Ubiquitination directly enhances activity of the deubiquitinating enzyme ataxin‐3

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102210/1/emboj2008289-sup-0001.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/102210/2/emboj2008289.pd

Ubiquitin-Specific Protease 25 Functions in Endoplasmic Reticulum-Associated Degradation

Endoplasmic Reticulum (ER)-associated degradation (ERAD) discards abnormal proteins synthesized in the ER. Through coordinated actions of ERAD components, misfolded/anomalous proteins are recognized, ubiquitinated, extracted from the ER and ultimately delivered to the proteasome for degradation. It is not well understood how ubiquitination of ERAD substrates is regulated. Here, we present evidence that the deubiquitinating enzyme Ubiquitin-Specific Protease 25 (USP25) is involved in ERAD. Our data support a model where USP25 counteracts ubiquitination of ERAD substrates by the ubiquitin ligase HRD1, rescuing them from degradation by the proteasome

Maternal Antibody Transmission in Relation to Mother Fluctuating Asymmetry in a Long-Lived Colonial Seabird: The Yellow-Legged Gull Larus michahellis

Female birds transfer antibodies to their offspring via the egg yolk, thus possibly providing passive immunity against infectious diseases to which hatchlings may be exposed, thereby affecting their fitness. It is nonetheless unclear whether the amount of maternal antibodies transmitted into egg yolks varies with female quality and egg laying order. In this paper, we investigated the transfer of maternal antibodies against type A influenza viruses (anti-AIV antibodies) by a long-lived colonial seabird, the yellow-legged gull (Larus michahellis), in relation to fluctuating asymmetry in females, i.e. the random deviation from perfect symmetry in bilaterally symmetric morphological and anatomical traits. In particular, we tested whether females with greater asymmetry transmitted fewer antibodies to their eggs, and whether within-clutch variation in yolk antibodies varied according to the maternal level of fluctuating asymmetry. We found that asymmetric females were in worse physical condition, produced fewer antibodies, and transmitted lower amounts of antibodies to their eggs. We also found that, within a given clutch, yolk antibody level decreased with egg laying order, but this laying order effect was more pronounced in clutches laid by the more asymmetric females. Overall, our results support the hypothesis that maternal quality interacts with egg laying order in determining the amount of maternal antibodies transmitted to the yolks. They also highlight the usefulness of fluctuating asymmetry as a sensitive indicator of female quality and immunocompetence in birds

Isoleucine 44 Hydrophobic Patch Controls Toxicity of Unanchored, Linear Ubiquitin Chains through NF-κB Signaling

Ubiquitination is a post-translational modification that regulates cellular processes by altering the interactions of proteins to which ubiquitin, a small protein adduct, is conjugated. Ubiquitination yields various products, including mono- and poly-ubiquitinated substrates, as well as unanchored poly-ubiquitin chains whose accumulation is considered toxic. We previously showed that transgenic, unanchored poly-ubiquitin is not problematic in Drosophila melanogaster. In the fruit fly, free chains exist in various lengths and topologies and are degraded by the proteasome; they are also conjugated onto other proteins as one unit, eliminating them from the free ubiquitin chain pool. Here, to further explore the notion of unanchored chain toxicity, we examined when free poly-ubiquitin might become problematic. We found that unanchored chains can be highly toxic if they resemble linear poly-ubiquitin that cannot be modified into other topologies. These species upregulate NF-κB signaling, and modulation of the levels of NF-κB components reduces toxicity. In additional studies, we show that toxicity from untethered, linear chains is regulated by isoleucine 44, which anchors a key interaction site for ubiquitin. We conclude that free ubiquitin chains can be toxic, but only in uncommon circumstances, such as when the ability of cells to modify and regulate them is markedly restricted

Evaluating Phenotypic and Transcriptomic Responses Induced by Low-Level VOCs in Zebrafish: Benzene as an Example

Urban environments are plagued by complex mixtures of anthropogenic volatile organic compounds (VOCs), such as mixtures of benzene, toluene, ethylene, and xylene (BTEX). Sources of BTEX that drive human exposure include vehicle exhaust, industrial emissions, off-gassing of building material, as well as oil spillage and leakage. Among the BTEX mixture, benzene is the most volatile compound and has been linked to numerous adverse health outcomes. However, few studies have focused on the effects of low-level benzene on exposure during early development, which is a susceptible window when hematological, immune, metabolic, and detoxification systems are immature. In this study, we used zebrafish to conduct a VOC exposure model and evaluated phenotypic and transcriptomic responses following 0.1 and 1 ppm benzene exposure during the first five days of embryogenesis (n = 740 per treatment). The benzene body burden was 2 mg/kg in 1 ppm-exposed larval zebrafish pools and under the detection limit in 0.1 ppm-exposed fish. No observable phenotypic changes were found in both larvae except for significant skeletal deformities in 0.1 ppm-exposed fish (p = 0.01) compared with unexposed fish. Based on transcriptomic responses, 1 ppm benzene dysregulated genes that were implicated with the development of hematological system, and the regulation of oxidative stress response, fatty acid metabolism, immune system, and inflammatory response, including apob, nfkbiaa, serpinf1, foxa1, cyp2k6, and cyp2n13 from the cytochrome P450 gene family. Key genes including pik3c2b, pltp, and chia.2 were differentially expressed in both 1 and 0.1 ppm exposures. However, fewer transcriptomic changes were induced by 0.1 ppm compared with 1 ppm. Future studies are needed to determine if these transcriptomic responses during embryogenesis have long-term consequences at levels equal to or lower than 1 ppm

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

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 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

<i>Drosophila</i> as a Model of Unconventional Translation in Spinocerebellar Ataxia Type 3

RNA toxicity contributes to diseases caused by anomalous nucleotide repeat expansions. Recent work demonstrated RNA-based toxicity from repeat-associated, non-AUG-initiated translation (RAN translation). RAN translation occurs around long nucleotide repeats that form hairpin loops, allowing for translation initiation in the absence of a start codon that results in potentially toxic, poly-amino acid repeat-containing proteins. Discovered in Spinocerebellar Ataxia Type (SCA) 8, RAN translation has been documented in several repeat-expansion diseases, including in the CAG repeat-dependent polyglutamine (polyQ) disorders. The ATXN3 gene, which causes SCA3, also known as Machado–Joseph Disease (MJD), contains a CAG repeat that is expanded in disease. ATXN3 mRNA possesses features linked to RAN translation. In this paper, we examined the potential contribution of RAN translation to SCA3/MJD in Drosophila by using isogenic lines that contain homomeric or interrupted CAG repeats. We did not observe unconventional translation in fly neurons or glia. However, our investigations indicate differential toxicity from ATXN3 protein-encoding mRNA that contains pure versus interrupted CAG repeats. Additional work suggests that this difference may be due in part to toxicity from homomeric CAG mRNA. We conclude that Drosophila is not suitable to model RAN translation for SCA3/MJD, but offers clues into the potential pathogenesis stemming from CAG repeat-containing mRNA in this disorder