Molecular and cellular approaches for the detection of protein-protein interactions: latest techniques and current limitations

Homotypic and heterotypic protein interactions are crucial for all levels of cellular function, including architecture, regulation, metabolism, and signaling. Therefore, protein interaction maps represent essential components of post-genomic toolkits needed for understanding biological processes at a systems level. Over the past decade, a wide variety of methods have been developed to detect, analyze, and quantify protein interactions, including surface plasmon resonance spectroscopy, NMR, yeast two-hybrid screens, peptide tagging combined with mass spectrometry and fluorescence-based technologies. Fluorescence techniques range from co-localization of tags, which may be limited by the optical resolution of the microscope, to fluorescence resonance energy transfer-based methods that have molecular resolution and can also report on the dynamics and localization of the interactions within a cell. Proteins interact via highly evolved complementary surfaces with affinities that can vary over many orders of magnitude. Some of the techniques described in this review, such as surface plasmon resonance, provide detailed information on physical properties of these interactions, while others, such as two-hybrid techniques and mass spectrometry, are amenable to high-throughput analysis using robotics. In addition to providing an overview of these methods, this review emphasizes techniques that can be applied to determine interactions involving membrane proteins, including the split ubiquitin system and fluorescence-based technologies for characterizing hits obtained with high-throughput approaches. Mass spectrometry-based methods are covered by a review by Miernyk and Thelen (2008; this issue, pp. 597–609 ). In addition, we discuss the use of interaction data to construct interaction networks and as the basis for the exciting possibility of using to predict interaction surfaces.     

Using Co-Cultures Expressing Fluorescence Resonance Energy Transfer Based Protein Biosensors to Simultaneously Image Caspase-3 and Ca<Superscript>2+</Superscript> Signaling

Fluorescence resonance energy transfer (FRET)-based protein biosensors allow the spatial and temporal imaging of signaling events in living cells. However, the simultaneous correlation of multiple events of a signaling pathway is hindered by the spectral cross-talk between fluorescent proteins. Here, we show, for signaling pathways that progress synchronously, multiple events can be correlated by using co-cultures expressing different FRET-based protein biosensors. As a demonstration, we investigated the simultaneous caspase-3 and Ca²⁺ signaling events involved in cell death of COS-7 cells induced by 10 mM H₂O₂. Interestingly, this H₂O₂ stimulus induced synchronous caspase-3 activation and Ca²⁺ signaling. In parallel to caspase-3 activation, cytosolic Ca²⁺ concentration, [Ca²⁺]_c, gradually rises to its peak and then slowly drops. As cell shrinkage and rounding ensues, [Ca²⁺]_c again gradually rises to its peak and then reaches a plateau. These observations reveal the relative timing and location of these signaling events in cell death induced by this stimulus of H₂O₂. Finally, our approach offers an exciting opportunity for spatial and temporal imaging of multiple events in a signaling pathway in living cells.     

Combining protein complementation assays with resonance energy transfer to detect multipartner protein complexes in living cells

A variety of fluorescent proteins with different spectral properties have been created by mutating green fluorescent protein. When these proteins are split in two, neither fragment is fluorescent per se, nor can a fluorescent protein be reconstituted by co-expressing the complementary N- and C-terminal fragments. However, when these fragments are genetically fused to proteins that associate with each other in cellulo, the N- and C-terminal fragments of the fluorescent protein are brought together and can reconstitute a fluorescent protein. A similar protein complementation assay (PCA) can be performed with two complementary fragments of various luciferase isoforms. This makes these assays useful tools for detecting the association of two proteins in living cells. Bioluminescence resonance energy transfer (BRET) or fluorescence resonance energy transfer (FRET) occurs when energy from, respectively, a luminescent or fluorescent donor protein is non-radiatively transferred to a fluorescent acceptor protein. This transfer of energy can only occur if the proteins are within 100 Å of each other. Thus, BRET and FRET are also useful tools for detecting the association of two proteins in living cells. By combining different protein fragment complementation assays (PCA) with BRET or FRET it is possible to demonstrate that three or more proteins are simultaneous parts of the same protein complex in living cells. As an example of the utility of this approach, we show that as many as four different proteins are simultaneously associated as part of a G protein-coupled receptor signalling complex.     

Epidermal growth factor induces changes of interaction between epidermal growth factor receptor and actin in intact cells

The epidermal growth factor receptor (EGFR) is a cyto-skeleton-binding protein. Although purified EGFR can interact with acting in vitro and normally at least 10% of EGFR exist in the insoluble cytoskeleton fraction of A431 cells, interaction of cytosolic EGFR with actin can only be visualized by fluorescence resonance energy transfer when epidermal growth factor presents in the cell medium. Results indicate that the correct orientation between EGFR and actin is important in the signal transduction process.     

Applications of BRET to study dynamic G-protein coupled receptor interactions in living cells

Summary Protein-protein interactions are fundamental processes for many biological systems including those involving the superfamily of G-protein coupled receptors (GPCRs). When addressing key questions concerning the regulation of GPCR-protein complexes and their functional significance, the development and refinement of non-invasive techniques to study these interactions will be of great value. One such technique, bioluminescence resonance energy transfer (BRET), is a recently described biophysical method that represents a powerful tool with which to measure protein-protein interactions in live cells, in real time. This minireview highlights the impact that evolving techniques such as BRET have had on the study of dynamic protein interactions involving GPCRs. In particular, the application of BRET to the study of protein interactions involving the receptors for hypothalamic peptide hormones, thyrotropin-releasing hormone (TRH) and gonadotropin-releasing hormone (GnRH), will be discussed. Using these receptors, BRET has successfully been used to demonstrate formation of both agonist-dependent and independent GPCR-GPCR complexes (oligomerization) and the agonist-dependent interaction of GPCRs with their intracellular adaptor protein partners, the arrestins. In summary, BRET is a highly snnsitive method that will not only aid in advancing our understanding of GPCR signalling and trafficking bout coud also potentially lead to the development of novel therapeutics that target these GPCR-protein complexes.     

Applications of BRET to study dynamic G-protein coupled receptor interactions in living cells

Protein-protein interactions are fundamental processes for manybiological systems including those involving the superfamily ofG-protein coupled receptors (GPCRs). When addressing keyquestions concerning the regulation of GPCR-protein complexes andtheir functional significance, the development and refinement ofnon-invasive techniques to study these interactions will be ofgreat value. One such technique, bioluminescence resonanceenergy transfer (BRET), is a recently described biophysicalmethod that represents a powerful tool with which to measureprotein-protein interactions in live cells, in real time. Thisminireview highlights the impact that evolving techniques such asBRET have had on the study of dynamic protein interactionsinvolving GPCRs. In particular, the application of BRET to thestudy of protein interactions involving the receptors forhypothalamic peptide hormones, thyrotropin-releasing hormone(TRH) and gonadotropin-releasing hormone (GnRH), will bediscussed. Using these receptors, BRET has successfully beenused to demonstrate formation of both agonist-dependent andindependent GPCR-GPCR complexes (oligomerization) and theagonist-dependent interaction of GPCRs with their intracellularadaptor protein partners, the arrestins. In summary, BRET is ahighly sensitive method that will not only aid in advancing ourunderstanding of GPCR signalling and trafficking but could alsopotentially lead to the development of novel therapeutics thattarget these GPCR-protein complexes.     

Unraveling molecular effects of ADAR1 overexpression in HEK293T cells by label-free quantitative proteomics

ADAR1 is a double-stranded RNA (dsRNA) editing enzyme that specifically converts adenosine to inosine. ADAR1 is ubiquitously expressed in eukaryotes and participate in various cellular processes such as differentiation, proliferation and immune responses. We report here a new proteomics study of HEK293T cells with and without ADAR1 overexpression. The up- and down-regulated proteins by ADAR1 overexpression are identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by label-free protein quantification. Totally 1,495 proteins (FDR < 0.01) are identified, among which 211 are up- and 159 are down-regulated for at least 1.5-fold (n = 3, p < 0.05). Gene ontology analysis reveals that these ADAR1-regulated proteins are involved in protein translation and cell cycle regulation. Bioinformatics analysis identifies a closely related network consistent for the protein translation machinery and a tightly connected network through proliferating cell nuclear antigen (PCNA)-interactions. Up-regulation of the proteins in the PCNA-mediated cell proliferation network is confirmed by Western blotting. In addition, ADAR1 overexpression is confirmed to increase cell proliferation in HEK293T cells and A549 cells. We conclude that ADAR1 overexpression modulates the protein translation and cell cycle networks through PCNA-mediated protein-protein interaction to promote cell proliferation in HEK293 cells.     

RELT induces cellular death in HEK 293 epithelial cells

RELT is a recently identified Tumor Necrosis Factor Receptor that possess two homologues in humans named RELL1 and RELL2. We investigated whether RELT and its homologues could induce cellular death when transiently transfected into HEK 293 epithelial cells. Transfection of RELT family members into HEK 293 epithelial cells induced cell death characterized by rounding and lifting of cells accompanied by DNA fragmentation, characteristics that are consistent with the activation of an apoptotic pathway. Overexpression of RELT in COS-7 cells resulted in cell rounding and lifting without DNA fragmentation, suggesting that the effects of RELT signaling may vary among different cell types. In summary, we report that overexpression of RELT or its homologues RELL1 and RELL2 in HEK 293 epithelial cells results in cell death with morphological characteristics consistent with the activation of an apoptotic pathway.     

Detection of an interaction between prion protein and neuregulin I-β1 by fluorescence resonance energy transfer analysis

Cellular prion protein (PrP) copurifies with neuregulin type I-β1 (NRG I-β1), but no interaction has been detected by a general immunoprecipitation study. We speculate that PrP interacts with NRG I-β1. Here, the interaction of PrP with NRG I-β1 was detected by measuring fluorescence resonance energy transfer (FRET) between enhanced blue (EBFP) and enhanced green (EGFP) fluorescent protein-fusion proteins. Full-length PrP interacted with EGFP in addition to NRG I-β1. From this result, we deduced that PrP interacts with EGFP through its unstructured N-terminal domain. We therefore detected FRET between PrP deleting the N-terminal domain and NRG I-β1. In contrast, the C-terminal domain of PrP interacted with NRG I-β1 and the proteins dissociated completely in the presence of sodium chloride. This interaction occurs at the nanomolar level, which is important for the reaction to be functional in organisms. We concluded that PrP interacted with NRG I-β1 through its C-terminal domain.     

Microtubule-mediated and microtubule-independent transport of adenovirus type 5 in HEK293 cells

Adenovirus serotypes 2 and 5 are taken into cells by receptor-mediated endocytosis, and following release from endosomes, destabilized virions travel along microtubules to accumulate around the nucleus. The entry process culminates in delivery of the viral genome through nuclear pores. This model is based on studies with conventional cell lines, such as HeLa and HEp-2, but in HEK293 cells, which are routinely used in this laboratory because they are permissive for replication of multiple adenovirus serotypes, a different trafficking pattern has been observed. Nuclei of 293 cells have an irregular shape, with an indented region, and virions directly labeled with carboxyfluorescein accumulate in a cluster within that indented region. The clusters, which form in close proximity to the microtubule organizing center (MTOC) and to the Golgi apparatus, are remarkably stable; a fluorescent signal can be seen in the MTOC region up to 16 h postinfection. Furthermore, if cells are infected and then undergo mitosis after the cluster is formed, the signal is found at each spindle pole. Despite the sequestration of virions near the MTOC, 293 cells are no less sensitive than other cells to productive infection with adenovirus. Even though cluster formation depends on intact microtubules, infectivity is not compromised by disruption of microtubules with either nocodazole or colchicine, as determined by expression of an enhanced green fluorescent protein reporter gene inserted in the viral genome. These results indicate that virion clusters do not represent the infectious pathway and suggest an alternative route to the nucleus that does not depend on nocodazole-sensitive microtubules.     

Splicing of scorpion toxin gene BmKK2 in HEK 293T cells

Using GFP as a reporter gene, splicing of scorpion toxin gene BmKK2 was investigated in cultured HEK 293T cells. The results of RT-PCR and western blotting showed that BmKK2's intron could be recognized and spliced in cultured HEK 293T cells. At the same time, a cryptic splicing site of BmKK2 gene was found at the 91st nucleotide site of the second exon, which is a typical form of alternative splicing. For the first time, alternative splicing would partially explain the diversity of scorpion toxins at the gene level. Moreover, replacing BmKK2's intron with BmP03's intron (an artificial BmKK2-BmP03 mosaic gene) did not affect the intron's recognition and splicing, but increased the expression of the toxin-GFP fusion protein by fluorescence imaging, which indicated that both introns may regulate the expression of toxin-GFP fusion protein. The artificial BmKK2-BmP03 mosaic gene was also spliced into two kinds of mRNA molecules, which showed that sequence of intron was not absolutely conserved. The results suggested that introns of scorpion toxin genes BmKK2 and BmP03 increase the diversity of scorpion toxins and regulate the expression of their genes.     

Functional characteristics of TRPC4 channels expressed in HEK 293 cells

The classical type of transient receptor potential (TRPC) channel is a molecular candidate for Ca²⁺-permeable cation channels in mammalian cells. Because TRPC4 and TRPC5 belong to the same subfamily of TRPC, they have been assumed to have the same physiological properties. However, we found that TRPC4 had its own functional characteristics different from those of TRPC5. TRPC4 channels had no constitutive activity and were activated by muscarinic stimulation only when a muscarinic receptor was co-expressed with TRPC4 in human embryonic kidney (HEK) cells. Endogenous muscarinic receptor appeared not to interact with TRPC4. TPRC4 activation by GTPγS was not desensitized. TPRC4 activation by GTPγS was not inhibited by either Rho kinase inhibitor or MLCK inhibitor. TRPC4 was sensitive to external pH with pK _a of 7.3. Finally, TPRC4 activation by GTPγS was inhibited by the calmodulin inhibitor W-7. We conclude that TRPC4 and TRPC5 have different properties and their own physiological roles. These authors contributed equally to this work.     

Structural changes and molecular interactions of hydrophobin SC3 in solution and on a hydrophobic surface

The hydrophobin SC3 belongs to a class of small proteins functioning in the growth and development of fungi. Its unique amphipathic property and remarkable surface activity make it interesting not only for biological studies but also for medical and industrial applications. Biophysical studies have revealed that SC3 possesses at least three distinct conformations, named "soluble-state SC3" for the protein in solution, and "alpha-helical-state SC3" and "beta-sheet-state SC3" for the different states of the protein associated at a hydrophobic-water interface. The present fluorescence study shows that the microenvironment of the dansyl-labeled N terminus of soluble-state SC3 is relatively hydrophobic, whereas it is hydrophilic for alpha-helical-state and beta-sheet-state SC3. Fluorescence collisional quenching indicates that the N terminus of soluble-state SC3 is more solvent-accessible than those of alpha-helical-state and beta-sheet-state SC3, with Stern-Volmer constants for acrylamide of 4.63, 0.02, and 0.2 M(-1) for the different states, respectively. Fluorescence resonance energy transfer measurements show that soluble-state SC3 tends to associate in solution but dissociates in TFA. Fluorescence energy transfer was eliminated by conversion of soluble-state SC3 to alpha-helical-state SC3 on a hydrophobic surface, indicating a spatial separation of the molecules in this state. By inducing the beta-sheet state, structural changes were observed, both by CD and by fluorescence, that could be fit to two exponentials with lifetimes of about 10 min and 4 h. Molecules in the beta-sheet state also underwent a slow change in spatial proximity on the hydrophobic surface, as revealed by the reappearance of fluorescence resonance energy transfer in time.     

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.     

Protein-protein interactions as a tool for site-specific labeling of proteins

Probing structures and dynamics within biomolecules using ensemble and single-molecule fluorescence resonance energy transfer requires the conjugation of fluorophores to proteins in a site-specific and thermodynamically nonperturbative fashion. Using single-molecule fluorescence-aided molecular sorting and the chymotrypsin inhibitor 2-subtilisin BPN' complex as an example, we demonstrate that protein-protein interactions can be exploited to afford site-specific labeling of a recombinant double-cysteine variant of CI2 without the need for extensive and time-consuming chromatography. The use of protein-protein interactions for site-specific labeling of proteins is compatible with and complementary to existing chemistries for selective labeling of N-terminal cysteines, and could be extended to label multiple positions within a given polypeptide chain.     

Effects of Wnt proteins on cell proliferation and apoptosis in HEK293 cells

Wnt proteins and Wnt signalings have been implicated in a variety of development and cell processes, while aberrant activation of Wnt signaling is linked to a range of cancers in many tissues. In this study, we used the HEK293 cell line to investigate the effects of Wnt3a and Wnt5a on proliferation and apoptosis in a serum starvation culture. After Wnt3a and Wnt5a proteins were expressed, they both promoted the proliferation of HEK293 cells under serum starvation. After 48h of serum starvation, both Wnt3a and Wnt5a inhibited serum starvation-induced apoptosis of HEK293 cells and continued up to 96h. We demonstrated that Wnt3a and Wnt5a can promote proliferation of HEK293 cells and inhibit serum starvation-induced apoptosis, which implies that Wnt3a and Wnt5a can maintain the survival of HEK293 cells under stress, and also provide a novel insight into the role of Wnt3a and Wnt5a and their related signalings in carcinogenesis.     

G蛋白偶联受体与G蛋白相互作用的最新研究进展

传统观念认为,在激动剂作用下,G蛋白偶联受体(GPCRs)能够激活G蛋白的α亚基,从而使Gα亚基与Gβγ亚基分离,被激活的Gα亚基通过信号转导进一步参与细胞的生理过程。但是,最新研究发现GPCRs和G蛋白存在多种偶联关系,GPCRs不仅能够激活Gα亚基,还可以与Gβγ亚基相互靠近,甚至会使G蛋白亚基构象发生重排而不分离,这对于疾病发病机制的研究及新的药物靶点的发现具有重要意义。就GPCRs与G蛋白之间的相互作用以及最新研究技术作一简要综述。     

Inositol depletion regulates phospholipid metabolism and activates stress signaling in HEK293T cells

Inositol plays a significant role in cellular function and signaling. Studies in yeast have demonstrated an “inositol-less death” phenotype, suggesting that inositol is an essential metabolite. In yeast, inositol synthesis is highly regulated, and inositol levels have been shown to be a major metabolic regulator, with its abundance affecting the expression of hundreds of genes. Abnormalities in inositol metabolism have been associated with several human disorders. Despite its importance, very little is known about the regulation of inositol synthesis and the pathways regulated by inositol in human cells. The current study aimed to address this knowledge gap. Knockout of ISYNA1 (encoding myo-inositol-3-P synthase 1) in HEK293T cells generated a human cell line that is deficient in de novo inositol synthesis. ISYNA1-KO cells exhibited inositol-less death when deprived of inositol. Lipidomic analysis identified inositol deprivation as a global regulator of phospholipid levels in human cells, including downregulation of phosphatidylinositol (PI) and upregulation of the phosphatidylglycerol (PG)/cardiolipin (CL) branch of phospholipid metabolism. RNA-Seq analysis revealed that inositol deprivation induced substantial changes in the expression of genes involved in cell signaling, including extracellular signal-regulated kinase (ERK), and genes controlling amino acid transport and protein processing in the endoplasmic reticulum (ER). This study provides the first in-depth characterization of the effects of inositol deprivation on phospholipid metabolism and gene expression in human cells, establishing an essential role for inositol in maintaining cell viability and regulating cell signaling and metabolism.     

橙色荧光蛋白的研究进展及其应用

荧光蛋白(Fluorescent protein,FPs)可作为探针用以探究细胞内分子间相互作用,追踪特定代谢物的代谢途径,对活细胞内的各种代谢过程和细胞通路进行详细、准确的描述。目前已有的FPs几乎已经覆盖了从紫外光到远红外光的所有光谱波段,这些FPs借助高分辨率显微技术应用于生命科学的诸多领域,为生物学的发展作出巨大贡献。橙色FPs通常指光谱区间在540–570nm的FPs,近几年来关于橙色FPs的研究进展较快,并且其作为标记蛋白以及荧光共振能量转移技术(Fluorescence resonance energy transfer,FRET)中的荧光受体在生物学及医学领域得到较多的应用。文中综述了近15年橙色FPs领域的相关研究,重点聚焦橙色FPs的发展和应用,为今后橙色FPs的研究提供依据。     

HeLa、HEK293、SH-SY5Y细胞中的Tau蛋白

通过间接免疫荧光测定了HeLa、HEK-293、SH-SY5Y细胞内Tau蛋白的分布,观察到在细胞间期单克隆抗体Tau-1的荧光信号分布于细胞质和胞核中．特别是HeLa细胞,其胞核内具有相对较高的Tau蛋白免疫荧光信号．通过分离SH-SY5Y的细胞核,更为清楚地显示了Tau蛋白在细胞核中的分布,并且免疫荧光信号与DNA的Hoechst33258染色信号相重合．Western blotting的测定结果进一步证明了SH-SY5Y细胞的胞质和胞核中均含有Tau蛋白的不同异构体．以上结果提示,Tau蛋白不仅存在于神经、肌肉等细胞内,也存在于肿瘤细胞系,并且分布于间期的胞核中．