Analyzing Protein–Protein Interaction Networks

The advent of the “omics” era in biology research has brought new challenges and requires the development of novel strategies to answer previously intractable questions. Molecular interaction networks provide a framework to visualize cellular processes, but their complexity often makes their interpretation an overwhelming task. The inherently artificial nature of interaction detection methods and the incompleteness of currently available interaction maps call for a careful and well-informed utilization of this valuable data. In this tutorial, we aim to give an overview of the key aspects that any researcher needs to consider when working with molecular interaction data sets and we outline an example for interactome analysis. Using the molecular interaction database IntAct, the software platform Cytoscape, and its plugins BiNGO and clusterMaker, and taking as a starting point a list of proteins identified in a mass spectrometry-based proteomics experiment, we show how to build, visualize, and analyze a protein–protein interaction network

Coverage of HIPPIE and overlap by three technique specific datasets.

<p>Left: proteins. Right: PPIs. Y2H is yeast-two-hybrid, Coprep is anti-bait coimmunoprecipitation and MS is affinity capture mass spectrometry. The protein numbers show that Y2H can focus on many proteins that have not been targeted by the other two techniques. Together the three techniques already cover 80% of all proteins currently considered in HIPPIE (i.e. 80% of all proteins in HIPPIE participate in at least one Y2H, Coprep or MS experiment). However, the overlap in PPIs between these datasets and to the remainder of HIPPIE is much smaller indicating that PPI detection is technique specific. Nevertheless, one can appreciate that similar techniques have a bias towards detecting similar PPIs, here illustrated by the significant overlap between Coprep and MS and by the little overlap of Y2H to the other two techniques.</p

Distribution of HIPPIE confidence scores.

<p>Interactions with scores above 0.73 (black bars) constitute only 25% of all and could be considered high-confidence interactions. According to the design of the scoring function, such score implies that the interaction is supported by multiple evidence.</p

Scores for experiment types.

<p>Scores for experiment types.</p

PPI data sources integrated in HIPPIE.

<p>PPI data sources integrated in HIPPIE.</p

Coverage of HIPPIE by novel datasets.

<p>Coverage of HIPPIE by novel datasets.</p

Importance of Dynamic Interactions for Circadian Rhythmicity.

<p>(A) Systematic RNAi-mediated silencing of circadian clock core and regulatory components. RNAi constructs were lentivirally delivered into U2OS cells harboring a <i>Bmal1</i>-promoter luciferase reporter and oscillation dynamics were monitored for several days (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen-1003398-g003" target="_blank">Figure 3</a>). Circles represent the difference in period (± s.e.m.; n = 3 independent experiments) relative to non-silencing controls (n>10) for two RNAi constructs (if available). Filled circles show additional amplitude and/or damping phenotypes. Cells were classified as arrhythmic (ar) if the fit to a cosine function resulted in a low correlation coefficient (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398.s013" target="_blank">Text S1</a>). Period deviations of more than 2 hours are given (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398.s004" target="_blank">Figure S4A</a>). (B) Systematic overexpression of circadian clock core and regulatory components. Experiments were performed with lentivirally delivered overexpression constructs as described in (A) (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398.s013" target="_blank">Text S1</a>). <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#s2" target="_blank">Results</a> of three independent experiments (± s.e.m.) are given (also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398.s004" target="_blank">Figure S4B</a>). (C) Correlation of circadian phenotype with number of dynamic interactions. The combined phenotypic score from silencing and overexpression experiments is significantly different for components with many dynamic interactions (≥5) compared to those with few (<5) (t-test: ** p<0.01; Mann Whitney test: ** p<0.01).</p

Systematic Interaction Mapping between 46 Circadian Clock Proteins and Associated Components.

<p>(A) Matrix based high-throughput yeast-two-hybrid interaction screen. (B) CLOCK interactors: Mating controls (top left); upon PPI reporter genes are activated (top middle: HIS, URA for growth selection, top right: lacZ for β-galactosidase activity). Bottom: Detected interactions with CLOCK; red lines: interactions previously discovered in yeast (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398.s001" target="_blank">Figure S1</a>). (C) Clock protein interaction matrix. Circles: interactions between two components not differentiating between bait and prey configuration. (D) Validation of new CLOCK and BMAL1 interactions in mammalian cells. HEK293 cells expressing CLOCK- or BMAL1-luciferase fusions were transfected with MYC-tagged components. Luciferase activity in anti-MYC co-immunoprecipitates is presented for one representative result of at least two independent experiments with similar results (for method and input controls also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398.s002" target="_blank">Figure S2</a>). MYC-β-galcactosidase fusions served as negative, MYC-BMAL1 and MYC-CRY1 as positive controls, respectively.</p

Network Neighborhood Contains Clock Modulating Components.

<p>Systematic RNAi-mediated downregulation of network neighborhood genes in dexamethasone-synchronized U2OS cells harboring a <i>Bmal1</i>-promoter luciferase reporter. Shown are altered oscillation dynamics (red dots with corresponding fit lines) for 16 genes achieved by individual RNAi constructs (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398.s009" target="_blank">Table S2</a>). For twelve genes, two RNAi constructs resulted in similar phenotypes, for nine genes only one construct was available in our laboratory library. Black dots with corresponding fit lines are controls representing the mean values of at least 80 irrelevant constructs. Period deviations from controls are shown.</p

Protein Phosphatase 1 Modulates CLOCK/BMAL1 Function.

<p>(A) CLOCK and BMAL1 interactors identified in yeast and their paralogs were co-transfected with CLOCK/BMAL1 and an E-box containing luciferase reporter (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398.s007" target="_blank">Figure S7A</a>). Shown are means ± s.d. of CLOCK/BMAL1 modifiers (n = 3; *** p<0.001, t-test). (B) PPP1CA dose-dependently reduces CLOCK/BMAL1 transactivation (n = 3; means ± SD.). (C) PPP1CA is present in the CLOCK/BMAL1 complex. Murine livers were harvested at indicated times. Dashed lines: longer exposure. (LC: light chain; HC: heavy chain). (D) PPP1CA destabilizes BMAL1 protein. Left: Stability is reported by the change of EGFP to DsRed ratio <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398-Yen1" target="_blank">[30]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398-Yen2" target="_blank">[31]</a>. Right: PPP1CA co-expression with BMAL1, CLOCK or short-lived EGFP fusion proteins in U2OS cells reduces BMAL1 stability (mean ± s.d.; ***p<0.001; t-test; n = 3; (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398.s007" target="_blank">Figure S7B, S7C</a>). (E) Endogenous BMAL1 levels are reduced upon PPP1CA overexpression in U2OS cells. Depicted are two independent experiments. (F) PPP1CA reduces BMAL1 stability. U2OS cells stably expressing PPP1CA or GFP were harvested at the indicated time points after cycloheximide (CHX) application and protein levels were analyzed by Western blot. Shown is one representative of two independently performed experiments (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003398#pgen.1003398.s007" target="_blank">Figure S7D</a>).</p