一种利用荧光蛋白mNeonGreen2鉴定EI24蛋白拓扑结构的方法（英文）
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摘要
方便且精准地检测跨膜蛋白拓扑结构,尤其是跨膜片段的氨基(N-)和羧基(C-)端的朝向,有利于发现新的蛋白质与蛋白质之间的相互作用,并进一步揭示蛋白质重要的生物学功能.自组装荧光蛋白已被广泛用于观察蛋白质与蛋白质之间的相互作用、标记细胞内源蛋白质并实现mRNA定位的可视化.本文扩展了自组装荧光蛋白的应用,将自组装荧光蛋白mNeonGreen2与定点标记技术相结合,以确定跨膜蛋白的拓扑结构.通过该方法,第一次清楚地证明了EI24的N端和C端均朝向细胞质方向.此外,该方法可用于确定定位于其他细胞器且结构尚未解析的跨膜蛋白的拓扑结构.
Convenient, reliable detection of transmembrane protein topology, especially the orientation of the amino(N-) and carboxyl(C-) termini of a membrane-spanning segment, may aid in identifying protein-protein interactions and clarifying the important biological functions of proteins. Self-complementing split fluorescent proteins have been widely used to image protein-protein interactions, label endogenous proteins and visualize mR NA localization. Here, we expand this toolset and develop an efficient method combining a self-complementing split mN eonGreen2 with site-directed labeling(SSDL) to identify the topology of transmembrane proteins. With SSDL, for the first time, we clearly demonstrate that both the N-and C-termini of etoposide-induced protein 2.4, which localizes in the endoplasmic reticulum, have a cytosolic orientation. This method can be useful for determining the topology of other organelle-based transmembrane proteins that have insufficient structural information.
Convenient, reliable detection of transmembrane protein topology, especially the orientation of the amino(N-) and carboxyl(C-) termini of a membrane-spanning segment, may aid in identifying protein-protein interactions and clarifying the important biological functions of proteins. Self-complementing split fluorescent proteins have been widely used to image protein-protein interactions, label endogenous proteins and visualize mR NA localization. Here, we expand this toolset and develop an efficient method combining a self-complementing split mN eonGreen2 with site-directed labeling(SSDL) to identify the topology of transmembrane proteins. With SSDL, for the first time, we clearly demonstrate that both the N-and C-termini of etoposide-induced protein 2.4, which localizes in the endoplasmic reticulum, have a cytosolic orientation. This method can be useful for determining the topology of other organelle-based transmembrane proteins that have insufficient structural information.
引文
[1]Tsirigos K D,Govindarajan S,Bassot C,et al.Topology of membrane proteins-predictions,limitations and variations.Curr Opin Struct Biol,2017,50:9-17(DOI:10.1016/j.sbi.2017.10.003)
[2]Earl L A,Falconieri V,Milne J L,et al.Cryo-EM:beyond the microscope.Curr Opin Struct Biol,2017,46:71-78(DOI:10.1016/j.sbi.2017.06.002)
[3]Goder V,Spiess M.Topogenesis of membrane proteins:determinants and dynamics.FEBS Lett,2001,504(3):87-93
[4]Preston G M,Jung J S,Guggino W B,et al.Membrane topology of aquaporin CHIP.Analysis of functional epitope-scanning mutants by vectorial proteolysis.J Biol Chem,1994,269(3):1668-1673
[5]Van Geest M,Lolkema J S.Membrane topology and insertion of membrane proteins:search for topogenic signals.Microbiol Mol Biol Rev,2000,64(1):13-33
[6]Voeltz G K,Prinz W A,Shibata Y,et al.A class of membrane proteins shaping the tubular endoplasmic reticulum.Cell,2006,124(3):573-586
[7]Lieu K G,Shim E H,Wang J,et al.The p53-induced factor EI24inhibits nuclear import through an importin beta-binding-like domain.J Cell Biol,2014,205(3):301-312
[8]Lorenz H,Hailey D W,Lippincott-Schwartz J.Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization.Nat Methods,2006,3(3):205-210
[9]Waldo G S,Standish B M,Berendzen J,et al.Rapid protein-folding assay using green fluorescent protein.Nature Biotechnology,1999,17(7):691-695
[10]Chen S,Song Y,Cao J,et al.Localization and function of calmodulin in live-cells of Aspergillus nidulans.Fungal Genetics and Biology,2010,47(3):268-278
[11]Kondoh G,Gao X H,Nakano Y,et al.Tissue‐inherent fate of GPIrevealed by GPI-anchored GFP transgenesis.FEBS Letters,1999,458(3):299-303
[12]Feng S,Sekine S,Pessino V,et al.Improved split fluorescent proteins for endogenous protein labeling.Nat Commun,2017,8(1):370
[13]Voeltz G K,Rolls M M,Rapoport T A.Structural organization of the endoplasmic reticulum.EMBO Rep,2002,3(10):944-950
[14]Walter P,Ron D.The unfolded protein response:from stress pathway to homeostatic regulation.Science,2011,334(6059):1081-1086
[15]Mekahli D,Bultynck G,Parys J B,et al.Endoplasmic-reticulum calcium depletion and disease.Cold Spring Harb Perspect Biol,2011,3(6):pii:a004317.doi:10.1101/cshperspect.a004317
[16]Shaner N C,Lambert G G,Chammas A,et al.A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum.Nat Methods,2013,10(5):407-409
[17]Manikandan P,Nagini S.Cytochrome P450 structure,function and clinical significance:a review.Curr Drug Targets,2018,19(1):38-54
[18]Renault H,De Marothy M,Jonasson G,et al.Gene duplication leads to altered membrane topology of a cytochrome P450 enzyme in seed plants.Mol Biol Evol,2017,34(8):2041-2056
[19]Williams P A,Cosme J,Ward A,et al.Crystal structure of human cytochrome P450 2C9 with bound warfarin.Nature,2003,424(6947):464-468
[20]Schrag J D,Bergeron J J,Li Y,et al.The Structure of calnexin,an ER chaperone involved in quality control of protein folding.Mol Cell,2001,8(3):633-644
[21]Lakkaraju A K,Van Der Goot F G.Calnexin controls the STAT3-mediated transcriptional response to EGF.Mol Cell,2013,51(3):386-396
[22]Lehar S M,Nacht M,Jacks T,et al.Identification and cloning of EI24,a gene induced by p53 in etoposide-treated cells.Oncogene,1996,12(6):1181-1187
[23]Zhao X,Ayer R E,Davis S L,et al.Apoptosis factor EI24/PIG8 is a novel endoplasmic reticulum-localized Bcl-2-binding protein which is associated with suppression of breast cancer invasiveness.Cancer Res,2005,65(6):2125-2129
[24]Zhao Y G,Zhao H,Miao L,et al.The p53-induced gene Ei24 is an essential component of the basal autophagy pathway.J Biol Chem,2012,287(50):42053-42063
[25]Gu Z,Flemington C,Chittenden T,et al.ei24,a p53 response gene involved in growth suppression and apoptosis.Mol Cell Biol,2000,20(1):233-241
[26]Yuan L,Wang H,Liu Q,et al.Etoposide-induced protein 2.4functions as a regulator of the calcium ATPase and protects pancreatic beta cell survival.J Biol Chem,2018,293(26):10128-10140
[27]Zhang W,Bogdanov M,Pi J,et al.Reversible topological organization within a polytopic membrane protein is governed by a change in membrane phospholipid composition.J Biol Chem,2003,278(50):50128-50135
[2]Earl L A,Falconieri V,Milne J L,et al.Cryo-EM:beyond the microscope.Curr Opin Struct Biol,2017,46:71-78(DOI:10.1016/j.sbi.2017.06.002)
[3]Goder V,Spiess M.Topogenesis of membrane proteins:determinants and dynamics.FEBS Lett,2001,504(3):87-93
[4]Preston G M,Jung J S,Guggino W B,et al.Membrane topology of aquaporin CHIP.Analysis of functional epitope-scanning mutants by vectorial proteolysis.J Biol Chem,1994,269(3):1668-1673
[5]Van Geest M,Lolkema J S.Membrane topology and insertion of membrane proteins:search for topogenic signals.Microbiol Mol Biol Rev,2000,64(1):13-33
[6]Voeltz G K,Prinz W A,Shibata Y,et al.A class of membrane proteins shaping the tubular endoplasmic reticulum.Cell,2006,124(3):573-586
[7]Lieu K G,Shim E H,Wang J,et al.The p53-induced factor EI24inhibits nuclear import through an importin beta-binding-like domain.J Cell Biol,2014,205(3):301-312
[8]Lorenz H,Hailey D W,Lippincott-Schwartz J.Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization.Nat Methods,2006,3(3):205-210
[9]Waldo G S,Standish B M,Berendzen J,et al.Rapid protein-folding assay using green fluorescent protein.Nature Biotechnology,1999,17(7):691-695
[10]Chen S,Song Y,Cao J,et al.Localization and function of calmodulin in live-cells of Aspergillus nidulans.Fungal Genetics and Biology,2010,47(3):268-278
[11]Kondoh G,Gao X H,Nakano Y,et al.Tissue‐inherent fate of GPIrevealed by GPI-anchored GFP transgenesis.FEBS Letters,1999,458(3):299-303
[12]Feng S,Sekine S,Pessino V,et al.Improved split fluorescent proteins for endogenous protein labeling.Nat Commun,2017,8(1):370
[13]Voeltz G K,Rolls M M,Rapoport T A.Structural organization of the endoplasmic reticulum.EMBO Rep,2002,3(10):944-950
[14]Walter P,Ron D.The unfolded protein response:from stress pathway to homeostatic regulation.Science,2011,334(6059):1081-1086
[15]Mekahli D,Bultynck G,Parys J B,et al.Endoplasmic-reticulum calcium depletion and disease.Cold Spring Harb Perspect Biol,2011,3(6):pii:a004317.doi:10.1101/cshperspect.a004317
[16]Shaner N C,Lambert G G,Chammas A,et al.A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum.Nat Methods,2013,10(5):407-409
[17]Manikandan P,Nagini S.Cytochrome P450 structure,function and clinical significance:a review.Curr Drug Targets,2018,19(1):38-54
[18]Renault H,De Marothy M,Jonasson G,et al.Gene duplication leads to altered membrane topology of a cytochrome P450 enzyme in seed plants.Mol Biol Evol,2017,34(8):2041-2056
[19]Williams P A,Cosme J,Ward A,et al.Crystal structure of human cytochrome P450 2C9 with bound warfarin.Nature,2003,424(6947):464-468
[20]Schrag J D,Bergeron J J,Li Y,et al.The Structure of calnexin,an ER chaperone involved in quality control of protein folding.Mol Cell,2001,8(3):633-644
[21]Lakkaraju A K,Van Der Goot F G.Calnexin controls the STAT3-mediated transcriptional response to EGF.Mol Cell,2013,51(3):386-396
[22]Lehar S M,Nacht M,Jacks T,et al.Identification and cloning of EI24,a gene induced by p53 in etoposide-treated cells.Oncogene,1996,12(6):1181-1187
[23]Zhao X,Ayer R E,Davis S L,et al.Apoptosis factor EI24/PIG8 is a novel endoplasmic reticulum-localized Bcl-2-binding protein which is associated with suppression of breast cancer invasiveness.Cancer Res,2005,65(6):2125-2129
[24]Zhao Y G,Zhao H,Miao L,et al.The p53-induced gene Ei24 is an essential component of the basal autophagy pathway.J Biol Chem,2012,287(50):42053-42063
[25]Gu Z,Flemington C,Chittenden T,et al.ei24,a p53 response gene involved in growth suppression and apoptosis.Mol Cell Biol,2000,20(1):233-241
[26]Yuan L,Wang H,Liu Q,et al.Etoposide-induced protein 2.4functions as a regulator of the calcium ATPase and protects pancreatic beta cell survival.J Biol Chem,2018,293(26):10128-10140
[27]Zhang W,Bogdanov M,Pi J,et al.Reversible topological organization within a polytopic membrane protein is governed by a change in membrane phospholipid composition.J Biol Chem,2003,278(50):50128-50135