in their monomeric form.However,some GPCRs can form heteromers,revealing complexity in their function...G protein-coupled receptors(GPCRs),crucial for diverse physiological responses,have traditionally been investigated in their monomeric form.However,some GPCRs can form heteromers,revealing complexity in their functional characteristics such as ligand binding properties,downstream signaling pathways,and trafficking.Understanding GPCR heteromers is crucial in both physiological contexts and drug development.Here,we review the methodologies for investigating physical interactions in GPCR heteromers,including co-immunoprecipitation,proximity ligation assays,interfering peptide approaches,and live cell imaging techniques based on resonance energy transfer and bimolecular fluorescence complementation.In addition,we discuss recent advances in live cell imaging techniques for exploring functional features of GPCR heteromers,for example,circularly permuted fluorescent protein-based GPCR biosensors,TRUPATH,and nanobody-based GPCR biosensors.These advanced biosensors and live cell imaging technologies promise a deeper understanding of GPCR heteromers,urging a reassessment of their physiological importance and pharmacological relevance.展开更多
Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid （MIA） anti- cancer agents： vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid an...Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid （MIA） anti- cancer agents： vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid and shikimate pathways, respectively. Geranyl diphosphate （GPP）, the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate （IPP） and dimethylallyl diphosphate （DMAPP） by GPP synthase （GPPS）. Here, we report three genes encoding proteins with sequence similarity to large subunit （CrGPPS.LSU） and small subunit （CrGPPS.SSU） of heteromeric GPPSs, and a homomeric GPPSs. CrGPPS.LSU is a bifunctional enzyme producing both GPP and geranyl geranyl diphosphate （GGPP）, CrGPPS.SSU is inactive, whereas CrGPPS is a homomeric enzyme forming GPP. Co-expression of both subunits in Escherichia coil resulted in heteromeric enzyme with enhanced activity producing only GPR While CrGPPS.LSU and CrGPPS showed higher expression in older and younger leaves, respectively, CrGPPS.SSU showed an increasing trend and decreased gradually. Methyl jasmonate （MelA） treatment of leaves sig- nificantly induced the expression of only CrGPPS.SSU. GFP localization indicated that CrGPPS.SSU is plastidial whereas CrGPPS is mitochondrial. Transient overexpression of AmGPPS.SSU in C. roseus leaves resulted in increased vindoline, immediate monomeric precursor of vinblastine and vincristine. Although C. roseus has both heteromeric and homomeric GPPS enzymes, our results implicate the involvement of only heteromeric GPPS with CrGPPS.SSU regulating the GPP flux for MIA biosynthesis.展开更多
Interleukin-17 （IL-17 or IL-17A） production is a hallmark of TH17 cells, a new unique lineage of CD4^＋ T lymphocytes contributing to the pathogenesis of multiple autoimmune and inflammatory diseases. IL-17 receptor...Interleukin-17 （IL-17 or IL-17A） production is a hallmark of TH17 cells, a new unique lineage of CD4^＋ T lymphocytes contributing to the pathogenesis of multiple autoimmune and inflammatory diseases. IL-17 receptor （IL-17R or IL-17RA） is essential for IL-17 biological activity. Emerging data suggest that the formation of a heteromeric and/or homomeric receptor complex is required for IL-17 signaling. Here we show that the orphan receptor IL-17RD （Sef, similar expression to FGF genes or IL-17RLM） is associated and colocalized with IL-17R. Importantly, IL-17RD mediates IL-17 signaling, as evaluated using a luciferase reporter driven by the native promoter of 24p3, an IL-17 target gene. In addition, an IL-17RD mutant lacking the intraeellnlar domain dominant-negatively suppresses IL-17R- mediated IL-17 signaling. Moreover, IL-17RD as well as IL-17R is associated with TRAF6, an IL-17R downstream molecule. These results indicate that IL-17RD is a part of the IL-17 receptor signaling complex, therefore providing novel evidence for IL-17 signaling through a heteromeric and/or homomeric receptor complex.展开更多
Dimerization is an effective strategy for designing antimicrobial peptides that combine the advantages of different native peptides. In this study, we explored the effects of different linker amino acids, including le...Dimerization is an effective strategy for designing antimicrobial peptides that combine the advantages of different native peptides. In this study, we explored the effects of different linker amino acids, including leucine, proline and aminocaproic acid, on the anticancer, antimicrobial and hemolytic activities of the heteromeric antimicrobial peptides AM-1, AM-2, and AM-3. Proline and aminocaproic acid are ideal linkers for increasing the potency and selectivity of heteromeric antimicrobial peptides. The results of MD simulations provided a rationalization for this observation. Both AM-2, which had a proline linker,and AM-3, which had an aminocaproic acid linker, adopted a compact conformation in water and a bent conformation in membranes. This change in the flexible structures of AM-2 and AM-3 could have resulted in decreased binding of these peptides to zwitterionic lipid bilayers and increased damage to mixed lipid bilayers containing acidic phospholipids. In short, these findings obtained via assessing the effects of linker amino acids will contribute to the design of ideal heteromeric antimicrobial peptides with high selectivity and potency.展开更多
基金supported by grants from the National Basic Research Development Program of China(2010CB912002)the National Natural Science Foundation of China(30730038 and 81171164)
文摘N-Methyl-D-aspartate receptors（NMDARs） play a central role in various physiological and pathological processes in the central nervous system.And they are commonly composed of four subunits,two GluN1 subunits and two GluN2 or GluN3 subunits.The different subunit compositions make NMDARs a heterogeneous population with distinct electrophysiological and pharmacological properties and thus with different abilities to conduct neuronal activities.The subunit composition,assembly process,and final structure of assembled NMDARs have been studied for years but no consensus has been achieved.In this study,we investigated the role of the amino terminal domain（ATD） of GluN2A in regulating NMDAR assembly.The ATD of GluN2A was first expressed in heterogeneous cells and the homodimer formation was investigated by fluorescent resonance energy transfer and non-reducing SDS- PAGE electrophoresis.Each of the three cysteine residues located in the ATD was mutated into alanine,and the homodimerization of the ATD or GluN2A,as well as the heteromeric assembly of NMDARs was assessed by non-reducing SDS- PAGE electrophoresis,co-immunoprecipitation and immunocytochemistry.We found that two cysteine residues,C87 and C320,in the ATD of the GluN2A subunit were required for the formation of disulfide bonds and GluN2A ATD homodimers.Furthermore,the disruption of GluN2A ATD domain dimerization had no influence on the assembly and surface expression of NMDARs.These results suggest that the two ATD domains of GluN2A are structurally adjacent in fully-assembled NMDARs.However,unlike GluN1,the homomerization of the ATD domain of GluN2A is not required for the assembly of NMDARs,implying that GluN2A and GluN1 play unequal roles in NMDAR assembly.
基金supported by the National Research Foundation of Korea(NRF)grant RS-2023-00227950,RS-2024-00338426,RS-2024-00403094,New Faculty Startup Fund from Seoul National University,and Seoul National University Bundang Hospital(J.S.).
文摘G protein-coupled receptors(GPCRs),crucial for diverse physiological responses,have traditionally been investigated in their monomeric form.However,some GPCRs can form heteromers,revealing complexity in their functional characteristics such as ligand binding properties,downstream signaling pathways,and trafficking.Understanding GPCR heteromers is crucial in both physiological contexts and drug development.Here,we review the methodologies for investigating physical interactions in GPCR heteromers,including co-immunoprecipitation,proximity ligation assays,interfering peptide approaches,and live cell imaging techniques based on resonance energy transfer and bimolecular fluorescence complementation.In addition,we discuss recent advances in live cell imaging techniques for exploring functional features of GPCR heteromers,for example,circularly permuted fluorescent protein-based GPCR biosensors,TRUPATH,and nanobody-based GPCR biosensors.These advanced biosensors and live cell imaging technologies promise a deeper understanding of GPCR heteromers,urging a reassessment of their physiological importance and pharmacological relevance.
文摘Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid （MIA） anti- cancer agents： vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid and shikimate pathways, respectively. Geranyl diphosphate （GPP）, the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate （IPP） and dimethylallyl diphosphate （DMAPP） by GPP synthase （GPPS）. Here, we report three genes encoding proteins with sequence similarity to large subunit （CrGPPS.LSU） and small subunit （CrGPPS.SSU） of heteromeric GPPSs, and a homomeric GPPSs. CrGPPS.LSU is a bifunctional enzyme producing both GPP and geranyl geranyl diphosphate （GGPP）, CrGPPS.SSU is inactive, whereas CrGPPS is a homomeric enzyme forming GPP. Co-expression of both subunits in Escherichia coil resulted in heteromeric enzyme with enhanced activity producing only GPR While CrGPPS.LSU and CrGPPS showed higher expression in older and younger leaves, respectively, CrGPPS.SSU showed an increasing trend and decreased gradually. Methyl jasmonate （MelA） treatment of leaves sig- nificantly induced the expression of only CrGPPS.SSU. GFP localization indicated that CrGPPS.SSU is plastidial whereas CrGPPS is mitochondrial. Transient overexpression of AmGPPS.SSU in C. roseus leaves resulted in increased vindoline, immediate monomeric precursor of vinblastine and vincristine. Although C. roseus has both heteromeric and homomeric GPPS enzymes, our results implicate the involvement of only heteromeric GPPS with CrGPPS.SSU regulating the GPP flux for MIA biosynthesis.
文摘Interleukin-17 （IL-17 or IL-17A） production is a hallmark of TH17 cells, a new unique lineage of CD4^＋ T lymphocytes contributing to the pathogenesis of multiple autoimmune and inflammatory diseases. IL-17 receptor （IL-17R or IL-17RA） is essential for IL-17 biological activity. Emerging data suggest that the formation of a heteromeric and/or homomeric receptor complex is required for IL-17 signaling. Here we show that the orphan receptor IL-17RD （Sef, similar expression to FGF genes or IL-17RLM） is associated and colocalized with IL-17R. Importantly, IL-17RD mediates IL-17 signaling, as evaluated using a luciferase reporter driven by the native promoter of 24p3, an IL-17 target gene. In addition, an IL-17RD mutant lacking the intraeellnlar domain dominant-negatively suppresses IL-17R- mediated IL-17 signaling. Moreover, IL-17RD as well as IL-17R is associated with TRAF6, an IL-17R downstream molecule. These results indicate that IL-17RD is a part of the IL-17 receptor signaling complex, therefore providing novel evidence for IL-17 signaling through a heteromeric and/or homomeric receptor complex.
基金the National Natural Science Foundation of China(Nos. 81773566, 21602092, 81473095)the Fundamental Research Funds for the Central Universities(Nos. lzujbky-2017-134, lzujbky-2017-120, lzujbky-2016-21)
文摘Dimerization is an effective strategy for designing antimicrobial peptides that combine the advantages of different native peptides. In this study, we explored the effects of different linker amino acids, including leucine, proline and aminocaproic acid, on the anticancer, antimicrobial and hemolytic activities of the heteromeric antimicrobial peptides AM-1, AM-2, and AM-3. Proline and aminocaproic acid are ideal linkers for increasing the potency and selectivity of heteromeric antimicrobial peptides. The results of MD simulations provided a rationalization for this observation. Both AM-2, which had a proline linker,and AM-3, which had an aminocaproic acid linker, adopted a compact conformation in water and a bent conformation in membranes. This change in the flexible structures of AM-2 and AM-3 could have resulted in decreased binding of these peptides to zwitterionic lipid bilayers and increased damage to mixed lipid bilayers containing acidic phospholipids. In short, these findings obtained via assessing the effects of linker amino acids will contribute to the design of ideal heteromeric antimicrobial peptides with high selectivity and potency.
