Inactivation of Effector Caspases through Nondegradative Polyubiquitylation

Ubiquitin-mediated inactivation of caspases has long been postulated to contribute to the regulation of apoptosis. However, detailed mechanisms and functional consequences of caspase ubiquitylation have not been demonstrated. Here we show that the Drosophila Inhibitor of Apoptosis 1, DIAP1, blocks effector caspases by targeting them for polyubiquitylation and nonproteasomal inactivation. We demonstrate that the conjugation of ubiquitin to drICE suppresses its catalytic potential in cleaving caspase substrates. Our data suggest that ubiquitin conjugation sterically interferes with substrate entry and reduces the caspase’s proteolytic velocity. Disruption of drICE ubiquitylation, either by mutation of DIAP1’s E3 activity or drICE’s ubiquitin-acceptor lysines, abrogates DIAP1’s ability to neutralize drICE and suppress apoptosis in vivo. We also show that DIAP1 rests in an “inactive” conformation that requires caspase-mediated cleavage to subsequently ubiquitylate caspases. Taken together, our findings demonstrate that effector caspases regulate their own inhibition through a negative feedback mechanism involving DIAP1 “activation” and nondegradative polyubiquitylation

Drosophila IAP1-Mediated Ubiquitylation Controls Activation of the Initiator Caspase DRONC Independent of Protein Degradation

Ubiquitylation targets proteins for proteasome-mediated degradation and plays important roles in many biological processes including apoptosis. However, non-proteolytic functions of ubiquitylation are also known. In Drosophila, the inhibitor of apoptosis protein 1 (DIAP1) is known to ubiquitylate the initiator caspase DRONC in vitro. Because DRONC protein accumulates in diap1 mutant cells that are kept alive by caspase inhibition (“undead” cells), it is thought that DIAP1-mediated ubiquitylation causes proteasomal degradation of DRONC, protecting cells from apoptosis. However, contrary to this model, we show here that DIAP1-mediated ubiquitylation does not trigger proteasomal degradation of full-length DRONC, but serves a non-proteolytic function. Our data suggest that DIAP1-mediated ubiquitylation blocks processing and activation of DRONC. Interestingly, while full-length DRONC is not subject to DIAP1-induced degradation, once it is processed and activated it has reduced protein stability. Finally, we show that DRONC protein accumulates in “undead” cells due to increased transcription of dronc in these cells. These data refine current models of caspase regulation by IAPs

Evolutionary Loss of Activity in De-Ubiquitylating Enzymes of the OTU Family.

Understanding function and specificity of de-ubiquitylating enzymes (DUBs) is a major goal of current research, since DUBs are key regulators of ubiquitylation events and have been shown to be mutated in human diseases. Most DUBs are cysteine proteases, relying on a catalytic triad of cysteine, histidine and aspartate to cleave the isopeptide bond between two ubiquitin units in a poly-ubiquitin chain. We have discovered that the two Drosophila melanogaster homologues of human OTUD4, CG3251 and Otu, contain a serine instead of a cysteine in the catalytic OTU (ovarian tumor) domain. DUBs that are serine proteases instead of cysteine- or metallo-proteases have not been described. In line with this, neither CG3251 nor Otu protein were active to cleave ubiquitin chains. Re-introduction of a cysteine in the catalytic center did not render the enzymes active, indicating that further critical features for ubiquitin binding or cleavage have been lost in these proteins. Sequence analysis of OTUD4 homologues from various other species showed that within this OTU subfamily, loss of the catalytic cysteine has occurred frequently in presumably independent events, as well as gene duplications or triplications, suggesting DUB-independent functions of OTUD4 proteins. Using an in vivo RNAi approach, we show that CG3251 might function in the regulation of Inhibitor of Apoptosis (IAP)-antagonist-induced apoptosis, presumably in a DUB-independent manner

Knock-down of <i>CG3251</i> disturbs IAP-antagonist induced apoptosis in the <i>Drosophila</i> eye.

<p>An intact wild type <i>Drosophila</i> eye is shown in <b>(A)</b>. Rpr and Hid as well as RNAi constructs were expressed using the UAS-<i>Gal4</i> system with the <i>glass multimer reporter</i> (<i>GMR</i>)-<i>Gal4</i> driver, which drives expression mainly in the developing eye and leads to a small, rough eye phenotype due to excessive apoptosis (<b>B,E</b>). Knock-down of <i>CG3251</i> suppressed Hid-induced cell death in the eye (<b>C,D</b>) and enhanced Rpr-induced cell death (<b>F,G</b>). Genotypes are: (<b>B</b>) <i>GMR</i>-<i>hid</i>, <i>GMR</i>-<i>Gal4</i>/<i>cyo</i> (<b>C</b>) <i>GMR</i>-<i>hid</i>, <i>GMR</i>-<i>Gal4</i>/<i>CG3251-RNAi-a</i> (<b>D</b>) <i>GMR</i>-<i>hid</i>, <i>GMR</i>-<i>Gal4</i>/<i>CG3251-RNAi-b</i> (<b>E</b>) <i>GMR</i>-<i>rpr</i>, <i>GMR</i>-<i>Gal4</i>/<i>TM6b</i>,<i>Tb</i><b>(F)</b><i>CG3251-RNAi-a</i>/<i>+; GMR</i>-<i>rpr</i>, <i>GMR</i>-<i>Gal4</i>/<i>+</i><b>(G)</b><i>CG3251-RNAi-b</i>/<i>+; GMR</i>-<i>rpr</i>, <i>GMR</i>-<i>Gal4</i>/<i>+</i>.</p

Re-mutation of S40 to cysteine does not restore catalytic activity of CG3251 and Otu protein.

<p><b>(A)</b> To test whether mutation of S40 to cysteine restored activity, wild type and S40C-mutated CG3251 protein from <i>E</i>.<i>coli</i> was incubated with di-ubiquitin molecules of all linkage types and potential ubiquitin cleavage assessed by WB. Cleavage activity was not observed. Input levels of GST-CG3251 or -CG3251 S40C are shown by anti-GST WB. Full-length GST-CG3251 is indicated by arrow, while also multiple truncated forms are present in the protein sample. <b>(B)</b> Otu and Otu S40C are inactive DUBs. An NH₂-terminal fragment encoding the OTU domain of the Otu protein (aa 1–149) was expressed in <i>E</i>. <i>coli</i> as GST-fusion protein. Wild type and an S40C-mutated form were used. Cleavage of K48- or K63-linked ubiquitin chains was determined by anti-ubiquitin WB and the input of recombinant proteins was controlled by anti-GST WB.</p

Occurrence of Cys-to-Ser mutations in proteins of the CG3251/OTUD4 family during <i>Drosophila</i> evolution.

<p>To compare the occurrence of Cys-to-Ser mutations in the catalytic triad of the OTU domain, protein sequences of proteins of the OTUD4 family were analysed as described in the main text. Shown is the most likely order of gene amplifications and independent Cys-to-Ser mutations in relation to <i>Drosophila</i> evolution.</p

(A) Schematic domain structure of the <i>Drosophila</i> proteins CG3251 and Otu. CG3251 is a 495 amino acid (aa) protein, with an OTU domain (position 29–150) and a Tudor domain (“TUD”, 296–361). The residues of the predicted catalytic triad are D37, S40 and H143. The Otu protein carries an OTU domain with identical features and also a Tudor domain (336–396). The full-length protein consists of 853 amino acids (not drawn to scale). The presence of Ser instead of Cys in the catalytic triad (S40) is highlighted by red colour. (B) Alignment of protein sequence of OTU domains of OTUD4 (human), aa 34–155, CG3251 (Q9VR20), aa 29–150 and Otu, aa 29–150. Fully conserved residues are marked by “*”, “:” denotes strongly similar residues and “.” weakly similar residues, according to Clustal Omega analysis [15]. The catalytic triad is highlighted in yellow and conserved areas required for catalytic activity are denoted as Cys-, His- and V(ariable)-loop, respectively.

<p>(A) Schematic domain structure of the <i>Drosophila</i> proteins CG3251 and Otu. CG3251 is a 495 amino acid (aa) protein, with an OTU domain (position 29–150) and a Tudor domain (“TUD”, 296–361). The residues of the predicted catalytic triad are D37, S40 and H143. The Otu protein carries an OTU domain with identical features and also a Tudor domain (336–396). The full-length protein consists of 853 amino acids (not drawn to scale). The presence of Ser instead of Cys in the catalytic triad (S40) is highlighted by red colour. (B) Alignment of protein sequence of OTU domains of OTUD4 (human), aa 34–155, CG3251 (Q9VR20), aa 29–150 and Otu, aa 29–150. Fully conserved residues are marked by “*”, “:” denotes strongly similar residues and “.” weakly similar residues, according to Clustal Omega analysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143227#pone.0143227.ref015" target="_blank">15</a>]. The catalytic triad is highlighted in yellow and conserved areas required for catalytic activity are denoted as Cys-, His- and V(ariable)-loop, respectively.</p

Ohgata, the Single Drosophila Ortholog of Human Cereblon, Regulates Insulin Signaling-dependent Organismic Growth

Cereblon (CRBN) is a substrate receptor of the E3 ubiquitin ligase complex that is highly conserved in animals and plants. CRBN proteins have been implicated in various biological processes such as development, metabolism, learning, and memory formation, and their impairment has been linked to autosomal recessive non-syndromic intellectual disability and cancer. Furthermore, human CRBN was identified as the primary target of thalidomide teratogenicity. Data on functional analysis of CRBN family members in vivo, however, are still scarce. Here we identify Ohgata (OHGT), the Drosophila ortholog of CRBN, as a regulator of insulin signaling-mediated growth. Using ohgt mutants that we generated by targeted mutagenesis, we show that its loss results in increased body weight and organ size without changes of the body proportions. We demonstrate that ohgt knockdown in the fat body, an organ analogous to mammalian liver and adipose tissue, phenocopies the growth phenotypes. We further show that overgrowth is due to an elevation of insulin signaling in ohgt mutants and to the down-regulation of inhibitory cofactors of circulating Drosophila insulin-like peptides (DILPs), named acid-labile subunit and imaginal morphogenesis protein-late 2. The two inhibitory proteins were previously shown to be components of a heterotrimeric complex with growth-promoting DILP2 and DILP5. Our study reveals OHGT as a novel regulator of insulin-dependent organismic growth in Drosophila