A general and rapid cell-free approach for the interrogation of protein-protein, protein-DNA, and protein-RNA interactions and their antagonists utilizing split-protein reporters

Split-protein reporters have emerged as a powerful methodology for imaging biomolecular interactions which are of much interest as targets for chemical intervention. Herein we describe a systematic evaluation of split-proteins, specifically the green fluorescent protein, beta-lactamase, and several luciferases, for their ability to function as reporters in completely cell-free systems to allow for the extremely rapid and sensitive determination of a wide range of biomolecular interactions without the requirement for laborious transfection, cell culture, or protein purification (12-48 h). We demonstrate that the cell-free split-luciferase system in particular is amenable for directly interrogating protein-protein, protein-DNA, and protein-RNA interactions in homogeneous assays with very high sensitivity (22-1800 fold) starting from the corresponding mRNA or DNA. Importantly, we show that the cell-free system allows for the rapid (2 h) identification of target-site specificity for protein-nucleic acid interactions and in evaluating antagonists of protein-protein and protein-peptide complexes circumventing protein purification bottlenecks. Moreover, we show that the cell-free split-protein system is adaptable for analysis of both protein-protein and protein-nucleic acid interactions in artificial cell systems comprising water-in-oil emulsions. Thus, this study provides a general and enabling methodology for the rapid interrogation of a wide variety of biomolecular interactions and their antagonists without the limitations imposed by current in vitro and in vivo approaches.     

Gene transfection into adherent cells using electroporation on a dendrimer-modified gold electrode

Gene transfection into adherent cells from plasmid DNA (pDNA)-arrayed substrates known as gene transfection arrays appears to be a promising tool for the high-throughput analysis of gene functions and protein-protein interaction networks. We tested the ability of electric pulse-stimulated gene transfection from a substrate to overcome low expression efficiency and cross contamination between spots on arrays. We prepared the electrodes used for electric pulse-stimulated gene transfection by sequentially loading a gold thin layer, a self-assembled monolayer of a carboxylic acid-terminated alkanethiol (COOH-SAM), and poly(amidoamine) (PAMAM) dendrimers, either through electrostatic interactions or by covalent linkage to COOH-SAM and then to pDNA. When dendrimers were loaded onto the electrode using electrostatic interactions, the gene-expression efficiency of adherent cells increased as the generation numbers of the dendrimers that we used increased. Gene expression was rarely observed in adherent cells when dendrimers were covalently immobilized onto the electrode. Additionally, we successfully demonstrated site-specific gene transfer using a dendrimer-array electrode with no cross contamination between spots on the electrode.     

A versatile amino acid analogue of the solvatochromic fluorophore 4-N,N-dimethylamino-1,8-naphthalimide: a powerful tool for the study of dynamic protein interactions

We have developed a new unnatural amino acid based on the solvatochromic fluorophore 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN) for application in the study of protein-protein interactions. The fluorescence quantum yield of this chromophore is highly sensitive to changes in the local solvent environment, demonstrating "switch-like" emission properties characteristic of the dimethylaminophthalimide family of fluorophores. In particular, this new species possesses a number of significant advantages over related fluorophores, including greater chemical stability under a wide range of conditions, a longer wavelength of excitation (408 nm), and improved synthetic accessibility. This amino acid has been prepared as an Fmoc-protected building block and may readily be incorporated into peptides via standard solid-phase peptide synthesis. A series of comparative studies are presented to demonstrate the advantageous properties of the 4-DMN amino acid relative to those of the previously reported 4-N,N-dimethylaminophthalimidoalanine and 6-N,N-dimethylamino-2,3-naphthalimidoalanine amino acids. Other commercially available solvatochromic fluorophores are also include in these studies. The potential of this new probe as a tool for the study of protein-protein interactions is demonstrated by introducing it into a peptide that is recognized by calcium-activated calmodulin. The binding interaction between these two components yields an increase in fluorescence emission greater than 900-fold.     

Imaging protein-protein interactions inside living cells via interaction-dependent fluorophore ligation

We report a new method, Interaction-Dependent PRobe Incorporation Mediated by Enzymes, or ID-PRIME, for imaging protein-protein interactions (PPIs) inside living cells. ID-PRIME utilizes a mutant of Escherichia coli lipoic acid ligase, LplA(W37V), which can catalyze the covalent ligation of a coumarin fluorophore onto a peptide recognition sequence called LAP1. The affinity between the ligase and LAP1 is tuned such that, when each is fused to a protein partner of interest, LplA(W37V) labels LAP1 with coumarin only when the protein partners to which they are fused bring them together. Coumarin labeling in the absence of such interaction is low or undetectable. Characterization of ID-PRIME in living mammalian cells shows that multiple protein-protein interactions can be imaged (FRB-FKBP, Fos-Jun, and neuroligin-PSD-95), with as little as 10 min of coumarin treatment. The signal intensity and detection sensitivity are similar to those of the widely used fluorescent protein complementation technique (BiFC) for PPI detection, without the disadvantage of irreversible complex trapping. ID-PRIME provides a powerful and complementary approach to existing methods for visualization of PPIs in living cells with spatial and temporal resolution.     

Discovery of antagonist peptides against bacterial helicase-primase interaction in B. stearothermophilus by reverse yeast three-hybrid

Developing small-molecule antagonists against protein-protein interactions will provide powerful tools for mechanistic/functional studies and the discovery of new antibacterials. We have developed a reverse yeast three-hybrid approach that allows high-throughput screening for antagonist peptides against essential protein-protein interactions. We have applied our methodology to the essential bacterial helicase-primase interaction in Bacillus stearothermophilus and isolated a unique antagonist peptide. This peptide binds to the primase, thus excluding the helicase and inhibiting an essential interaction in bacterial DNA replication. We provide proof of principle that our reverse yeast three-hybrid method is a powerful "one-step" screen tool for direct high-throughput antagonist peptide selection against any protein-protein interaction detectable by traditional yeast two-hybrid systems. Such peptides will provide useful "leads" for the development of new antibacterials.     

Delivery of bioactive,gel-isolated proteins into live cells

The delivery of proteins into live cells is a promising strategy for the targeted modulation of protein-protein interactions and the manipulation of specific cellular functions. Cellular delivery can be facilitated by complexing the protein of interest with carrier molecules. Recently, an amphipatic peptide was identified, Pep-1 (KETWWETWWTE WSQPKKKRKV), which crosses the plasma membrane of many cell types to carry and deliver proteins as large as antibodies. Pep-1 effectively delivers proteins in solution; but Pep-1 is not suitable for delivering sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) isolated proteins because Pep-1 complexes with cargo proteins are destroyed by SDS. Here, we report cellular delivery of SDS-PAGE-isolated proteins, without causing cellular damage, by using a nonionic detergent, Triton X-100, as carrier. To determine the specificity of our method, we separated antibodies against different intracellular targets by nonreducing SDS-PAGE. Following electrophoresis, the antibody bands were detected by zinc-imidazole reverse staining, excised, in-gel refolded with Triton X-100, and eluted in detergent-free phosphate-buffered saline. When overlaid on cultured NIH 3T3 cells, the antibodies penetrated the cells localizing to their corresponding intracellular targets. These results are proof-of-principle for the delivery of gel-isolated bioactive proteins into cultured cells and suggest new ways for experimental protein therapy and for studying protein-protein interactions using gel-isolated protein.     

氢氘交换质谱技术在蛋白质和蛋白复合物结构研究中的应用进展

蛋白质是生命功能的执行者,其功能的发挥受自身结构动态变化、与其他生物分子的相互作用及修饰等因素的调节。因此,对蛋白质及蛋白复合物结构的研究有助于揭示重要生命过程中的分子机理与机制。氢氘交换质谱(Hydrogen deuterium exchange mass spectrometry,HDX-MS)是研究蛋白质结构、动态变化和相互作用的强有力工具,也是传统生物物理手段的重要补充。该文综述了HDX-MS的基本原理、机制、实验方法和研究最新进展,并从蛋白质自身动态变化、蛋白质-小分子相互作用、蛋白质-蛋白质相互作用3个方面介绍了近年来HDX-MS在蛋白及蛋白复合物研究中的应用进展。     

Deciphering In-vivo Cross-linking Mass Spectrometry Data for Dynamic Protein Structure Analysis

Protein structure and protein-protein interactions(PPIs) are crucial for regulating cellular activities required for cell viability and homeostasis. Chemical cross-linking coupled with mass spectrometry(CXMS) has become a versatile tool providing insights into both protein structure with distance restraints and protein-protein interactions with interface sites. Cross-links as the most information-rich data in a CXMS experiment are responsible for the structural model validation and integrative modeling with high throughput and sensitivity. In this work, ensemble refinement of the existing protein structure against the in-vivo cross-linking distance restraints was performed for dynamic protein structure modeling and protein interaction binding interface building in the intracellular environment. These results indicate great potential of in-vivo CXMS data for providing a molecular basis of protein structural dynamics exploration and function performance.     

Virtual screening for compounds that mimic protein-protein interface epitopes

Modulation of protein-protein interactions (PPI) has emerged as a new concept in rational drug design. Here, we present a computational protocol for identifying potential PPI inhibitors. Relevant regions of interfaces (epitopes) are predicted for three-dimensional protein models and serve as queries for virtual compound screening. We present a computational screening protocol that incorporates two different pharmacophore models. One model is based on the mathematical concept of autocorrelation vectors and the other utilizes fuzzy labeled graphs. In a proof-of-concept study, we were able to identify serine protease inhibitors using a predicted trypsin epitope as query. Our virtual screening framework may be suited for rapid identification of PPI inhibitors and suggesting bioactive tool compounds.     

Peptide Assemblies in Living Cells. Methods for Detecting Protein-Protein Interactions†

In mammalian cells, protein-protein interactions constitute essential regulatory steps that modulate the activity of signaling pathways. To understand the complicated mechanisms of the interactions in living cells, chemical crosslinking or immunoprecipitation has extensively been used. However, such biochemical methods require cell disruption and do not necessarily preserve all interactions intact. In recent years, several elegant approaches have been developed towards understanding the interactions. A common advantage of these new approaches is direct observation of the interaction in living cells without the need for disrupting the cells. We describe herein recent advances of those methods including our recent works based on protein splicing for detecting protein-protein interactions in vivo and highlight some potential applications of these techniques.     

Quantitative chemical proteomics approach to identify post-translational modification-mediated protein-protein interactions

Post-translational modifications (PTMs) (e.g., acetylation, methylation, and phosphorylation) play crucial roles in regulating the diverse protein-protein interactions involved in essentially every cellular process. While significant progress has been made to detect PTMs, profiling protein-protein interactions mediated by these PTMs remains a challenge. Here, we report a method that combines a photo-cross-linking strategy with stable isotope labeling in cell culture (SILAC)-based quantitative mass spectrometry to identify PTM-dependent protein-protein interactions. To develop and apply this approach, we focused on trimethylated lysine-4 at the histone H3 N-terminus (H3K4Me(3)), a PTM linked to actively transcribed gene promoters. Our approach identified proteins previously known to recognize this modification and MORC3 as a new protein that binds H3M4Me(3). This study indicates that our cross-linking-assisted and SILAC-based protein identification (CLASPI) approach can be used to profile protein-protein interactions mediated by PTMs, such as lysine methylation.     

Phosphopeptide selective coordination complexes as promising SRC homology 2 domain mimetics

Src Homology 2 (SH2) domains are the paradigm of phosphotyrosine (pY) protein recognition modules and mediate numerous cancer-promoting protein-protein complexes. Effective SH2 domain mimicry with pY-binding coordination complexes offers a promising route to new and selective disruptors of pY-mediated protein-protein interactions. We herein report the synthesis and in vitro characterization of a library of coordination complex SH2 domain proteomimetics. Compounds were designed to interact with phosphopeptides via a two-point interaction, principally with pY, and to make secondary interactions with pY+2/3, thereby achieving sequence-selective discrimination. Here, we report that lead mimetics demonstrated high target phosphopeptide affinity (K(a) ～ 10(7) M(-1)) and selectivity. In addition, biological screening in various tumor cells for anticancer effects showed a high degree of variability in cytotoxicity among receptors, which supported the proposed two-point binding mode. Several receptors potently disrupted cancer cell viability in breast cancer, prostate cancer, and acute myeloid leukemia cell lines.     

Application of a green fluorescent fusion protein to study protein-protein interactions by electrophoretic methods

A screening procedure for protein-protein interactions in cellular extracts using a green fluorescent protein (GFP) and affinity capillary electrophoresis (ACE) was established. GFP was fused as a fluorescent indicator to the C-terminus of a cyclophilin (rDmCyp20) from Drosophila melanogaster. Cyclophilins (Cyps) belong to the ubiquitously distributed enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases) and are well known as cellular targets of the immunosuppressive drug cyclosporin A (CsA). The PPlase activity of the GFP fused rDmCyp20 as well as the high affinity to CsA remain intact. Using native gel electrophoresis and ACE mobility-shift assays, it was demonstrated that the known moderate affinity of Cyp20 to the capsid protein p24 of HIV-1 was detectable in the case of rDmCyp20 fused to the fluorescent tag. For the p24 / rDmCyp20-GFP binding an ACE method was established which allowed to determine a dissociation constant of Kd = 20+/-1.5 x 10(-6) M. This result was verified by size-exclusion chromatography and is in good agreement with published data for the nonfused protein. Moreover the fusion protein was utilized to screen rDmCyp20-protein interactions by capillary electrophoresis in biological matrices. A putative ligand of rDmCyp20 in crude extracts of embryonic D. melanogaster was discovered by mobility-shift assays using native gel electrophoresis with fluorescence imaging and ACE with laser-induced fluorescence detection. The approach seems applicable to a wide range of proteins and offers new opportunities to screen for moderate protein-protein interactions in biological samples.     

A novel mechanism-based mammalian cell assay for the identification of SH2-domain-specific protein-protein inhibitors

Background: Many intracellular signal-transduction pathways are regulated by specific protein-protein interactions. These interactions are mediated by structural domains within signaling proteins that modulate a protein's cellular location, stability or activity. For example, Src-homology 2 (SH2) domains mediate protein-protein interactions through short contiguous amino acid motifs containing phosphotyrosine. As SH2 domains have been recognized as key regulatory molecules in a variety of cellular processes, they have become attractive drug targets.Results: We have developed a novel mechanism-based cellular assay to monitor specific SH2-domain-dependent protein-protein interactions. The assay is based on a two-hybrid system adapted to function in mammalian cells where the SH2 domain ligand is phosphorylated, and binding to a specific SH2 domain can be induced and easily monitored. As examples, we have generated a series of mammalian cell lines that can be used to monitor SH2-domain-dependent activity of the signaling proteins ZAP-70 and Src. We are utilizing these cell lines to screen for immunosuppressive and anti-osteoclastic compounds, respectively, and demonstrate here the utility of this system for the identification of small-molecule, cell-permeant SH2 domain inhibitors.Conclusions: A mechanism-based mammalian cell assay has been developed to identify inhibitors of SH2-domain-dependent protein-protein interactions. Mechanism-based assays similar to that described here might have general use as screens for cell-permeant, nontoxic inhibitors of protein-protein interactions.     

Protein tango: the toolbox to capture interacting partners

The evaluation of protein function in the context of the whole cell is crucial for understanding of living systems. In this context, the identification and modulation of protein-protein interactions in and outside cells is of ample importance. Several methods have been developed in the past years to detect and/or actively induce protein-protein interactions in living cells. As a result, tools are now available to manipulate intracellular events by reversible or irreversible cross-linking of proteins in a specific manner. These techniques open many new doors and enable the dissection of complicated protein networks. Herein we describe which cross-linkers and inducers of dimerization are out there and how to make use of this great toolbox.     

MimoDB: a new repository for mimotope data derived from phage display technology

Peptides selected from phage-displayed random peptide libraries are valuable in two aspects. On one hand, these peptides are candidates for new diagnostics, therapeutics and vaccines. On the other hand, they can be used to predict the networks or sites of protein-protein interactions. MimoDB, a new repository for these peptides, was developed, in which 10,716 peptides collected from 571 publications were grouped into 1,229 sets. Besides peptide sequences, other important information, such as the target, template, library and complex structure, was also included. MimoDB can be browsed and searched through a user-friendly web interface. For computational biologists, MimoDB can be used to derive customized data sets and benchmarks, which are useful for new algorithm development and tool evaluation. For experimental biologists, their results can be searched against the MimoDB database to exclude possible target-unrelated peptides. The MimoDB database is freely accessible at http://immunet.cn/mimodb/.     

Studying protein-protein interactions using peptide arrays

Screening of arrays and libraries of compounds is well-established as a high-throughput method for detecting and analyzing interactions in both biological and chemical systems. Arrays and libraries can be composed from various types of molecules, ranging from small organic compounds to DNA, proteins and peptides. The applications of libraries for detecting and characterizing biological interactions are wide and diverse, including for example epitope mapping, carbohydrate arrays, enzyme binding and protein-protein interactions. Here, we will focus on the use of peptide arrays to study protein-protein interactions. Characterization of protein-protein interactions is crucial for understanding cell functionality. Using peptides, it is possible to map the precise binding sites in such complexes. Peptide array libraries usually contain partly overlapping peptides derived from the sequence of one protein from the complex of interest. The peptides are attached to a solid support using various techniques such as SPOT-synthesis and photolithography. Then, the array is incubated with the partner protein from the complex of interest. Finally, the detection of the protein-bound peptides is carried out by using immunodetection assays. Peptide array screening is semi-quantitative, and quantitative studies with selected peptides in solution are required to validate and complement the screening results. These studies can improve our fundamental understanding of cellular processes by characterizing amino acid patterns of protein-protein interactions, which may even develop into prediction algorithms. The binding peptides can then serve as a basis for the design of drugs that inhibit or activate the target protein-protein interactions. In the current review, we will introduce the recent work on this subject performed in our and in other laboratories. We will discuss the applications, advantages and disadvantages of using peptide arrays as a tool to study protein-protein interactions.     

Targeting the protein-protein interactions of the HIV lifecycle

The human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS), relies heavily on protein-protein interactions in almost every step of its lifecycle. Targeting these interactions, especially those between virus and host proteins, is increasingly viewed as an ideal avenue for the design and development of new therapeutics. In this tutorial review, we outline the lifecycle of HIV and describe some of the protein-protein interactions that control and regulate each step of this process, also detailing efforts to develop therapies that target these interactions.