Participation of the extracellular domain in (pro)renin receptor dimerization

The (pro)renin receptor [(P)RR] induces the catalytic activation of prorenin, as well as the activation of the mitogen-activated protein kinase (MAPK) signaling pathway; as such, it plays an important regulatory role in the renin–angiotensin system. (P)RR is known to form a homodimer, but the region participating in its dimerization is unknown. Using glutathione S-transferase (GST) as a carrier protein and a GST pull-down assay, we investigated the interaction of several (P)RR constructs with full-length (FL) (P)RR in mammalian cells. GST fusion proteins with FL (P)RR (GST-FL), the C-terminal M8-9 fragment (GST-M8-9), the extracellular domain (ECD) of (P)RR (GST-ECD), and the (P)RR ECD with a deletion of 32 amino acids encoded by exon 4 (GST-ECDd4) were retained intracellularly, whereas GST alone was efficiently secreted into the culture medium when transiently expressed in COS-7 cells. Immunofluorescence microscopy showed prominent localization of GST-ECD to the endoplasmic reticulum. The GST pull-down analysis revealed that GST-FL, GST-ECD, and GST-ECDd4 bound FLAG-tagged FL (P)RR, whereas GST-M8-9 showed little or no binding when transiently co-expressed in HEK293T cells. Furthermore, pull-down analysis using His-tag affinity resin showed co-precipitation of soluble (P)RR with FL (P)RR from a stable CHO cell line expressing FL h(P)RR with a C-terminal decahistidine tag. These results indicate that the (P)RR ECD participates in dimerization.     

Liquid chemiluminescent DNA pull-down assay to measure nuclear receptor-DNA binding in solution

Electrophoretic mobility shift assays (EMSAs) are commonly used to investigate protein-DNA binding in vitro. However, EMSA can generate considerable amounts of undesirable waste, particularly when toxic compounds are examined. We therefore developed a novel in vitro protein-DNA binding assay called liquid chemiluminescent DNA pull-down assay, which is based on solution hybridization between digoxigenin-labeled DNA and glutathione S-transferase (GST)-fused DNA binding protein bound to glutathione-Sepharose beads.     

Expression in Escherichia coli and purification of human recombinant connexin-43, a four-pass transmembrane protein

We have recently shown, using a well-defined in vitro model, that connexin 43 (Cx43) is directly involved in human cytotrophoblastic cell fusion into a multinucleated syncytiotrophoblast. Cx43 appears to interact with partner proteins within a fusogenic complex, in a multi factorial and dynamic process. This fusogenic complex remains to be characterized and constituent proteins need to be identified. In order to identify proteins interacting with the entire Cx43 molecule (extracellular, transmembrane and intracellular domains), we produced and purified full-length recombinant Cx43 fused to glutathione S-transferase (GST-Cx43) and used it as "bait" in GST pull-down experiments. Cx43 cDNA was first cloned into the pDEST15 vector in order to construct a GST-fusion protein, using the Gateway system. The fusion protein GST-Cx43 was then expressed in Escherichia coli strain BL21-AI? and purified by glutathione-affinity chromatography. The purified fusion protein exhibited the expected size of 70 kDa on SDS-PAGE, western blot and GST activity. A GST pull-down assay was used to show the capacity of the full-length recombinant protein to interact with known partners. Our results suggest that this method has the capacity to produce sufficient full-length recombinant protein for investigations aimed at identifying Cx43 partner proteins.     

Interaction of receptor-activity-modifying protein1 with tubulin

Receptor-activity-modifying protein (RAMP) 1 is an accessory protein of the G protein-coupled calcitonin receptor-like receptor (CLR). The CLR/RAMP1 heterodimer defines a receptor for the potent vasodilatory calcitonin gene-related peptide. A wider tissue distribution of RAMP1, as compared to that of the CLR, is consistent with additional biological functions. Here, glutathione S-transferase (GST) pull-down, coimmunoprecipitation and yeast two-hybrid experiments identified beta-tubulin as a novel RAMP1-interacting protein. GST pull-down experiments indicated interactions between the N- and C-terminal domains of RAMP1 and beta-tubulin. Yeast two-hybrid experiments confirmed the interaction between the N-terminal region of RAMP1 and beta-tubulin. Interestingly, alpha-tubulin was co-extracted with beta-tubulin in pull-down experiments and immunoprecipitation of RAMP1 coprecipitated alpha- and beta-tubulin. Confocal microscopy indicated colocalization of RAMP1 and tubulin predominantly in axon-like processes of neuronal differentiated human SH-SY5Y neuroblastoma cells. In conclusion, the findings point to biological roles of RAMP1 beyond its established interaction with G protein-coupled receptors.     

A型流感病毒PB1-F2蛋白与MOAP-1的相互作用

【目的】探讨A型流感病毒PB1-F2蛋白和人类凋亡调节因子1(MOAP-1)之间的相互作用。【方法】构建pACT2-MOAP-1重组质粒,与pGBKT7-PB1-F2质粒共转化酵母AH109,检测转化菌在四缺培养基的生长情况及β半乳糖苷酶报告基因的活性;利用GST pull-down和免疫共沉淀(Co-IP)技术进一步验证PB1-F2与宿主细胞蛋白MOAP-1的相互作用;通过过表达PB1-F2和MOAP-1,检测PB1-F2对MOAP-1蛋白表达水平的影响。【结果】酵母双杂交结果表明,PB1-F2和MOAP-1可以在酵母细胞内特异性结合。GST pull-down和Co-IP实验也进一步证实了这两种蛋白的相互作用,而且PB1-F2可上调外源MOAP-1的蛋白水平。【结论】流感病毒PB1-F2与MOAP-1存在相互作用,PB1-F2可能通过与MOAP-1的相互作用参与调控细胞生长及凋亡过程。     

应用蛋白组方法筛选血浆中乙肝病毒PreS1结合蛋白

目的筛选血浆中乙型肝炎病毒PreS1结合蛋白。方法表达纯化了PreS1-谷胱甘肽-S-转移酶（glutathione—S-transferase,GST）融合蛋白,利用该蛋白与血浆进行Pull—down实验,并设立GST与血浆Pull—down,GST、PreS1-GST与PBS Pull—down对照,Pull-down产物进行双向电泳分离（2-DE）,差异蛋白点通过质谱鉴定。结果成功表达纯化出PreS1-GST融合蛋白,通过双向电泳分析发现一个PreS1特异结合蛋白,经质谱鉴定为含锚蛋白重复序列的蛋白57（ANKRD57）。结论锚蛋白重复序列的主要功能是介导蛋白质与蛋白质之间的相互作用,ANKRD57与PreS1特异结合后的生理功能值得深入研究。     

Characterizing the interaction between the Rab6 GTPase and Mint3 via flow cytometry based FRET analysis

In extension to previously applied techniques like yeast two-hybrid and GST pull-down assays, we successfully established a FACS-based FRET analysis to investigate the interaction of the Mint3 adaptor protein and the small Rab GTPase Rab6A in living mammalian cells. A Mint3 mutant containing only the PTB domain (Mint3Δ6) is able to interact with the constitutively active form of Rab6A. Mint3Δ4, a mutant lacking part of the PTB domain was unable to interact with Rab6A in GST pull-down analysis and did not produce FRET signals, when co-expressed with active Rab6A.We demonstrate that this FACS-based FRET analysis is a suitable method for interaction studies between two proteins in living cells.     

Novel cytosolic binding partners of the neural cell adhesion molecule: mapping the binding domains of PLC gamma, LANP, TOAD-64, syndapin, PP1, and PP2A

The neural cell adhesion molecule (NCAM) is implicated in important functions during development and maintenance of the nervous system. Two of the three major isoforms, NCAM 140 and NCAM 180, are transmembrane glycoproteins with large cytoplasmic domains of different length. The purpose of this study was to identify novel intracellular binding partners of NCAM 140 and NCAM 180. We expressed both cytoplasmic domains, as well as cytoplasmic fragments of NCAM, as fusion proteins in Escherichia coli and used them for ligand affinity chromatography or glutathione S-transferase (GST) pull-down assays. By peptide mass fingerprinting Western blot analysis, or both, we identified PLCgamma, LANP, syndapin, PP1, and PP2A as binding partners for both NCAM 140 and NCAM 180, whereas TOAD-64 was identified as a NCAM 180-specific interacting protein. Furthermore, we were able to show that binding of these novel binding proteins, as well as the previously described interaction partners ROK alpha (rho A binding kinase alpha) and alpha- and beta-tubulin, bind to specific cytosolic sequences of NCAM. For this purpose, we performed GST pull-down experiments using cytosolic fragments of NCAM as GST-fusion proteins and cytosolic- or cytoskeleton-enriched protein fractions of rat brain.     

Septin 8 is an interaction partner and in vitro substrate of MK5

AIM:To identify novel substrates for the mitogen-activated protein kinase-activated protein kinase 5(MK5).METHODS:Yeast two-hybrid screening with MK5 as bait was used to identify novel possible interaction partners.The binding of putative partner was further examined by glutathione S-transferase(GST) pull-down,co-immunoprecipitation and fluorescence resonance energy transfer(FRET) analysis.In vitro kinase and peptide array assays were used to map MK5 phosphoacceptor sites on the new partner.Confocal microscopy was performed to study the subcellular localization of MK5 and its partners.RESULTS:Septin 8 was identified as a novel interaction partner for MK5 by yeast two-hybrid screening.This interaction was confirmed by GST pull-down,coimmunoprecipitation and FRET analysis.Septin 5,which can form a complex with septin 8,did not interact with MK5.Serine residues 242 and 271 on septin 8 were identified as in vitro MK5 phosphorylation sites.MK5 and septin 8 co-localized in the perinuclear area and in cell protrusions.Moreover,both proteins co-localized with vesicle marker synaptophysin.     

mRNA decay during herpes simplex virus (HSV) infections: protein-protein interactions involving the HSV virion host shutoff protein and translation factors eIF4H and eIF4A

During lytic infections, the virion host shutoff (Vhs) protein of herpes simplex virus accelerates the degradation of both host and viral mRNAs. In so doing, it helps redirect the cell from host to viral protein synthesis and facilitates the sequential expression of different viral genes. Vhs interacts with the cellular translation initiation factor eIF4H, and several point mutations that abolish its mRNA degradative activity also abrogate its ability to bind eIF4H. In addition, a complex containing bacterially expressed Vhs and a glutathione S-transferase (GST)-eIF4H fusion protein has RNase activity. eIF4H shares a region of sequence homology with eIF4B, and it appears to be functionally similar in that both stimulate the RNA helicase activity of eIF4A, a component of the mRNA cap-binding complex eIF4F. We show that eIF4H interacts physically with eIF4A in the yeast two-hybrid system and in GST pull-down assays and that the two proteins can be coimmunoprecipitated from mammalian cells. Vhs also interacts with eIF4A in GST pull-down and coimmunoprecipitation assays. Site-directed mutagenesis of Vhs and eIF4H revealed residues of each that are important for their mutual interaction, but not for their interaction with eIF4A. Thus, Vhs, eIF4H, and eIF4A comprise a group of proteins, each of which is able to interact directly with the other two. Whether they interact simultaneously as a tripartite complex or sequentially is unclear. The data suggest a mechanism for linking the degradation of an mRNA to its translation and for targeting Vhs to mRNAs and to regions of translation initiation.     

Glutamate receptor-interacting protein 1 protein binds to the microtubule-associated protein

To identify the interaction proteins for the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit glutamate receptor-interacting protein 1 (GRIP1), GRIP1 interactions with microtubule-associated protein (MAP)-1B light chain (LC) were investigated. GRIP1 interacts with MAP-1A and MAP-1B in the yeast two-hybrid assay, as is indicated also by glutathione S-transferase (GST) pull-down and coimmunoprecipitation with MAP-1B LC antibody in brain fractions. These results suggest a novel mechanism for localizing AMPA receptors to synaptic sites.     

Functional interaction of heterogeneous nuclear ribonucleoprotein C with poliovirus RNA synthesis initiation complexes

We had previously demonstrated that a cellular protein specifically interacts with the 3' end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione S-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3D(pol), as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1DeltaC) diminished these protein-protein interactions. When GST/hnRNP C1DeltaC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.     

大肠杆菌YggG与结合蛋白Era的相互作用研究

Era是细菌生长必须的一高度保守的GTPase。yggG是从大肠杆菌全基因组文库中钓取并克隆的Era结合蛋白基因,进一步的研究表明该基因在大肠杆菌中的表达与环境应激相关,提示yggG基因产物参与细菌的应激调控。为了阐明YggG蛋白与Era蛋白间的相互关系,利用所构建的双启动子表达载体pDH2-YggG-Ptac-Era在同一细胞中同时表达YggG与Era蛋白,并通过免疫共沉淀实验检测细菌裂解产物YggG与Era蛋白间的相互作用;在此基础上,构建并表达纯化了GST融合的Era蛋白氨基端截短肽和Era羧基端截短肽,通过GST Pull-down检测了Era不同功能区域与YggG蛋白间的相互作用。结果显示, Era/YggG 复合物仅存在于同时过表达Era和YggG蛋白的细菌细胞内,不诱导Era或者不诱导YggG蛋白过表达,均检测不到Era/YggG 复合物存在;纯化的GST不能Pull-down YggG蛋白,而纯化的GST融合的Era蛋白、Era氨基端截短肽及Era羧基端截短肽均可以Pull-down YggG蛋白;GST融合Era氨基端截短肽和GST融合的Era蛋白对YggG蛋白结合作用明显高于GST融合的Era蛋白羧基端截短肽。上述结果说明,YggG是一大肠杆菌Era结合蛋白,YggG与Era的氨基端和羧基端的结合活性存在差异。     

Glutamate Receptor-interacting Protein 1 Protein Binds to the Microtubule-associated Protein

To identify the interaction proteins for the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit glutamate receptor-interacting protein 1 (GRIP1), GRIP1 interactions with microtubule-associated protein (MAP)-1B light chain (LC) were investigated. GRIP1 interacts with MAP-1A and MAP-1B in the yeast two-hybrid assay, as is indicated also by glutathione S-transferase (GST) pull-down and coimmunoprecipitation with MAP-1B LC antibody in brain fractions. These results suggest a novel mechanism for localizing AMPA receptors to synaptic sites.     

The EICP22 protein of equine herpesvirus 1 physically interacts with the immediate-early protein and with itself to form dimers and higher-order complexes

The EICP22 protein (EICP22P) of Equine herpesvirus 1 (EHV-1) is an early protein that functions synergistically with other EHV-1 regulatory proteins to transactivate the expression of early and late viral genes. We have previously identified EICP22P as an accessory regulatory protein that has the ability to enhance the transactivating properties and the sequence-specific DNA-binding activity of the EHV-1 immediate-early protein (IEP). In the present study, we identify EICP22P as a self-associating protein able to form dimers and higher-order complexes during infection. Studies with the yeast two-hybrid system also indicate that physical interactions occur between EICP22P and IEP and that EICP22P self-aggregates. Results from in vitro and in vivo coimmunoprecipitation experiments and glutathione S-transferase (GST) pull-down studies confirmed a direct protein-protein interaction between EICP22P and IEP as well as self-interactions of EICP22P. Analyses of infected cells by laser-scanning confocal microscopy with antibodies specific for IEP and EICP22P revealed that these viral regulatory proteins colocalize in the nucleus at early times postinfection and form aggregates of dense nuclear structures within the nucleoplasm. Mutational analyses with a battery of EICP22P deletion mutants in both yeast two-hybrid and GST pull-down experiments implicated amino acids between positions 124 and 143 as the critical domain mediating the EICP22P self-interactions. Additional in vitro protein-binding assays with a library of GST-EICP22P deletion mutants identified amino acids mapping within region 2 (amino acids [aa] 65 to 196) and region 3 (aa 197 to 268) of EICP22P as residues that mediate its interaction with IEP.     

Hepatitis C virus envelope proteins bind lactoferrin.

Hepatitis C virus (HCV) has two envelope proteins, E1 and E2, which form a heterooligomer. During dissection of interacting regions of HCV E1 and E2, we found the presence of an interfering compound or compounds in skim milk. Here we report that human as well as bovine lactoferrin, a multifunctional immunomodulator, binds two HCV envelope proteins. As determined by far-Western blotting, the bacterially expressed E1 and E2 could bind lactoferrin in human milk directly separated or immunopurified and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The bindings of lactoferrin and HCV envelope proteins in vitro were confirmed by another method, the pull-down assay, with immunoprecipitated lactoferrin-bound protein A resin. By the same assay, mammal-expressed recombinant E1 and E2 were also demonstrated to bind human lactoferrin efficiently in vitro. Direct interaction between E2 and lactoferrin was proved in vivo, since anti-human lactoferrin antibody efficiently coimmunoprecipitated with secreted and intracellular forms of the E2 protein, but not glutathione S-transferase (GST), from lysates of HepG2 cells transiently cotransfected with the expression plasmids of human lactoferrin and gE2t-GST (the N-terminal two-thirds of E2 fused to GST) or GST. The N-terminal loop of lactoferrin, the region important for the antibacterial activity, has only a little role in the binding ability to HCV E2 but affected the secretion or stability of lactoferrin. Taken together, these results indicate the specific interaction between lactoferrin and HCV envelope proteins in vivo and in vitro.     

α-Synuclein与14-3-3蛋白在体外分子间相互作用的鉴定

帕金森病(Parkinson’s disease, PD)涉及两种蛋白——α-synuclein蛋白(SNCP)与14-3-3蛋白.通过重组,将这两种蛋白在大肠杆菌DH5α中表达, 通过谷胱甘肽- Sepharose 4B亲和层析将其纯化,得到GST-14-3-3蛋白;利用凝血酶对纯化的融合蛋白GST-SNCP切割,再经谷胱甘肽 Sepharose 4B亲和层析获得SNCP.通过免疫共沉淀、GST pull down和ELISA等技术,证明SNCP能够与14-3-3蛋白结合;为了进一步证明SNCP也与在脑组织中的天然14-3-3蛋白发生作用,利用His pull down方法进行实验.结果证明,SNCP能够和脑组织中的14-3-3蛋白发生相互作用.这些结果从分子水平提供了SNCP与14-3-3蛋白相互作用的实验证据,为进一步了解SNCP的结构和功能,及其在中枢神经系统退行性疾病的作用提供了必要的实验基础.     

ETRAP (efficient trapping and purification) of target protein polyclonal antibodies from GST-protein immune sera

Recombinant GST (glutathione transferase) proteins are widely used as immunogens to generate polyclonal antibodies. Advantages of using GST proteins include: commercially available cloning vectors, vast literature for protein expression in Escherichia coli, the ease of protein purification, immunogen can be used as an ELISA standard and GST can be removed in some systems. However, there are disadvantages: GST oligomerization, inclusion body formation and target protein insolubility after GST removal. Perhaps the most detrimental is the significant generation of anti-GST antibodies by the host animal. A two-column procedure using a glutathione-GST column and a glutathione-(GST-protein) column can yield affinity-purified anti-(GST-protein) polyclonal antibody. Several passes over the first column are often required, though, to completely extract the anti-GST antibodies from the immune sera. We reasoned that knowledge of the target protein linear epitope(s) would allow construction of a peptide affinity resin for a single-pass 'one and done' purification termed ETRAP (efficient trapping and purification). In the present paper, we describe our efforts and present data on rabbits and sheep immunized with GST proteins having target protein molecular masses of ~8, 21 and 33?kDa. The titre and purity of the target antibodies using the ETRAP protocol were comparable to the more laborious multi-column purifications but with a considerable saving in time.