USP25 inhibits degradation of the ERAD substrate CD3δ

<p>. A) Western blots of whole cell lysates. Top: HEK-293 cells were transfected as indicated and treated with the proteasome inhibitor MG132 where noted (15 µM, 6 hours) before harvesting. Bottom: semi-quantification of bands from western blots shown above and other similar, independent experiments. CD3δ protein levels were normalized to loading control. Shown are means +/− standard deviations. USP25(WT): common isoform of USP25; USP25(m): muscle-specific isoform of USP25. P values from Student T-tests are shown below histograms. B) Top: HEK-293 cells were transfected with the indicated constructs and harvested 48 hours later. Shown are western blots of whole cell lysates probed with the indicated antibodies. WT: wild type USP25, C178S: the catalytic cysteine of USP25 was replaced by a serine residue <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Denuc1" target="_blank">[18]</a>, ΔUBA: UBA deleted, ΔUIM: both UIMs deleted. Bottom: semi-quantification of data from the top and two other independent experiments. CD3δ protein levels were normalized to loading control. Shown are means +/− standard deviations. P values from Student T-tests are shown below histograms. C) Top: HEK-293 cells were transfected as indicated. 48 hours post-transfection cells were treated for the indicated periods of time with 75 µg/ml cycloheximide to inhibit synthesis of new protein. Bottom: semi-quantification of western blots from the top and three other, independent experiments. CD3δ levels were normalized to loading control. Shown are means +/− standard deviations. P values are from Student T-tests of USP25 compared to vector control. D and E) HEK-293 cells were transfected with the indicated constructs. 48 hours later tagged constructs were immunopurified with bead-bound antibodies and probed as indicated.</p

USP25 and HRD1 have opposing effects on CD3

<p>δ <b>protein levels and ubiquitination.</b> A) HEK-293 cells were transfected as indicated and harvested 48 hours later. Western blots are from whole cell lysates. HRD1(WT): normal HRD1; HRD1(CA): catalytically inactive HRD1, in which the catalytic cysteine is substituted by an alanine residue <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Kikkert1" target="_blank">[7]</a>. Histograms on the right: semi-quantification of data from the left and other independent experiments. Shown are means +/− standard deviations. CD3δ levels were normalized to loading control. P values from Student T tests are shown below histograms. B and C) HEK-293 cells were transfected with the indicated constructs. 48 hours post transfection, cells were treated for 6 hours with MG132 (15 µM) and HA-CD3δ was immunopurified using bead-bound anti-HA antibody after a stringent denature/renature step (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#s4" target="_blank">Materials and Methods</a> for details). Histograms: semi-quantification of bracketed ubiquitin smears from the experiment on the left and other similar, independent experiments. Shown are means +/− standard deviations. P values for panel C are from Student T-tests. </p

USP25 regulates protein levels of the ERAD substrates APP and CFTRΔF508.

<p>A) Left: whole cell lysates of HEK-293 cells transfected with the indicated constructs. USP25 (WT) and USP25(m) are both catalytically active isoforms. Where noted, cells were treated with the proteasome inhibitor MG132 (15 µM, 6 hrs) before harvesting. Right: histograms show semi-quantification of APP signal from the left portion and other similar, independent experiments. Bracket: APP bands were quantified separately, added and normalized to loading control. Shown are means +/− standard deviations. P values from Student T-tests are shown above histograms. No statistically significant differences were observed when cells were treated with MG132. B) Left: whole cells lysates of HEK-293 cells transfected as indicated and treated 48 hours later with cycloheximide to inhibit translation of new protein. Right: semi-quantification of western blots from the right and two other independent experiments. Shown are means +/− standard deviations. APP levels were normalized to loading control. P values are from Student T-tests where APP levels in the presence of USP25(WT) were compared to APP levels in presence of vector control. C) Left: HEK-293 cells were transfected with shRNA constructs targeting different portions of endogenous USP25 (RNAi-1, 2) or scramble RNA (RNAscr-1, 2). Cells were harvested 48 hours post-transfection and probed as indicated in western blots. Trials with 72 hour-long transfections yielded similar results (not shown). Right: semi-quantification of signal from the left and other similar, independent experiments. Bracket: APP bands were quantified separately, added together and normalized to loading control. Asterisks: P<0.01 according to Student T-tests comparing RNAi-1 and RNAi-2 lanes to RNAi-scr lanes. D) HEK-293 cells were transfected with the indicated constructs and Myc-USP25 was co-immunoprecipitated 48 hours later. E and F) HEK-293 cells were transfected with the indicated constructs. Western blots of whole cell lysates. For panels D, E and F: similar results were obtained from COS-7 cells (not shown).</p

USP25 interacts with ERAD components.

<p>A) Schematics depict known domains of common (USP25(WT)) and muscle-specific (USP25(m)) isoforms of USP25 that are expressed in mammals <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Denuc1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Meulmeester1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Valero1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Valero2" target="_blank">[42]</a>. B) HEK-293 cells were transfected with HA-USP25. 48 hours later cells were fixed, probed as indicated and imaged with laser confocal microscopy. Panels IA-IC are single optical plane images (1 µM) of a cell immunolabeled for ER (KDEL, endogenous marker), HA-USP25 and nucleus (DAPI). Panel IC is the merged view of panels IA (green channel), IB (red channel) and DAPI (blue channel; not shown as a separate channel). Panels II and III are merged views of other cells stained similarly to panel I. Scale bars: 10 µM. C–G) HEK-293 cells were transfected as shown. Indicated constructs were immunopurified with bead-bound antibodies. Similar results were obtained from COS-7 cells for panels B–E (not shown). All USP25 constructs used in this figure were the common isoform (USP25(WT)).</p

Systematic Analysis of the Physiological Importance of Deubiquitinating Enzymes

<div><p>Deubiquitinating enzymes (DUBs) are proteases that control the post-translational modification of proteins by ubiquitin and in turn regulate diverse cellular pathways. Despite a growing understanding of DUB biology at the structural and molecular level, little is known about the physiological importance of most DUBs. Here, we systematically identify DUBs encoded by the genome of <em>Drosophila melanogaster</em> and examine their physiological importance <em>in vivo</em>. Through domain analyses we uncovered 41 <em>Drosophila</em> DUBs, most of which have human orthologues. Systematic knockdown of the vast majority of DUBs throughout the fly or in specific cell types had dramatic consequences for <em>Drosophila</em> development, adult motility or longevity. Specific DUB subclasses proved to be particularly necessary during development, while others were important in adults. Several DUBs were indispensable in neurons or glial cells during developmental stages; knockdown of others perturbed the homeostasis of ubiquitinated proteins in adult flies, or had adverse effects on wing positioning as a result of neuronal requirements. We demonstrate the physiological significance of the DUB family of enzymes in intact animals, find that there is little functional redundancy among members of this family of proteases, and provide insight for future investigations to understand DUB biology at the molecular, cellular and organismal levels.</p> </div

Differences in distribution and levels of ubiquitinated species in whole fly lysates.

<p>Shown are western blots of whole, newly-eclosed adult flies homogenized in SDS lysis buffer and electrophoresed in SDS-PAGE gels. Experimental groups were heterozygous for sqh-Gal4 and UAS-RNAi. Controls were heterozygous for sqh-Gal4 on the isogenic background of RNAi lines. Boxes highlight some areas with visible differences in ubiquitinated species. Western blots are representative of at least three independent repeats with similar results. Underlined: ubiquitous knockdown led to phenotype (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043112#pone-0043112-g003" target="_blank">Figure 3</a>).</p

Phenotypic distribution by developmental stage.

<p>Histograms show the number of DUBs whose knockdown led to phenotype at each stage. A) sqh-Gal4 (ubiquitous) driver; details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043112#pone-0043112-g003" target="_blank">Figure 3</a>. B) strong elav-Gal4 (pan-neuronal) driver; details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043112#pone-0043112-g005" target="_blank">Figure 5</a>. In cases where two different RNAi lines targeting the same DUB led to phenotype in different stages, only the earlier stage was counted. Instances where knockdown of a specific DUB by one RNAi line led to defects in two stages were counted for each stage (e.g. knockdown of CG3416 led to death in both larval and pupal stages).</p

Gene dosage effects with pan-neuronal knockdown.

<p>List of phenotypes observed when individual fly DUBs were knocked down by pan-neuronal drivers with different expression strengths.</p

Comprehensive list of fly DUBs and their physiological significance.

<p>Listed are all the fly DUBs that we identified, highlighting previously reported functions and our current findings. Not tested: DUBs that we did not examine either because of lack of reagents, too many non-specific targets from existing RNAi lines, or because the function of these DUBs is well characterized in flies. Empty cells: as of this publication, no information had been previously reported for these DUBs.</p

List of phenotypes when individual DUBs are knocked down throughout the fly.

<p>Listed are the strongest phenotypes associated with ubiquitous knockdown of individual fly DUBs. Diamonds highlight DUBs whose knockdown led to observable phenotypes. Experimental groups consisted of flies heterozygous for sqh-Gal4 and UAS-RNAi. Controls were heterozygous for sqh-Gal4 on the isogenic background of RNAi lines. “Dead soon after eclosion” category denotes flies that eclosed successfully from the pupal case but fell on food and died within a few hours. Late motility phenotype means it was first observed 20 or more days after eclosing from the pupal case.</p