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 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 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 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

<i>ck</i>/MyoVIIA function is modulated by Sqh and DMBS in adult JO.

<p>A: Genetic interactions of <i>ck</i>/MyoVIIA with actin pathways in the <i>Drosophila</i> wing (top panel) and in embryonic dorsal closure (bottom panel). Boxed area indicates the portion of the pathway investigated in panel B. Based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002115#pone.0002115-Winter1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002115#pone.0002115-Mizuno2" target="_blank">[36]</a>. B: <i>ck</i>/MyoVIIA genetically interacts with Sqh and DMBS in adult JO. Responses from flies with only one functional copy of <i>Drok</i>, <i>sqh</i>, <i>zip</i>/MyoII or <i>DMBS</i> were normalized to their respective, two-copy sibling controls, all in a sensitized <i>ck</i>/MyoVIIA background. Histograms show mean +/− SD.</p

<i>ck</i>/MyoVIIA is necessary for JO organization during development.

<p>A: Comparison of control (<i>ck¹³/CyO</i>; white labels) vs. mutant (<i>ck¹³/ck¹³</i>; yellow labels) developing JO. Green channel: GFP-NompA labelling dendritic caps. Red channel: Texas red-phalloidin labelling actin filaments. Block arrows: developing scolopale rods. Open block arrows: direction of a2/a3 boundary. Box indicates disorganization of cap alignment in JO. Inset in 14 hrs control: in some cases the cap shows an elongated profile by this time. Inset in 14 hrs mutant antenna: magnification of the globular profile of the cap. Scale bar = 20 µm. B: Similar staining and labelling as “A”. Note dendritic caps juxtaposed with the perimeter of a2 (dotted line) or extending into the space between a2/a3 in controls (arrow; white labels). In mutants (yellow labels) the caps remain distanced from the future a2/a3 joint.</p

Schematic of the antenna and JO.

<p>Drawing not to scale.</p

Scolopidia lacking <i>ck</i>/MyoVIIA are not attached to the a2/a3 joint during development.

<p>A: Wild type (<i>ck¹³/CyO</i>; white labels) and mutant (<i>ck¹³/ck¹³</i>; yellow labels) cultured antennal discs labelled as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002115#pone-0002115-g002" target="_blank">Figure 2</a> support our finding that <i>ck</i>/MyoVIIA is necessary for JO organization. B: Representative EM micrographs from wild type (26 hrs APF) and mutant (28 hrs APF) antennae. Black arrowheads: dendritic caps extending beyond the a2/a3 joint boundary in controls. White arrowheads: dendritic caps at the apical levels of the scolopale space. In <i>ck</i> mutants the cap is more compact than in controls. Inset: magnification of the boxed area. C: Wild type (<i>ck¹³/CyO</i>; white labels) and mutant (<i>ck¹³/ck¹³</i>; yellow labels) cultured antennal discs labelled with anti-cadherin antibody. Green channel: GFP-NompA. Red channel: anti-cadherin. Arrows: cellular junction “tracts” in mutant discs. Open block arrows: a2/a3 boundary. Inset: enlargement of the boxed area. Arrowhead: a dendritic cap extending beyond the a2/a3 boundary. Block arrows: cell-cell junctions.</p

hHR23A influences cleavage of polyUb chains by the Josephin domain.

<p><b>A)</b> Full length ataxin-3 (ataxin-3(Q22); 100 nM) was incubated with K63-Ub5 (250 nM) chains and either full length GST-tagged hHR23A (100–250 nM) or its GST-tagged UBL domain (100–250 nM). Fractions were collected at the indicated times. <b>B</b>) Isolated Josephin domain (ataxin-3(1-182); 100 nM) was incubated with K48-Ub5 chains (250 nM) and either GST-tagged hHR23A (100–250 nM) or its GST-tagged UBL domain (100–250 nM). Samples were collected at the indicated times. Block arrows highlight differences in cleavage activity among samples. <b>C</b>) Full length ataxin-3 (ataxin-3Q22(WT); 100 nM) or the isolated Josephin domain (ataxin-3(1-182); 100 nM) were incubated with mixed-linkage polyUb chains (K48-K63-K18-Ub4; 250 nM) and either full-length GST-hHR23A (100–250 nM) or its GST-tagged UBL domain (100–250 nM). Block arrows highlight differences in cleavage among samples.</p

Modelling of diUb complexes with Josephin.

<p><b>A</b>) Structural superposition of representative models of the Josephin complexes with K48- (left) and K63-linked (right) diUb. The structures are superposed on Josephin backbone atoms to enhance the similarities/differences of the Ub relative positions. The C-terminus (residue G76) of the Ub in site 1 is indicated by spheres. The side chains of the catalytic triad are shown in cyan. The side chains of the cross-linking lysines are also shown explicitly. <b>B</b>) Comparison of the Josephin/K48-linked diUb model and the known structures of polyUb chains in isolation and in a complex. Top line: the structures of diUb from Ub₂ (1aar in cyan, 2bgf in red) and from Ub₄ chains (2o6v in green, 1tbe in magenta). Bottom line: the structure diUb from a different crystal form (1f9j in purple); the structure of the UBA domain in complex with Ub₂ (1zo6 in gold) and the structure of the Josephin complex (2jri, in blue). Residues L8, I44 and V71, which are often involved in Ub interfaces, are indicated by green spheres to provide a direct comparison of the relative orientations of the two subunits. With the sole exception of 1f9j, which was suggested to provide evidence of an open-to-closed equilibrium, the Ub subunits are in a closed conformation when not in a complex. In the two complexes, the conformation is open with the linkers differently stretched to adapt to the binding sites.</p