Quantitative Proteomics Reveals Extensive Changes in the Ubiquitinome after Perturbation of the Proteasome by Targeted dsRNA-Mediated Subunit Knockdown in <i>Drosophila</i>

The ubiquitin–proteasome system (UPS), a highly regulated mechanism including the active marking of proteins by ubiquitin to be degraded, is critical in regulating proteostasis. Dysfunctioning of the UPS has been implicated in diseases such as cancer and neurodegenerative disorders. Here we investigate the effects of proteasome malfunctioning on global proteome and ubiquitinome dynamics using SILAC proteomics in <i>Drosophila</i> S2 cells. dsRNA-mediated knockdown of specific proteasome target subunits is used to inactivate the proteasome. Upon this perturbation, both the global proteome and the ubiquitinome become modified to a great extent, with the overall impact on the ubiquitinome being the most dramatic. The abundances of ∼10% of all proteins are increased, while the abundances of the far majority of over 14 000 detected diGly peptides are increased, suggesting that the pool of ubiquitinated proteins is highly dynamic. Remarkably, several proteins show heterogeneous ubiquitination dynamics, with different lysine residues on the same protein showing either increased or decreased ubiquitination. This suggests the occurrence of simultaneous and functionally different ubiquitination events. This strategy offers a powerful tool to study the response of the ubiquitinome upon interruption of normal UPS activity by targeted interference and opens up new avenues for the dissection of the mode of action of individual components of the proteasome. Because this is to our knowledge the first comprehensive ubiquitinome screen upon proteasome malfunctioning in a fruit fly cell system, this data set will serve as a valuable repository for the <i>Drosophila</i> community

Semi-quantitative proteomics data management and analysis.

<p>The SILAC-based mass spectrometry data from EMF exposed mammalian cells were analyzed by the MaxQuant/Andromeda software. The resulting peptide/protein identifications and quantifications were uploaded to the PIQMIe proteomics server, which integrated the EMF exposure data with protein information from UniProtKB and made the databases available for user-driven queries and statistical analyses.</p

Experimental setup to study proteome-wide biological responses to non-ionizing EMFs using semi-quantitative mass spectrometry.

<p>Triple-state (triplex) SILAC proteomics with reverse metabolic labeling of human fibroblasts (VH10), human osteosarcomas (U2OS) and mouse embryonic stem cells (IB10) exposed to different EMFs with extremely low (ELF) or radio frequencies (UMTS or WiFi). Cells were cultured in media containing “Light” (Arg-0/Lys-0), “Medium” (Arg-6/Lys-4) and “Heavy” (Arg-10/Lys-8) stable isotopes. Cultures were sham (denoted as L0, M0 and H0) or exposed (denoted as L1, M1 and H1) to EMFs. Two independent LC-MS experiments of mixtures of cell extracts were performed: two sham and one exposed extract in the mixture (L0+M0+H1, indicated as replicate 1), and two exposed extracts with one sham extract in the mixture (L1+M1+H0, indicated as replicate 2). Note that the L and M samples (duplicates) were treated equally in both experiments and could therefore be used as internal controls to quantify the experimental variation due to cell culturing, metabolic labeling and/or preparing the samples for mass spectrometry analysis. Further downstream bioinformatics analyses involved peptide/protein identification and quantitation, and the detection of differentially regulated proteins and perturbed cellular processes or pathways.</p

Quantitative immunoblot analysis of MLH1 expression in the VH10 cell line upon ELF exposure.

<p>(A) Immunoblot of control cell line HEK293T-Lα [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170762#pone.0170762.ref025" target="_blank">25</a>] in which expression of DNA mismatch repair protein MLH1 and its binding partner PMS2 is regulated by doxycycline. The presence (+) and absence (-) of doxycycline is indicated. (B) Immunoblot of extracts from untreated (SHAM) and exposed (ON) VH10 cells labeled with different isotopes (L, M and H) and used for semi-quantitative mass-spectrometry analysis. Proteins were visualized using antibodies against MLH1, its binding partner PMS2, and α-tubulin as loading control. (C) Relative MLH1 expression levels (mean ± SD) in extracts from untreated and exposed cells as determined from 6 immunoblot replicas of the MS samples. (D) Tabulated ratios for relative MLH1 expression levels in differentially labeled VH10 cells as determined by mass spectrometry (MS) and immunoblot (IB) analyses. (E) Immunoblot of VH10 cells, lysed using either urea or SDS, obtained from an independent exposure. (F) Relative MLH1 expression levels (mean ± SD) in extracts from an independent exposure as determined from 9 immunoblot replicas. (G) Tabulated ratios for relative MLH1 expression levels based on immunoblot analysis of extracts from the independent exposure.</p

Summary of (non-redundant) protein identifications/quantifications in human (VH10 and U2OS) and mouse (IB10) cells exposed to EMFs.

<p>Summary of (non-redundant) protein identifications/quantifications in human (VH10 and U2OS) and mouse (IB10) cells exposed to EMFs.</p

Variability of SILAC protein ratios in human U2OS cells upon ELF, UMTS and WiFi exposures.

<p>Median absolute deviation (MAD) is used as a robust measure of variability in SILAC protein ratios. Note that <i>unfiltered</i> refers to an unfiltered SILAC data set containing all quantitated protein groups; <i>(in)consistent</i> refers to a filtered SILAC data set of protein groups with (in)consistent ratios in both reverse labeling experiments, this fold-change filtering procedure is only possible because of the duplex SILAC design; <i>composite</i> refers to a filtered SILAC data set of protein groups with greater 'treated' ratios than 'control' ratios (in total there are four 'treated' ratios from exposed <i>versus</i> sham samples, and two 'control' ratios from exposed <i>versus</i> exposed samples and sham <i>versus</i> sham samples), this (composite) fold-change filtering procedure requires triplex SILAC design. The results for the human VH10 and mouse IB10 cells are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170762#pone.0170762.s005" target="_blank">S5 Fig</a>.</p

Correlation analysis of reciprocal SILAC protein quantifications in three mammalian cell lines exposed to three different EMFs.

<p>(A-C) Three scatterplots based on simulated SILAC protein ratios from reverse labeling experiments—with treated (H1 or L1) <i>versus</i> sham (L0 or H0) samples on both axes—illustrate three possible scenarios in which the scatter depends on the effect of a treatment (e.g. EMF exposure) <i>versus</i> SILAC reverse labeling: (A) the scatter is in the direction of the treatment, as indicated by a positive Pearson's correlation coefficient (<i>r</i>); (B) the scatter does not have an identifiable trend, as indicated by a value of <i>r</i> close to zero; (C) the scatter is in the direction of the SILAC reverse labeling, as indicated by a negative value of <i>r</i>. Each scatterplot is divided into four quadrants (I-IV): proteins with inconsistent SILAC reciprocal ratios are located in the I and III quadrants whereas proteins with consistent up- and down-regulation upon treatment are located in the II and IV quadrants, respectively. (D) The dot plot summarizes quantitative data from human (U2OS and VH10) and mouse (IB10) cell lines exposed to ELF, UMTS and WiFi (the individual scatterplots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170762#pone.0170762.s002" target="_blank">S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170762#pone.0170762.s004" target="_blank">S4</a> Figs); the <i>r</i> estimates including the error bars (95% confidence interval) are based on SILAC protein ratios from the H-M (in blue) and the H-L (in red) reverse labeling experiments. Note that the estimated <i>r</i> values are negative in all EMF exposures and hence correspond to the third scenario illustrated by the scatterplot (C).</p

Summary of mass spectrometry-based protein identifications in human and mouse proteomes exposed to EMFs.

<p>Summary of mass spectrometry-based protein identifications in human and mouse proteomes exposed to EMFs.</p

Differentially regulated proteins detected upon EMFs in human (VH10 and U2OS) and mouse (IB10) cells using an array of statistical methods.

<p>Differentially regulated proteins detected upon EMFs in human (VH10 and U2OS) and mouse (IB10) cells using an array of statistical methods.</p

Two- and three-way set comparisons of protein identifications from the EMF exposure experiments.

<p>Two- and three-way set comparisons of protein identifications from the EMF exposure experiments.</p