Surface plasmon resonance imaging-based protein arrays for high-throughput screening of protein-protein interaction inhibitors

The E7 protein produced by high-risk human papillomavirus (HPV) induces a degradation of the retinoblastoma tumor suppressor RB through direct interaction, which suggests that an inhibitor for the interaction can be a potential anticancer drug. A surface plasmon resonance (SPR) imaging-based protein array chip was developed for the high-throughput screening of inhibitor molecules targeting RB-E7 interaction. The glutathione S-transferase-fused E7 protein (GST-E7) was first layered onto a glutathionylated gold chip surface that had been designed to specifically bind to GST-fused proteins. Subsequently, a microarrayer was used to spot the hexa-histidine-tagged RB proteins (His(6)-RB) onto the GST-E7-layered gold chip surface, and the resulting SPR image was analyzed. Upon increased His(6)-RB concentration in the spotting solution, the SPR signal intensity increased proportionally, indicating that His(6)-RB bound to GST-E7 in a concentration-dependent manner. The His(6)-RB/GST-E7 interaction was challenged by spotting the His(6)-RB solution in the presence of a RB binding peptide (PepC) derived from a motif on E7. The SPR imaging data showed that PepC inhibited the His(6)-RB/GST-E7 interaction in a concentration-dependent manner. Our results show that the SPR imaging-based protein array chip can be applied to screen small molecule inhibitors that target protein-protein interaction.     

Surface plasmon resonance imaging analysis of protein-protein interactions using on-chip-expressed capture protein

A surface plasmon resonance (SPR) imaging system, combined with a microwell gold chip for on-chip cell cultivation, was used to monitor protein-protein interactions. In particular, we developed an on-chip microscale cell cultivation system that integrates cell culture and on-chip analysis of protein-protein interactions on a single microwell chip in a time- and labor-saving manner. To assess the performance of this system in the analysis of protein-protein interactions, we conducted a series of protein-protein interaction analyses by measuring the binding of the yeast GAL4 dimerization domain (GAL4DD) to the GAL11 protein (GAL11P). Our system was found to enable the simple and rapid analysis of protein-protein interactions, requiring no special cell culturing equipment or recombinant protein expression prior to the immobilization of the purified proteins onto the chip. Our results demonstrate that the combination of an on-chip cell cultivation system and an SPR imaging system can be a useful tool to study protein-protein interactions without the need for time-consuming and labor-intensive protein preparation steps as well as fluorescent or other labeling of the interactants.     

Differential binding studies applying functional protein microarrays and surface plasmon resonance

A variety of different in vivo and in vitro technologies provide comprehensive insights in protein-protein interaction networks. Here we demonstrate a novel approach to analyze, verify and quantify putative interactions between two members of the S100 protein family and 80 recombinant proteins derived from a proteome-wide protein expression library. Surface plasmon resonance (SPR) using Biacore technology and functional protein microarrays were used as two independent methods to study protein-protein interactions. With this combined approach we were able to detect nine calcium-dependent interactions between Arg-Gly-Ser-(RGS)-His6 tagged proteins derived from the library and GST-tagged S100B and S100A6, respectively. For the protein microarray affinity-purified proteins from the expression library were spotted onto modified glass slides and probed with the S100 proteins. SPR experiments were performed in the same setup and in a vice-versa approach reversing analytes and ligands to determine distinct association and dissociation patterns of each positive interaction. Besides already known interaction partners, several novel binders were found independently with both detection methods, albeit analogous immobilization strategies had to be applied in both assays.     

High-throughput analysis of GST-fusion protein expression and activity-dependent protein interactions on GST-fusion protein arrays with a spectral surface plasmon resonance biosensor

We modified gold arrays with a glutathione (GSH) surface, and investigated high-throughput protein interactions with a spectral surface plasmon resonance (SPR) biosensor. We fabricated the GSH exterior on gold surfaces by successive modification with aminoethanethiol, 4-maleimidobutyric acid N-hydroxysuccinimide ester and GSH. We immobilized GST-Rac1, GST-RhoA, the GST-Rho-binding domain of rhotekin and the GST-p21-binding domain of PAK1 onto the GSH surface, and observed specific antigen-antibody interactions on the GST-fusion protein arrays. We determined the expression of GST-fusion proteins in Escherichia coli on the GSH surface with the SPR biosensor. We then analyzed the interactions of tissue transglutaminase (tTGase), a Ca2+-dependent enzyme, with RhoA and Rac1 on the GST-fusion protein arrays with the SPR biosensor. We found that tTGase interacted with RhoA and Rac1 in a Ca2+-dependent manner, indicating that the interactions were dependent on tTGase activity. In addition, transamidation of Rac1 by tTGase was dependent on Ca2+ concentration. We obtained similar results with GST pull-down assays. Thus, protein arrays prepared on the GSH surface provide a useful system for the high-throughput analysis of GST-fusion protein expression and activity-dependent protein interactions with the spectral SPR biosensors.     

Development of a protein microarray using sequence-specific DNA binding domain on DNA chip surface

A protein microarray based on DNA microarray platform was developed to identify protein-protein interactions in vitro. The conventional DNA chip surface by 156-bp PCR product was prepared for a substrate of protein microarray. High-affinity sequence-specific DNA binding domain, GAL4 DNA binding domain, was introduced to the protein microarray as fusion partner of a target model protein, enhanced green fluorescent protein. The target protein was oriented immobilized directly on the DNA chip surface. Finally, monoclonal antibody of the target protein was used to identify the immobilized protein on the surface. This study shows that the conventional DNA chip can be used to make a protein microarray directly, and this novel protein microarray can be applicable as a tool for identifying protein-protein interactions.     

Adenoviral p53 effects and cell-specific E7 protein-protein interactions of human cervical cancer cells

We investigated the time-course tumor growth suppression effects of recombinant adenovirus expressing p53 on human cervical cancer cells and cell-specific E7 protein-protein interactions in cell lysates using surface plasmon resonance (SPR) biosensor. Six HPV-infected human cervical cancer cell lines (HPV 16-positive cells, CaSki and SiHa cells; HPV 18-positive cells, HeLa and HeLaS3 cells; and HPV negative C33A and HT3 cells) were used. After infection with AdCMVp53, the cell-specific growth inhibition was studied in vitro and in vivo. Also, we produced the recombinant E7 oncoprotein of HPV 16 type and tested chip-based protein-protein interactions with each cell lysate. For each cervical cancer cell, differential cell growth inhibitions were shown via cell count assay and MTT assay. Note that the same trend in suppression levels was shown in CaSki, HeLa and in SiHa, HeLaS3, respectively. In contrast, infection with AdCMVLacZ showed increased cell growth in a manner similar to the negative control group. The levels of p53 protein were notably expressed in CaSki and HeLa more than in SiHa and HeLaS3 for 4 days. In contrast, p53 expression was continually maintained in C33A and HT3 for 6 days. After transfection AdCMVp53 into CaSki- and SiHa-xenografted nude mice, the size of tumor was remarkably decreased in SiHa cells as compared to AdCMVLacZ transfection. The SPR sensor surface was successfully modified with the recombinant E7 oncoprotein and showed cell-specific interactions between E7 and its target proteins from cell lysates. The anti-tumor effects were accomplished via differential role of p53-specific apoptotic cell death, which is dependent upon the cervical cancer cell line. Also, a molecular level understanding of cell-dependent protein interaction effects of recombinant E7 was shown.     

Deciphering the mechanisms of HPV E6 mutations in the destabilization of E6/E6AP/p53 complex

In epithelial tumors, oncoprotein E6 binds with the ubiquitin ligase E6AP to form E6/E6AP heterodimer; then this heterodimer recruits p53 to form E6/E6AP/p53 heterotrimer and induces p53 degradation. Recent experiments demonstrated that three E6 single-site mutants (F47R, R102A, and L50E) can inhibit the E6/E6AP/p53 heterotrimer formation and rescue p53 from the degradation pathway. However, the molecular mechanism underlying mutation-induced heterotrimer inhibition remains largely elusive. Herein, we performed extensive molecular dynamics simulations (totally ～13 μs) on both heterodimer and heterotrimer to elucidate at an atomic level how each p53-degradation-defective HPV16 E6 mutant reduces the structural stabilities of the two complexes. Our simulations reveal that the three E6 mutations destabilize the structure of E6/E6AP/p53 complex through distinct mechanisms. Although F47R^E6 mutation has no effect on the structure of E6/E6AP heterodimer, it results in an electrostatic repulsion between R47^E6 and R290^p53, which is unfavorable for E6-p53 binding. R102A^E6 mutation destabilizes the structure of E6/E6AP heterodimer and significantly disrupts hydrophobic and cation-π interactions between F47^E6 and E286^p53/L298^p53/R290^p53. L50E^E6 mutation impairs both E6 interdomain interactions (especially F47-K108 cation-π interaction) and E6-E6AP intermolecular interactions important for the stabilization of E6/E6AP heterodimer. This study identifies the intra- and intermolecular interactions crucial for the complex stability, which may provide mechanistic insights into the inhibition of complex formation by the three HPV16 E6 mutations.     

Surface plasmon resonance imaging analysis of hexahistidine-tagged protein on the gold thin film coated with a calix crown derivative

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the hexahistidine-ubiquitin-tagged human parathyroid hormone fragment (His₆-Ub-hPTHF(1–34)) expressed inEscherichia coli. The hexahistidine-specific antibody was immobilized on a thin gold film coated with ProLinker^TM B, a novel calixcrown derivative with a bifunctional coupling property that permits efficient immobilization of capture proteins on solid matrices. The soluble and insoluble fractions of anE. coli cell lysate were spotted onto the antibody-coated gold chip, which was then washed with buffer (pH 7.4) solution and dried. SPR imaging measurements were carried out to detect the expressed His₆-Ub-hPTHF (1–34). There was no discernible protein image in the uninduced cell lysate, indicating that non-specific binding of contaminant proteins did not occur on the gold chip surface. It is expected that the approach used here to detect affinity-tagged recombinant proteins using an SPR imaging technique could be used as a powerful tool for the analyses of a number of proteins in a high-throughput mode.     

Kinetic analysis of the interactions of human papillomavirus E6 oncoproteins with the ubiquitin ligase E6AP using surface plasmon resonance

Cervical cancers evolve from lesions generated by genital human papillomaviruses (HPV). "Low-risk" genital HPVs cause benign proliferations whereas "high-risk" types have the potential to progress into cancer. High-risk HPV E6 oncoproteins interact with the ubiquitin ligase E6AP and target several cellular proteins, including p53 and proteins of the MAGI family, towards ubiquitin-mediated degradation. E6AP, like other E6 binding proteins such as E6BP, IRF-3 and paxillin, interacts with E6 via a consensus leucine-charged motif. Here we have investigated the kinetics of the interactions of a 15-mer peptide containing the LxxvarphiLsh motif of E6AP with E6. For this we have developed a Biacore assay based on antibody-capture on the sensor surface of GST- and/or MBP-E6AP peptide constructs followed by E6 protein injection. Our experiments show that E6 oncoproteins from four major high-risk (16, 18, 33 and 58) HPV types bind to E6AP with equilibrium dissociation constants in the low micromolar range. The kinetic dissociation parameters of these interactions are remarkably similar. On the other hand, low-risk HPV 11 E6 does not interact with E6AP even at relatively high concentrations. We also show that the two zinc-binding domains of E6 are required for E6AP recognition. Finally, we have analysed the binding properties of site-directed mutants of the E6AP-derived peptide. We demonstrate the importance for binding of conserved aliphatic side-chains and the moderate role of the global negative charge of the peptide. This work provides the first quantitative data on an HPV E6-mediated interaction, which support the current models of E6AP-mediated degradation.     

Comprehensive Analysis of Host Cellular Interactions with Human Papillomavirus E6 Proteins Identifies New E6 Binding Partners and Reflects Viral Diversity

We have begun to define the human papillomavirus (HPV)-associated proteome for a subset of the more than 120 HPV types that have been identified to date. Our approach uses a mass spectrometry-based platform for the systematic identification of interactions between human papillomavirus and host cellular proteins, and here we report a proteomic analysis of the E6 proteins from 16 different HPV types. The viruses included represent high-risk, low-risk, and non-cancer-associated types from genus alpha as well as viruses from four different species in genus beta. The E6 interaction data set consists of 153 cellular proteins, including several previously reported HPV E6 interactors such as p53, E6AP, MAML1, and p300/CBP and proteins containing PDZ domains. We report the genus-specific binding of E6s to either E6AP or MAML1, define the specific HPV E6s that bind to p300, and demonstrate several new features of interactions involving beta HPV E6s. In particular, we report that several beta HPV E6s bind to proteins containing PDZ domains and that at least two beta HPV E6s bind to p53. Finally, we report the newly discovered interaction of proteins of E6 of beta genus, species 2, with the Ccr4-Not complex, the first report of a viral protein binding to this complex. This data set represents a comprehensive survey of E6 binding partners that provides a resource for the HPV field and will allow continued studies on the diverse biology of the human papillomaviruses.     

A fusion protein expression analysis using surface plasmon resonance imaging

A surface plasmon resonance (SPR) imaging system was constructed and used to detect the affinity-tagged recombinant proteins expressed in Escherichia coli. With regards to model proteins, the hexahistidine-ubiquitin-tagged human growth hormone (His(6)-Ub-hGH), glutathione S-transferase-tagged human interleukin-6 (GST-hIL6), and maltose-binding protein-tagged human interleukin-6 (MBP-hIL6) expressed in E. coli were analyzed. The cell lysates were spotted on gold thin films coated with 11-mercaptoundecanol (MUOH)/dextran derivatized with Ni(II)-iminodiacetic acid (IDA-Ni(II)), glutathione, or cyclodextrin. After a brief washing of the gold chip, SPR imaging measurements were carried out in order to detect the bound affinity-tagged fusion proteins. Using this new approach, rapid high-throughput expression analysis of the affinity-tagged proteins were obtained. The SPR imaging protein chip system used to measure the expression of affinity-tagged proteins in a high-throughput manner is expected to be an attractive alternative to traditional laborious and time-consuming methods, such as SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blots.     

Biophysical characterization of the complex between human papillomavirus E6 protein and synapse-associated protein 97

The E6 protein of human papillomavirus (HPV) exhibits complex interaction patterns with several host proteins, and their roles in HPV-mediated oncogenesis have proved challenging to study. Here we use several biophysical techniques to explore the binding of E6 to the three PDZ domains of the tumor suppressor protein synapse-associated protein 97 (SAP97). All of the potential binding sites in SAP97 bind E6 with micromolar affinity. The dissociation rate constants govern the different affinities of HPV16 and HPV18 E6 for SAP97. Unexpectedly, binding is not mutually exclusive, and all three PDZ domains can simultaneously bind E6. Intriguingly, this quaternary complex has the same apparent hydrodynamic volume as the unliganded PDZ region, suggesting that a conformational change occurs in the PDZ region upon binding, a conclusion supported by kinetic experiments. Using NMR, we discovered a new mode of interaction between E6 and PDZ: a subset of residues distal to the canonical binding pocket in the PDZ(2) domain exhibited noncanonical interactions with the E6 protein. This is consistent with a larger proportion of the protein surface defining binding specificity, as compared with that reported previously.     

Identification of a second transforming function in bovine papillomavirus type 1 E6 and the role of E6 interactions with paxillin, E6BP, and E6AP

Papillomavirus E6 oncoproteins transform mammalian cells through interaction with cellular proteins. Bovine papillomavirus type 1 E6 (BE6) interacts with three previously described cellular targets: the E6AP E3 ubiquitin ligase, the calcium-binding protein E6BP (also known as ERC-55), and paxillin, which is a focal adhesion adapter protein. BE6 interacts strongly with each of these proteins in vitro, binding to similar peptide sequences found in E6AP, E6BP, and paxillin. To determine which BE6 interactions are necessary for transformation by BE6, we used a novel selection strategy for temperature-sensitive BE6 mutants in yeast that could discriminate in their interaction between E6AP, E6BP, and paxillin. All BE6 mutants that retained transforming ability retained association with paxillin, while some mutants that were transformation positive failed to interact with E6AP or E6BP. This study demonstrates that oncogene mutants that are temperature sensitive for transformation can be selected in yeast and that the induction of anchorage-independent cell proliferation by BE6 does not require strong association of BE6 with either E6AP or E6BP. Of particular interest is the identification of a BE6 mutant that interacts strongly with the acidic charged leucine motifs of E6AP, E6BP, and paxillin but is devoid of transformation activity, thereby genetically identifying a second essential transformation function in BE6 that is independent of interaction with acidic charged leucine motifs.     

Identification and proteomic analysis of distinct UBE3A/E6AP protein complexes

The E6AP ubiquitin ligase catalyzes the high-risk human papillomaviruses' E6-mediated ubiquitylation of p53, contributing to the neoplastic progression of cells infected by these viruses. Defects in the activity and the dosage of E6AP are linked to Angelman syndrome and to autism spectrum disorders, respectively, highlighting the need for precise control of the enzyme. With the exception of HERC2, which modulates the ubiquitin ligase activity of E6AP, little is known about the regulation or function of E6AP normally. Using a proteomic approach, we have identified and validated several new E6AP-interacting proteins, including HIF1AN, NEURL4, and mitogen-activated protein kinase 6 (MAPK6). E6AP exists as part of several different protein complexes, including the proteasome and an independent high-molecular-weight complex containing HERC2, NEURL4, and MAPK6. In examining the functional consequence of its interaction with the proteasome, we found that UBE3C (another proteasome-associated ubiquitin ligase), but not E6AP, contributes to proteasomal processivity in mammalian cells. We also found that E6 associates with the HERC2-containing high-molecular-weight complex through its binding to E6AP. These proteomic studies reveal a level of complexity for E6AP that has not been previously appreciated and identify a number of new cellular proteins through which E6AP may be regulated or functioning.     

On-chip Escherichia coli culture, purification, and detection of expressed proteins

In a recent study, we reported the results of a rapid high-throughput expression analysis of the affinity-tagged proteins present in total cell lysates, using a surface plasmon resonance (SPR) imaging protein chip system. In this paper, we describe a novel method, which is able to sequentially carry out a recombinant Escherichia coli culture, as well as the detection and purification of the expressed proteins on a single microwell chip, fabricated on a two-dimensional thin gold film. Following the induction of the protein on the microwell chip, the E. coli cells were lysed on the chip via the addition of lysozymes, and the expressed glutathione S-transferase-fused green fluorescent protein (GST–GFP) was then purified on the chip via affinity interaction with the glutathionylated gold surface of the chip. Finally, the expressed protein was directly detected using the surface plasmon resonance (SPR) imaging system. This system saves a substantial amount of time, experimental resources, and labor, by allowing for the complicated and labor-intensive procedures inherent to the production of recombinant proteins to be conducted on a single microwell chip, simply and economically.M. Kim and S. Y. Lee contributed equally to this work.     

Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53.

The E6 oncoproteins of the cancer-associated or high-risk human papillomaviruses (HPVs) target the cellular p53 protein. The association of E6 with p53 leads to the specific ubiquitination and degradation of p53 in vitro, suggesting a model by which E6 deregulates cell growth control by the elimination of the p53 tumor suppressor protein. Complex formation between E6 and p53 requires an additional cellular factor, designated E6-AP (E6-associated protein), which has a native and subunit molecular mass of approximately 100 kDa. Here we report the purification of E6-AP and the cloning of its corresponding cDNA, which contains a novel open reading frame encoding 865 amino acids. E6-AP, translated in vitro, has the following properties: (i) it associates with wild-type p53 in the presence of the HPV16 E6 protein and simultaneously stimulates the association of E6 with p53, (ii) it associates with the high-risk HPV16 and HPV18 E6 proteins in the absence of p53, and (iii) it induces the E6- and ubiquitin-dependent degradation of p53 in vitro.