Dynamic Protein-protein Interactions Research Articles

Proteomic and protein interaction network analysis of human T lymphocytes during cell‐cycle entry

Regulating the transition of cells such as T lymphocytes from quiescence (G(0)) into an activated, proliferating state involves initiation of cellular programs resulting in entry into the cell cycle (proliferation), the growth cycle (blastogenesis, cell size) and effector (functional) activation. We show the first proteomic analysis of protein interaction networks activated during entry into the first cell cycle from G(0). We also provide proof of principle that blastogenesis and proliferation programs are separable in primary human T cells. We employed a proteomic profiling method to identify large-scale changes in chromatin/nuclear matrix-bound and unbound proteins in human T lymphocytes during the transition from G(0) into the first cell cycle and mapped them to form functionally annotated, dynamic protein interaction networks. Inhibiting the induction of two proteins involved in two of the most significantly upregulated cellular processes, ribosome biogenesis (eIF6) and hnRNA splicing (SF3B2/SF3B4), showed, respectively, that human T cells can enter the cell cycle without growing in size, or increase in size without entering the cell cycle.

Fluorescence Correlation Spectroscopy to Monitor Kai Protein-based Circadian Oscillations in Real Time

Dynamic protein-protein interactions play an essential role in cellular regulatory systems. The cyanobacterial circadian clock is an oscillatory system that can be reconstituted in vitro by mixing ATP and three clock proteins: KaiA, KaiB, and KaiC. Association and dissociation of KaiB from KaiC-containing complexes are critical to circadian phosphorylation and dephosphorylation of KaiC. We developed an automated and noninvasive method to monitor dynamic complex formation in real time using confocal fluorescence correlation spectroscopy (FCS) and uniformly labeled KaiB as a probe. A nanomolar concentration of the labeled KaiB for FCS measurement did not interfere with the oscillatory system but behaved similarly to the wild-type one during the measurement period (>5 days). The fluorescent probe was stable against repeated laser exposure. As an application, we show that this detection system allowed analysis of the dynamics of both long term circadian oscillations and short term responses to temperature changes (∼10 min) in the same sample. This suggested that a phase shift of the clock with a high temperature pulse occurred just after the stimulus through dissociation of KaiB from the KaiC complex. This monitoring method should improve our understanding of the mechanisms underlying this cellular circadian oscillator and provide a means to assess dynamic protein interactions in biological systems characterized by rates similar to those observed with the Kai proteins.

Nanoscale Membrane Organization: Where Biochemistry Meets Advanced Microscopy

Understanding the molecular mechanisms that shape an effective cellular response is a fundamental question in biology. Biochemical measurements have revealed critical information about the order of protein-protein interactions along signaling cascades but lack the resolution to determine kinetics and localization of interactions on the plasma membrane. Furthermore, the local membrane environment influences membrane receptor distributions and dynamics, which in turn influences signal transduction. To measure dynamic protein interactions and elucidate the consequences of membrane architecture interplay, direct measurements at high spatiotemporal resolution are needed. In this review, we discuss the biochemical principles regulating membrane nanodomain formation and protein function, ranging from the lipid nanoenvironment to the cortical cytoskeleton. We also discuss recent advances in fluorescence microscopy that are making it possible to quantify protein organization and biochemical events at the nanoscale in the living cell membrane.

Fasting induces the expression of PGC-1α and ERR isoforms in the outer stripe of the outer medulla (OSOM) of the mouse kidney.

BackgroundPeroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α) is a member of the transcriptional coactivator family that plays a central role in the regulation of cellular energy metabolism under various physiological stimuli. During fasting, PGC-1α is induced in the liver and together with estrogen-related receptor a and γ (ERRαand ERRγ, orphan nuclear receptors with no known endogenous ligand, regulate sets of genes that participate in the energy balance program. We found that PGC-1α, ERRα and ERRγ was highly expressed in human kidney HK2 cells and that PGC-1α induced dynamic protein interactions on the ERRα chromatin. However, the effect of fasting on the expression of endogenous PGC-1α, ERRα and ERRγ in the kidney is not known.Methodology/Principal FindingsIn this study, we demonstrated by qPCR that the expression of PGC-1α, ERRα and ERRγ was increased in the mouse kidney after fasting. By using immunohistochemistry (IHC), we showed these three proteins are co-localized in the outer stripe of the outer medulla (OSOM) of the mouse kidney. We were able to collect this region from the kidney using the Laser Capture Microdissection (LCM) technique. The qPCR data showed significant increase of PGC-1α, ERRα and ERRγ mRNA in the LCM samples after fasting for 24 hours. Furthermore, the known ERRα target genes, mitochondrial oxidative phosphorylation gene COX8H and the tricarboxylic acid (TCA) cycle gene IDH3A also showed an increase. Taken together, our data suggest that fasting activates the energy balance program in the OSOM of the kidney.

Elevated Calcium Acutely Regulates Dynamic Interactions of NHERF2 and NHE3 Proteins in Opossum Kidney (OK) Cell Microvilli

The brush border (BB) Na(+)/H(+) exchanger NHE3 is rapidly activated or inhibited by changes in trafficking, which mimics renal and intestinal physiology. However, there is a paradox in that NHE3 has limited mobility in the BB due to its binding to the multi-PDZ domain containing the NHERF family. To allow increased endocytosis, as occurs with elevated intracellular Ca(2+), we hypothesized that NHE3 had to be, at least transiently, released from the BB cytoskeleton. Because NHERF1 and -2 are localized at the BB, where they bind NHE3 as well as the cytoskeleton, we tested whether either or both might dynamically interact with NHE3 as part of Ca(2+) signaling. We employed FRET to study close association of NHE3 and these NHERFs and fluorescence recovery after photobleaching to monitor NHE3 mobility in the apical domain in polarized opossum kidney cells. Under basal conditions, NHERF2 and NHE3 exhibited robust FRET signaling. Within 1 min of A23187 (0.5 μm) exposure, the NHERF2-NHE3 FRET signal was abolished, and BB NHE3 mobility was transiently increased. The dynamics in FRET signal and NHE3 mobility correlated well with a change in co-precipitation of NHE3 and NHERF2 but not NHERF1. We conclude the following. 1) Under basal conditions, NHE3 closely associates with NHERF2 in opossum kidney cell microvilli. 2) Within 1 min of elevated Ca(2+), the close association of NHE3-NHERF2 is abolished but is re-established in ∼60 min. 3) The change in NHE3-NHERF2 association is accompanied by an increased BB mobile fraction of NHE3, which contributes to inhibition of NHE3 transport activity via increased endocytosis.

Mapping a Dynamic Innate Immunity Protein Interaction Network Regulating Type I Interferon Production

To systematically investigate innate immune signaling networks regulating production of type I interferon, we analyzed protein complexes formed after microbial recognition. Fifty-eight baits were associated with 260 interacting proteins forming a human innate immunity interactome for type I interferon (HI5) of 401 unique interactions; 21% of interactions were modulated by RNA, DNA, or LPS. Overexpression and depletion analyses identified 22 unique genes that regulated NF-κB and ISRE reporter activity, viral replication, or virus-induced interferon production. Detailed mechanistic analysis defined a role for mind bomb (MIB) E3 ligases in K63-linked ubiquitination of TBK1, a kinase that phosphorylates IRF transcription factors controlling interferon production. Mib genes selectively controlled responses to cytosolic RNA. MIB deficiency reduced antiviral activity, establishing the role of MIB proteins as positive regulators of antiviral responses. The HI5 provides a dynamic physical and regulatory network that serves as a resource for mechanistic analysis of innate immune signaling.

Time-resolved FTIR spectroscopy for monitoring protein dynamics exemplified by functional studies of Ras protein bound to a lipid bilayer

Time-resolved Fourier transform infrared (FTIR) difference spectroscopy is a valuable tool for monitoring the dynamics of protein reactions and interactions. Absorbance changes can be monitored with time resolutions down to nanoseconds and followed for time periods that range over nine orders of magnitude. Membrane proteins bound to solid supported lipid bilayers can be investigated in near physiological conditions with the attenuated total reflection (ATR) technique. Here, we review the basics of time-resolved FTIR with a focus on Ras, a GTPase that is mutated in 25% of human tumors. We show the first time-resolved measurements of membrane anchored Ras and observed the switching between its activated and its inactivated state. We compared those measurements with measurements of the truncated Ras in solution. We found that both the kinetics and the functional groups involved were very similar. This suggested that the membrane did not have a major influence on the hydrolysis reaction.

An Integrated Computational Analysis of the Structure, Dynamics, and Ligand Binding Interactions of the Human Galectin Network

Galectins, a family of evolutionarily conserved animal lectins, have been shown to modulate signaling processes leading to inflammation, apoptosis, immunoregulation, and angiogenesis through their ability to interact with poly-N-acetyllactosamine-enriched glycoconjugates. To date 16 human galectin carbohydrate recognition domains have been established by sequence analysis and found to be expressed in several tissues. Given the divergent functions of these lectins, it is of vital importance to understand common and differential features in order to search for specific inhibitors of individual members of the human galectin family. In this work we performed an integrated computational analysis of all individual members of the human galectin family. In the first place, we have built homology-based models for galectin-4 and -12 N-terminus, placental protein 13 (PP13) and PP13-like protein for which no experimental structural information is available. We have then performed classical molecular dynamics simulations of the whole 15 members family in free and ligand-bound states to analyze protein and protein-ligand interaction dynamics. Our results show that all galectins adopt the same fold, and the carbohydrate recognition domains are very similar with structural differences located in specific loops. These differences are reflected in the dynamics characteristics, where mobility differences translate into entropy values which significantly influence their ligand affinity. Thus, ligand selectivity appears to be modulated by subtle differences in the monosaccharide binding sites. Taken together, our results may contribute to the understanding, at a molecular level, of the structural and dynamical determinants that distinguish individual human galectins.

Quantitative analysis of dynamic protein–protein interactions in planta by a floated‐leaf luciferase complementation imaging (FLuCI) assay using binary Gateway vectors

Dynamic protein-protein interactions are essential in all cellular and developmental processes. Protein-fragment complementation assays allow such protein-protein interactions to be investigated in vivo. In contrast to other protein-fragment complementation assays, the split-luciferase (split-LUC) complementation approach facilitates dynamic and quantitative in vivo analysis of protein interactions, as the restoration of luciferase activity upon protein-protein interaction of investigated proteins is reversible. Here, we describe the development of a floated-leaf luciferase complementation imaging (FLuCI) assay that enables rapid and quantitative in vivo analyses of protein interactions in leaf discs floating on a luciferin infiltration solution after transient expression of split-LUC-labelled interacting proteins in Nicotiana benthamiana. We generated a set of eight Gateway-compatible split-LUC destination vectors, enabling fast, and almost fail-safe cloning of candidate proteins to the LUC termini in all possible constellations. We demonstrate their functionality by visualizing the well-established homodimerization of the 14-3-3 regulator proteins. Quantitative interaction analyses of the molybdenum co-factor biosynthesis proteins CNX6 and CNX7 show that the luciferase-based protein-fragment complementation assay allows direct real-time monitoring of absolute values of protein complex assembly. Furthermore, the split-LUC assay is established as valuable tool to investigate the dynamics of protein interactions by monitoring the disassembly of actin filaments in planta. The new Gateway-compatible split-LUC destination vector system, in combination with the FLuCI assay, provides a useful means to facilitate quantitative analyses of interactions between large numbers of proteins constituting interaction networks in plant cells.

Visualization of single mRNAs reveals temporal association of proteins with microRNA-regulated mRNA

Although many proteins are known to function in microRNA (miRNA)-based translational repression, we lack a comprehensive understanding of temporal relationships between the mRNA, miRNA and their constituent proteins. To understand the dynamics of miRNA and protein interactions, we created a synthetic inducible miRNA system in mammalian cells. By visualizing single mRNAs and observing their co-localization with proteins over time, we produced a temporal association map of miRNA-associated factors. Argonaute2, Dcp1a, hedls and Rck co-localize with miRNA-regulated mRNA after 24 h of miRNA induction, and RNAi knockdown of any one of these proteins affected the co-localization of any of the other proteins with miRNA-regulated mRNA, demonstrating that these proteins could interact with each other in a complex. We identified Argonaute2 and hedls as proteins that co-localize and interact with miRNA-regulated mRNA, indicating that processing body components are involved in long-term storage of miRNA-regulated mRNA.

In vivo analysis of the cell cycle dependent association of the bovine papillomavirus E2 protein and ChlR1

It has been shown that the genomes of episomally maintained DNA viruses are tethered to host cell chromosomes during cell division, facilitating maintenance in dividing cells. The papillomavirus E2 protein serves this mechanism of viral genome persistence by simultaneously associating with chromatin and the viral genome during mitosis. Several host cell proteins are reported to be necessary for the association of E2 with chromatin including the cohesion establishment factor ChlR1. Here we use fluorescence resonance energy transfer (FRET) technology to confirm the interaction between BPV-1 E2 and ChlR1. Furthermore, we use synchronised live cells to study the temporal nature of this dynamic protein interaction and show that ChlR1 and E2 interact during specific phases of the cell cycle. These data provide evidence that the association of E2 with ChlR1 contributes to a loading mechanism during DNA replication rather than direct tethering during mitotic division.

Highly Reproducible Label Free Quantitative Proteomic Analysis of RNA Polymerase Complexes

The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports.

Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay

BackgroundProtein interactions control the regulatory networks underlying developmental processes. The understanding of developmental complexity will, therefore, require the characterization of protein interactions within their proper environment. The bimolecular fluorescence complementation (BiFC) technology offers this possibility as it enables the direct visualization of protein interactions in living cells. However, its potential has rarely been applied in embryos of animal model organisms and was only performed under transient protein expression levels.ResultsUsing a Hox protein partnership as a test case, we investigated the suitability of BiFC for the study of protein interactions in the living Drosophila embryo. Importantly, all BiFC parameters were established with constructs that were stably expressed under the control of endogenous promoters. Under these physiological conditions, we showed that BiFC is specific and sensitive enough to analyse dynamic protein interactions. We next used BiFC in a candidate interaction screen, which led to the identification of several Hox protein partners.ConclusionOur results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development.

Relating dynamic protein interactions of metallochaperones with metal transfer at the single-molecule level

Metallochaperones undertake specific interactions with their target proteins to deliver metal ions inside cells. Understanding how these protein interactions are coupled with the underlying metal transfer process is important, but challenging because they are weak and dynamic. Here we use a nanovesicle trapping scheme to enable single-molecule FRET measurements of the weak, dynamic interactions between the copper chaperone Hahl and the fourth metal binding domain (MBD4) of WDP. By monitoring the behaviors of single interacting pairs, we visualize their interactions in real time in both the absence and the presence of various equivalents of Cu(1+). Regardless of the proteins' metallation state, we observe multiple, interconverting interaction complexes between Hah1 and MBD4. Within our experimental limit, the overall interaction geometries of these complexes appear invariable, but their stabilities are dependent on the proteins' metallation state. In apo-holo Hah1-MBD4 interactions, the complexes are stabilized relative to that observed in the apo-apo interactions. This stabilization is indiscernible when Hah1's Cu(1+)-binding is eliminated or when both proteins have Cu(1+) loaded. The nature of this Cu(1+)-induced complex stabilization and of the interaction complexes are discussed. These Cu(1+)-induced effects on the Hah1-MBD4 interactions provide a step toward understanding how the dynamic protein interactions of copper chaperones are coupled with their metal transfer function.

The role of Hepatitis C Virus in the dynamic protein interaction networks of hepatocellular cirrhosis and carcinoma

As an important risk factor for Hepatocellular Carcinoma (HCC), Hepatitis C Virus (HCV) infection can induce cirrhosis and HCC. But, the molecular mechanisms of HCV-induced transformation remain largely unknown. In this study, first, we identified the dysfunctional protein interaction networks in cirrhosis and HCC based on the gene expression profiles of 19 normal, 58 cirrhotic and 47 HCC liver tissues. Then, the relationship between dysfunctional networks and HCV infection was analysed. Our results may help understand the mechanisms of HCV-induced malignant transformation and provide clues to cirrhosis and HCC therapy or prevention.

The Three Rs of Transcription: Recruit, Retain, and Recycle

The dynamic protein interactions required for transcription are functionally important yet poorly understood; in this issue of Molecular Cell, Zobeck etal. (2010) resolve the sequential recruitment and selective recycling of transcription factors at an actively transcribing locus in Drosophila.

Modulation of M4 muscarinic acetylcholine receptors by interacting proteins

Protein-protein interactions represent an important mechanism for posttranslational modifications of protein expression and function. In brain cells, surface-expressed and membrane-bound neurotransmitter receptors are common proteins that undergo dynamic protein-protein interactions between their intracellular domains and submembranous regulatory proteins. Recently, the Gα(i/o)-coupled muscarinic M4 receptor (M4R) has been revealed to be one of these receptors. Through direct interaction with the intracellular loops or C-terminal tails of M4Rs, M4R interacting proteins (M4RIPs) vigorously regulate the efficacy of M4R signaling. A synapse-enriched protein kinase, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), exemplifies a prototype model of M4RIPs, and is capable of binding to the second intracellular loop of M4Rs. Through an activity- and phosphorylation-dependent mechanism, CaMKII potentiates the M4R/Gα(i/o)-mediated inhibition of M4R efficacy in inhibiting adenylyl cyclase and cAMP production. In striatal neurons where M4Rs are most abundantly expressed, M4RIPs dynamically control M4R activity to maintain a proper cholinergic tone in these neurons. This is critical for maintaining the acetylcholine-dopamine balance in the basal ganglia, which determines the behavioral responsiveness to dopamine stimulation by psychostimulants.

Dynamic interaction of formin proteins and cytoskeleton in mouse oocytes during meiotic maturation

Formin-2 (Fmn2) nucleates actin filaments required for spindle migration during the metaphase of meiosis I in mouse oocytes. While recent studies showed that Fmn2 is involved in the formation of a dynamic actin meshwork on meiotic spindle and the migration of chromosomes, the precise location and the mechanism of action of Fmn2 in the mouse oocyte is not known. In this work, we show that Fmn2 is colocalized with spindle during metaphase I (MI) and this pattern is lost in nocodazole-treated oocytes. Fmn2 directly interacts with polymerized microtubules (MTs) in vitro via a well-conserved domain called formin homology 2 (FH2). Microinjection of mRNA encoding formin homology 1 (FH1)FH2 domains of Fmn2 into Fmn2-/- oocytes partially rescued the defect of polar body extrusion, while mRNAs encoding FH2 domain alone could not rescue the defect. mDia1 and mDia2, Diaphanous (Dia) subfamily of formin proteins, exhibit unique patterns of expression in mouse oocytes. While mDia1 is localized on meiotic spindle, mDia2 localization is confined in spindle poles similar to γ-tubulin. Collectively, our results suggest that the ability of Fmn2 to directly interact with MTs and to polymerize actins via the conserved FH1FH2 domains is crucial for chromosomal migration in MI oocytes. We also show that mDia1 and mDia2 are dynamic components of meiotic spindle and pole complex during meiotic maturation of oocytes.

DockAnalyse: an application for the analysis of protein-protein interactions

BackgroundIs it possible to identify what the best solution of a docking program is? The usual answer to this question is the highest score solution, but interactions between proteins are dynamic processes, and many times the interaction regions are wide enough to permit protein-protein interactions with different orientations and/or interaction energies. In some cases, as in a multimeric protein complex, several interaction regions are possible among the monomers. These dynamic processes involve interactions with surface displacements between the proteins to finally achieve the functional configuration of the protein complex. Consequently, there is not a static and single solution for the interaction between proteins, but there are several important configurations that also have to be analyzed.ResultsTo extract those representative solutions from the docking output datafile, we have developed an unsupervised and automatic clustering application, named DockAnalyse. This application is based on the already existing DBscan clustering method, which searches for continuities among the clusters generated by the docking output data representation. The DBscan clustering method is very robust and, moreover, solves some of the inconsistency problems of the classical clustering methods like, for example, the treatment of outliers and the dependence of the previously defined number of clusters.ConclusionsDockAnalyse makes the interpretation of the docking solutions through graphical and visual representations easier by guiding the user to find the representative solutions. We have applied our new approach to analyze several protein interactions and model the dynamic protein interaction behavior of a protein complex. DockAnalyse might also be used to describe interaction regions between proteins and, therefore, guide future flexible dockings. The application (implemented in the R package) is accessible.

Use of Fluorescence Microscopy to Study Intracellular Signaling in Bacteria

Following the introduction of fluorescent protein tags, the application of fluorescence microscopy in microbial cell biology has advanced the field dramatically. We now understand that bacterial cells are not simple bags of enzymes but have complex internal structures, and that specific intracellular organization plays an important role in a number of processes, including signal transduction. The quantitative nature and high temporal resolution of fluorescence microscopy make it particularly useful for studies of intracellular dynamic systems, such as signaling networks. Applications of fluorescence microscopy in signaling are not limited to studying localization. Several techniques allow researchers to follow real-time dynamics of protein interactions, at steady state or upon stimulation, and therefore to investigate signal propagation, amplification, and integration in the cell. Moreover, microscopy enables investigation of single-cell gene expression kinetics, bringing such concepts as cell individuality and robustness against stochasticity of gene expression to the forefront of signaling studies.