Endogenous Protein-protein Interactions Research Articles

Detection of Protein-Protein Interactions and Posttranslational Modifications Using the Proximity Ligation Assay: Application to the Study of the SUMO Pathway.

The detection of protein-protein interactions by imaging techniques often requires the overexpression of the proteins of interest tagged with fluorescent molecules, which can affect their biological properties and, subsequently, flaw experiment interpretations. The recent development of the proximity ligation assays (PLA) technology allows easy visualization of endogenous protein-protein interactions at the single molecule level. PLA relies on the use of combinations of antibodies coupled to complementary oligonucleotides that are amplified and revealed with a fluorescent probe, each spot representing a single protein-protein interaction. Another application of this technique is the detection of proteins posttranslational modifications to monitor their localization and dynamics in situ. Here, we describe the use of PLA to detect protein SUMOylation, a posttranslational modification related to ubiquitination, as well as interaction of SUMOylated substrates with other proteins, using both adherent and suspension cells.

A Pipeline for Determining Protein–Protein Interactions and Proximities in the Cellular Milieu

It remains extraordinarily challenging to elucidate endogenous protein-protein interactions and proximities within the cellular milieu. The dynamic nature and the large range of affinities of these interactions augment the difficulty of this undertaking. Among the most useful tools for extracting such information are those based on affinity capture of target bait proteins in combination with mass spectrometric readout of the co-isolated species. Although highly enabling, the utility of affinity-based methods is generally limited by difficulties in distinguishing specific from nonspecific interactors, preserving and isolating all unique interactions including those that are weak, transient, or rapidly exchanging, and differentiating proximal interactions from those that are more distal. Here, we have devised and optimized a set of methods to address these challenges. The resulting pipeline involves flash-freezing cells in liquid nitrogen to preserve the cellular environment at the moment of freezing; cryomilling to fracture the frozen cells into intact micron chunks to allow for rapid access of a chemical reagent and to stabilize the intact endogenous subcellular assemblies and interactors upon thawing; and utilizing the high reactivity of glutaraldehyde to achieve sufficiently rapid stabilization at low temperatures to preserve native cellular interactions. In the course of this work, we determined that relatively low molar ratios of glutaraldehyde to reactive amines within the cellular milieu were sufficient to preserve even labile and transient interactions. This mild treatment enables efficient and rapid affinity capture of the protein assemblies of interest under nondenaturing conditions, followed by bottom-up MS to identify and quantify the protein constituents. For convenience, we have termed this approach Stabilized Affinity Capture Mass Spectrometry. Here, we demonstrate that Stabilized Affinity Capture Mass Spectrometry allows us to stabilize and elucidate local, distant, and transient protein interactions within complex cellular milieux, many of which are not observed in the absence of chemical stabilization.

Using an in situ proximity ligation assay to systematically profile endogenous protein-protein interactions in a pathway network.

Signal transduction pathways in the cell require protein-protein interactions (PPIs) to respond to environmental cues. Diverse experimental techniques for detecting PPIs have been developed. However, the huge amount of PPI data accumulated from various sources poses a challenge with respect to data reliability. Herein, we collected ∼ 700 primary antibodies and employed a highly sensitive and specific technique, an in situ proximity ligation assay, to investigate 1204 endogenous PPIs in HeLa cells, and 557 PPIs of them tested positive. To overview the tested PPIs, we mapped them into 13 PPI public databases, which showed 72% of them were annotated in the Human Protein Reference Database (HPRD) and 8 PPIs were new PPIs not in the PubMed database. Moreover, TP53, CTNNB1, AKT1, CDKN1A, and CASP3 were the top 5 proteins prioritized by topology analyses of the 557 PPI network. Integration of the PPI-pathway interaction revealed that 90 PPIs were cross-talk PPIs linking 17 signaling pathways based on Reactome annotations. The top 2 connected cross-talk PPIs are MAPK3-DAPK1 and FAS-PRKCA interactions, which link 9 and 8 pathways, respectively. In summary, we established an open resource for biological modules and signaling pathway profiles, providing a foundation for comprehensive analysis of the human interactome.

Probing for protein-protein interactions during cell migration: limitations and challenges.

Cellular migration is a fundamental biological process occurring as early as embryogenesis to the pathological state of cancer metastasis. Nearly all cellular migrations involve an extracellular signal that is transduced internally by members of a signalling cascade. These signal transduction events are driven by protein-protein interactions (PPIs) that coordinate intracellular activities to enable a cell to migrate. Understanding these PPIs will provide valuable insight into how cellular migration can be modulated perhaps towards a therapeutic end. Histologically, not many techniques are available to demonstrate PPIs. Contrasting agents only demonstrate the presence of a particular protein, and perhaps its co-localisation with another protein. Yet, co-localisation need not necessarily indicate physical interaction between the two proteins. In this review, we highlight four commonly used methods that continue to expand our understanding of PPIs underlying cell migration: co-immunoprecipitation, bimolecular fluorescence complementation, proximity ligation assay and surface plasmon resonance. The methods discussed herein allow for the study of PPIs in a wide variety of biological samples, including cell lysates, live cells, fixed cells and tissues, enabling the quantification of endogenous PPIs and exploration of the nature of PPIs. We also include a rudimentary framework for researchers to decide which experimental method best suits their research goals.

PPIRank - an advanced method for ranking protein-protein interations in TAP/MS data

BackgroundTandem affinity purification coupled with mass-spectrometry (TAP/MS) analysis is a popular method for the identification of novel endogenous protein-protein interactions (PPIs) in large-scale. Computational analysis of TAP/MS data is a critical step, particularly for high-throughput datasets, yet it remains challenging due to the noisy nature of TAP/MS data.ResultsWe investigated several major TAP/MS data analysis methods for identifying PPIs, and developed an advanced method, which incorporates an improved statistical method to filter out false positives from the negative controls. Our method is named PPIRank that stands for PPI ranking in TAP/MS data. We compared PPIRank with several other existing methods in analyzing two pathway-specific TAP/MS PPI datasets from Drosophila.ConclusionExperimental results show that PPIRank is more capable than other approaches in terms of identifying known interactions collected in the BioGRID PPI database. Specifically, PPIRank is able to capture more true interactions and simultaneously less false positives in both Insulin and Hippo pathways of Drosophila Melanogaster.

Analysis of Protein-Protein Interactions in Cross-talk Pathways Reveals CRKL Protein as a Novel Prognostic Marker in Hepatocellular Carcinoma

Deciphering the network of signaling pathways in cancer via protein-protein interactions (PPIs) at the cellular level is a promising approach but remains incomplete. We used an in situ proximity ligation assay to identify and quantify 67 endogenous PPIs among 21 interlinked pathways in two hepatocellular carcinoma (HCC) cells, Huh7 (minimally migratory cells) and Mahlavu (highly migratory cells). We then applied a differential network biology analysis and determined that the novel interaction, CRKL-FLT1, has a high centrality ranking, and the expression of this interaction is strongly correlated with the migratory ability of HCC and other cancer cell lines. Knockdown of CRKL and FLT1 in HCC cells leads to a decrease in cell migration via ERK signaling and the epithelial-mesenchymal transition process. Our immunohistochemical analysis shows high expression levels of the CRKL and CRKL-FLT1 pair that strongly correlate with reduced disease-free and overall survival in HCC patient samples, and a multivariate analysis further established CRKL and the CRKL-FLT1 as novel prognosis markers. This study demonstrated that functional exploration of a disease network with interlinked pathways via PPIs can be used to discover novel biomarkers.

Abstract 5169: Flow-proteometric analysis of single signaling complex.

Abstract Signal transductions are critical for cell physiology functions and signaling complexes play major roles in carrying and delivering “signals. These processes occur mainly through protein-protein interaction (PPI) and protein-nucleic acid interaction (PNI). Conventional approaches are usually limited in identifying a specific cellular PPI, or PNI from large amount of a specific protein with limited relative quantification. However, multiple PPIs and PNIs actually occur within a single signaling complex in vivo in a dynamic manner and an effective methodology for accurately detecting these individual complexes for multiple targets are critical for further understanding the in depth mechanisms of cell signaling transduction. Herein we introduce a novel flow-proteometric technique named microchannel for Multi-parameters Analyzing Proteins in Single-complex (mMAPS), which integrates a microfluidics device into single-molecule spectroscopy with three different emission-wavelength detectors to directly detect and analyze each individual signaling complex. Briefly, proteins and/or nucleic acid were labeled by three separate wavelength fluorophores in individual complexes and each complex was examined when it flew through the detection spot in microchannel. Using this approach, we demonstrate various applications by analyzing multiple endogenous PPIs and PNIs in p53, EGFR, HER2, and NFκB complexes from cancer cell lines with the resolution of “single” signaling complex. For instance, using 50,000 cancer cells (HeLa) we have detected and quantified the interaction between three proteins including EGF, EGFR and STAT3. We identified the single protein complexes which contain all three targets (EGF/EGFR/STAT3) after EGF treatment for 30min. EGF/EGFR and EGFR/STAT3 were also been identified as well but we didn't identified EGF/STAT3 interaction. Interestingly, although 20% of detected EGFR were interacted with STAT3 after EGF stimulation, only 3% of EGFR found to interact with EGF in the EGFR complex. In addition, we have also detected the protein complexes containing both proteins and nucleic acid including endogenous p53, TBP1, and genomic DNA fragment and successfully identified p53, TBP1, and genomic DNA in a complex. Quantification results revealed that p53 and TBP1 interaction only occurred in the complexes with genomic DNA. In conclusion, mMAPS provides a powerful platform for direct investigating the composition of individual target protein complexes and reveals the actual quantitative results of specific PTMs, PPIs, and PNIs involved in the signaling complexes, which is important to dissect the detailed dynamics of signal transduction in vivo. Citation Format: Chao-Kai Chou, Heng-Huan Lee, Pei-Hsiang Tsou, Hirohito Yamaguchi, Ying-Nai Wang, Hong-Jen Lee, Jun Kameoka, Mien-Chie Hung. Flow-proteometric analysis of single signaling complex. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5169. doi:10.1158/1538-7445.AM2013-5169

The Mitochondrial Proteins NLRX1 and TUFM Form a Complex that Regulates Type I Interferon and Autophagy

The mitochondrial protein MAVS (also known as IPS-1, VISA, and CARDIF) interacts with RIG-I-like receptors (RLRs) to induce type I interferon (IFN-I). NLRX1 is a mitochondrial nucleotide-binding, leucine-rich repeats (NLR)-containing protein that attenuates MAVS-RLR signaling. Using Nlrx1(-/-) cells, we confirmed that NLRX1 attenuated IFN-I production, but additionally promoted autophagy during viral infection. This dual function of NLRX1 paralleled the previously described functions of the autophagy-related proteins Atg5-Atg12, but NLRX1 did not associate with Atg5-Atg12. High-throughput quantitative mass spectrometry and endogenous protein-protein interaction revealed an NLRX1-interacting partner, mitochondrial Tu translation elongation factor (TUFM). TUFM interacted with Atg5-Atg12 and Atg16L1 and has similar functions as NLRX1 by inhibiting RLR-induced IFN-I but promoting autophagy. In the absence of NLRX1, increased IFN-I and decreased autophagy provide an advantage for host defense against vesicular stomatitis virus. This study establishes a link between an NLR protein and the viral-induced autophagic machinery via an intermediary partner, TUFM.

Abstract 1976: Systematic identification of protein-protein interactions by Proximity Ligation Assay

Abstract Signal transduction pathways at cellular level require massive protein-protein interactions (PPIs) in response to different environmental cues. Diverse experimental techniques for detecting PPIs have been developed; however, the large amount of data accumulated from various sources has posed a grand challenge in data reliability for PPI. To evaluate the role of PPIs within signaling pathways, we have systematic profiled PPI residing in 28 KEGG pathways, for example mTOR, MAPK, Wnt, VEGF, and Hedgehog, and 3 cancer-related pathways stored in KEGG (http://www.genome.jp/kegg/). Of ∼60000 human PPI collected, 2786 PPI can be detected within targeted 14 pathways. To validate these PPIs, we used a newly developed Proximity Ligation Assay, which detects endogenous PPI through a pair of antibodies that bind to proteins in close proximity. Each PPI can be made visible through DNA amplification and quantified by counting fluorescent dots in the cell. Of 1,204 PPIs examined, 557 PPIs can be confirmed in HeLa cells. Specifically, 383 out of 557 validated PPIs residing within pathways in cancer and mTOR, Wnt and MAPK pathways are considered novel “links” based on annotations of KEGG. Moreover, 79 validated PPIs are cross-talk events among 35 signaling transduction and cancer related pathways. Surprisingly, only 16 PPIs (out of 557 confirmed PPIs) are highly correlated in 7 sets of cancer cells from NCI60 microarray dataset. In this large scale PPI profiling, 41 apoptosis related PPIs were further tested in 3 cancer cell lines to explore the possibility for drug screening. These systematic analyses of endogenous PPI pairs within and between pathways may set the foundation to disrupt the signaling pathways by targeting PPIs in cancer drug discovery. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1976. doi:10.1158/1538-7445.AM2011-1976

Abstract 4929: Dissection of protein-protein interaction-mediated cross-talk pathways in hepatocellular carcinoma

Abstract Multiple dys-regulated cell signaling pathways and extensive cross-talks, which can be depicted via protein-protein interactions (PPIs), among individual signaling pathways, characterize cancer development and progression. However, systematic analysis of the cross-talk events from distinct pathways via PPIs remains fragmentary, and the reliability of PPI datasets, particularly at the cellular level, remains to be vigorously examined. Here, we first build the cross-talk pathway map of heptocellular carcinoma (HCC) through overexpression patterns in HCC among 23 pathways. By detecting endogenous PPIs in two HCC cell lines, Huh7 (low invasive cells) and Mahlavu (high invasive cells), we validate 50 PPI events within pathway and 19 PPI-mediated cross-talk events among 23 pathways in HCC. Not surprisingly, many distinct PPIs are present between Huh7 and Mahlavu, providing the opportunity to characterize tumor invasion. By analyzing topology of cross-talk PPI network, CRKL has a pivotal role in cross-talk pathway. This initial attempt to map the cross-talk pathways in HCC may represent the first step in deciphering hepatocarcinogenesis at the systems level and might reveal a novel strategy for HCC treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4929. doi:10.1158/1538-7445.AM2011-4929

Precise measurement of protein interacting fractions with fluorescence lifetime imaging microscopy

Precise quantification of endogenous protein-protein interactions across live cells would be a major boon to biology. Such precise measurement is theoretically possible with fluorescence lifetime imaging microscopy (FLIM) but requires first properly addressing multiple biological, instrumental, statistical, and photophysical challenges. We present a detailed investigation of the last three FLIM-specific challenges. Using an efficient, highly accurate analysis code for time-domain FLIM data that accounts for all significant instrumental artifacts (in part, through use of a parametrized model for the instrument response function) and is rigorously based on both conventional statistics (full lifetime histogram fitting by χ(2) minimization) and novel statistics (single pixel fitting of lifetime populations using "maximum fidelity"), we address multiple photophysical challenges, including the proper side-by-side statistical comparison of fluorophore monoexponentiality, the precise assessment of fluorophore lifetimes and lifetime photostability, and the determination of acceptor dark state fractions. Finally, we demonstrate the feasibility of precise measurement of the interacting fraction of a protein across live cells with FLIM.

A truncated IFN-regulatory factor-8\IFN consensus sequence-binding protein acts as dominant-negative, interferes with endogenous protein-protein interactions and leads to apoptosis of immune cells.

IFN consensus sequence-binding protein (ICSBP) is a member of the IFN-regulatory factors (IRF) and is thus also called IRF-8. Its expression is restricted to hematopoietic cells and IRF-8\ICSBP(-/-) mice are defective in myeloid cell differentiation. This factor exerts its transcriptional activity through interaction with other transcription factors, which leads to either repression or activation. In this paper, we describe the use of a dominant-negative (DN) mutant of IRF-8\ICSBP designed to serve as a molecular tool to dissociate the role of the various protein-protein interactions. This DN-ICSBP is truncated at the DNA-binding domain and can still associate with other factors, but the heterocomplexes produced are incapable of binding to the DNA. We show that the DN-ICSBP is able to compete for the interaction of IRF-8\ICSBP with either IRF or non-IRF members such as PU.1. Accordingly, this DN construct was able to inhibit the PU.1-dependent expression of the IgLlambda in the plasmacytoma cell line J558L. However, stable expression of this DN-ICSBP led to apoptosis of only hematopoietic cells. The data suggests that DN-ICSBP can form heterocomplexes with an as-yet unidentified survival factor for hematopoietic cells.