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Inhibitors of apoptosis proteins (IAPs) suppress cell death by inactivating proapoptotic regulators, and therefore play important roles in controlling apoptosis in normal and malignant cells. Many IAPs are ubiquitin ligases, and their activity is mediated via ubiquitination and subsequent degradation of their targets. Here we corroborate a previous observation that DIAP1 ( Drosophila IAP1) can be degraded via a two-step mechanism: (i) limited caspase-mediated cleavage and (ii) degradation of the released fragment via the ubiquitin N-end rule pathway. Yet, we demonstrate that this pathway is not the only one involved in DIAP1 degradation, and the intact protein can be degraded independent of prior caspase cleavage. Importantly, this mode of degradation does not require the RING-finger-mediated autoubiquitinating activity of DIAP1, believed to target many RING-finger E3s for self-destruction. Our preliminary data suggest that DIAP2 mediates DIAP1 degradation, suggesting a novel regulatory loop within the apoptotic pathway. Studying the role of the autoubiquitinating activity of DIAP1, we demonstrate that it does not involve formation of Lys48-based polyubiquitin chains, but probably chains linked via Lys63. Our preliminary data suggest that the autoubiquitination serves to attenuate the ligase activity of DIAP1 towards its exogenous substrates.

Active caspases execute apoptosis to eliminate superfluous or harmful cells in animals. In Drosophila , living cells prevent uncontrolled caspase activation through an inhibitor of apoptosis protein (IAP) family member, dIAP1, and apoptosis is preceded by the expression of IAP-antagonists, such as Reaper, Hid and Grim. Strong genetic modifiers of this pathway include another IAP family gene encoding an E2 ubiquitin conjugating enzyme domain, dBruce . Although the genetic effects of dBruce mutants are well documented, molecular targets of its encoded protein have remained elusive. Here, we report that dBruce targets Reaper for ubiquitination through an unconventional mechanism. Specifically, we show that dBruce physically interacts with Reaper, dependent upon Reaper's IAP-binding (IBM) and GH3 motifs. Consistently, Reaper levels were elevated in a dBruce −/− background. Unexpectedly, we found that dBruce also affects the levels of a mutant form of Reaper without any internal lysine residues, which normally serve as conventional ubiquitin acceptor sites. Furthermore, we were able to biochemically detect ubiquitin conjugation on lysine-deficient Reaper proteins, and knockdown of dBruce significantly reduced the extent of this ubiquitination. Our results indicate that dBruce inhibits apoptosis by promoting IAP-antagonist ubiquitination on unconventional acceptor sites.

Irradiated or injured cells enter apoptosis, and in turn, promote proliferation of surrounding unaffected cells. In Drosophila , apoptotic cells have an active role in proliferation, where the caspase Dronc and p53 induce mitogen expression and growth in the surrounding tissues. The Drosophila p53 gene structure is conserved and encodes at least two protein isoforms: a full-length isoform (Dp53) and an N-terminally truncated isoform (DΔNp53). Historically, DΔNp53 was the first p53 isoform identified and was thought to be responsible for all p53 biological activities. It was shown that DΔNp53 induces apoptosis by inducing the expression of IAP antagonists, such as Reaper. Here we investigated the roles of Dp53 and DΔNp53 in apoptosis and apoptosis-induced proliferation. We found that both isoforms were capable of activating apoptosis, but that they each induced distinct IAP antagonists. Expression of DΔNp53 induced Wingless (Wg) expression and enhanced proliferation in both ‘undead cells’ and in ‘genuine’ apoptotic cells. In contrast to DΔNp53, Dp53 did not induce Wg expression in the absence of the endogenous p53 gene. Thus, we propose that DΔNp53 is the main isoform that regulates apoptosis-induced proliferation. Understanding the roles of Drosophila p53 isoforms in apoptosis and in apoptosis-induced proliferation may shed new light on the roles of p53 isoforms in humans, with important implications in cancer biology.

Cell death in higher organisms is negatively regulated by Inhibitor of Apoptosis Proteins (IAPs), which contain a ubiquitin ligase motif, but how ubiquitin-mediated protein degradation is regulated during apoptosis is poorly understood. Here, we report that Drosophila melanogaster IAP1 (DIAP1) auto-ubiquitination and degradation is actively regulated by Reaper (Rpr) and UBCD1. We show that Rpr, but not Hid (head involution defective), promotes significant DIAP1 degradation. Rpr-mediated DIAP1 degradation requires an intact DIAP1 RING domain. Among the mutations affecting ubiquitination, we found ubcD1 , which suppresses rpr -induced apoptosis. UBCD1 and Rpr specifically bind to DIAP1 and stimulate DIAP1 auto-ubiquitination in vitro . Our results identify a novel function of Rpr in stimulating DIAP1 auto-ubiquitination through UBCD1, thereby promoting its degradation.

During the development of multicellular organisms, gene expression must be tightly regulated, both spatially and temporally. One set of transcription factors that are important in animal development is encoded by the homeotic (Hox) genes, which govern the choice between alternative developmental pathways along the anterior-posterior axis. Hox proteins, such as Drosophila Ultrabithorax, have low DNA-binding specificity by themselves but gain affinity and specificity when they bind together with the homeoprotein Extradenticle (or Pbx1 in mammals). To understand the structural basis of Hox-Extradenticle pairing, we determine here the crystal structure of an Ultrabithorax-Extradenticle-DNA complex at 2.4 angstrom resolution, using the minimal polypeptides that form a cooperative heterodimer. The Ultrabithorax and Extradenticle homeodomains bind opposite faces of the DNA, with their DNA-recognition helices almost touching each other. However, most of the cooperative interactions arise from the YPWM amino-acid motif of Ultrabithorax--located amino-terminally to its homeodomain--which forms a reverse turn and inserts into a hydrophobic proteins on the Extradenticle homeodomain surface. Together, these protein-DNA and protein-protein interactions define the general principles by which homeotic proteins interact with Extradenticle (or Pbx1) to affect development along the anterior-posterior axis of animals.

During development, tissue growth is controlled by coordinated cellular growth and apoptosis. A series of recent papers describes a newly identified Drosophila gene, hippo , that restricts excessive growth during development by both limiting cell cycle progression and promoting susceptibility to apoptosis. Therefore, hippo may coordinate the regulation of cell division and cell death.

The fungal pellets were formed from the surface thermodynamic balance between fungal cell and liquid media. The Gibbs free energy of pellet formation of the initial culture media (-73 -81 ergs/cm2) were increased to -13 -46 ergs/cm2 at 48 h. FTIR analysis showed that factors inducing pellet formation simultaneously increased the cell wall hydrophobicity of Aspergillus niger.[PUBLICATION ABSTRACT]

Dual blockade of PD-L1 and VEGF has enhanced anticancer immunity through multiple mechanisms and augmented antitumour activity in multiple malignancies. We aimed to assess the efficacy and safety of atezolizumab (anti-PD-L1) alone and combined with bevacizumab (anti-VEGF) in patients with unresectable hepatocellular carcinoma. GO30140 is an open-label, multicentre, multiarm, phase 1b study that enrolled patients at 26 academic centres and community oncology practices in seven countries worldwide. The study included five cohorts, and the two hepatocellular carcinoma cohorts, groups A and F, are described here. Inclusion criteria for these two groups included age 18 years and older; histologically, cytologically, or clinically (per American Association for the Study of Liver Diseases criteria) confirmed unresectable hepatocellular carcinoma that was not amenable to curative treatment; no previous systemic treatment; and Eastern Cooperative Oncology Group performance status of 0 or 1. In group A, all patients received atezolizumab (1200 mg) and bevacizumab (15 mg/kg) intravenously every 3 weeks. In group F, patients were randomly assigned (1:1) to receive intravenous atezolizumab (1200 mg) plus intravenous bevacizumab (15 mg/kg) every 3 weeks or atezolizumab alone by interactive voice-web response system using permuted block randomisation (block size of two) and stratification factors of geographical region; macrovascular invasion, extrahepatic spread, or both; and baseline α-fetoprotein concentration. Primary endpoints were confirmed objective response rate in all patients who received the combination treatment for group A and progression-free survival in the intention-to-treat population in group F, both assessed by an independent review facility according to Response Evaluation Criteria in Solid Tumors version 1.1. In both groups, safety was assessed in all patients who received at least one dose of any study treatment. This study is registered with ClinicalTrials.gov, NCT02715531, and is closed to enrolment. In group A, 104 patients were enrolled between July 20, 2016, and July 31, 2018, and received atezolizumab plus bevacizumab. With a median follow-up of 12·4 months (IQR 8·0–16·2), 37 (36%; 95% CI 26–46) of 104 patients had a confirmed objective response. The most common grade 3–4 treatment-related adverse events were hypertension (13 [13%]) and proteinuria (seven [7%]). Treatment-related serious adverse events occurred in 25 (24%) patients and treatment-related deaths in three (3%) patients (abnormal hepatic function, hepatic cirrhosis, and pneumonitis). In group F, 119 patients were enrolled and randomly assigned (60 to atezolizumab plus bevacizumab; 59 to atezolizumab monotherapy) between May 18, 2018, and March 7, 2019. With a median follow-up of 6·6 months (IQR 5·5–8·5) for the atezolizumab plus bevacizumab group and 6·7 months (4·2–8·2) for the atezolizumab monotherapy group, median progression-free survival was 5·6 months (95% CI 3·6–7·4) versus 3·4 months (1·9–5·2; hazard ratio 0·55; 80% CI 0·40–0·74; p=0·011). The most common grade 3–4 treatment-related adverse events in group F were hypertension (in three [5%] patients in the atezolizumab plus bevacizumab group; none in the atezolizumab monotherapy group) and proteinuria (in two [3%] patients in the atezolizumab plus bevacizumab group; none in the atezolizumab monotherapy group). Treatment-related serious adverse events occurred in seven (12%) patients in the atezolizumab plus bevacizumab group and two (3%) patients in the atezolizumab monotherapy group. There were no treatment-related deaths. Our study shows longer progression-free survival with a combination of atezolizumab plus bevacizumab than with atezolizumab alone in patients with unresectable hepatocellular carcinoma not previously treated with systemic therapy. Therefore, atezolizumab plus bevacizumab might become a promising treatment option for these patients. This combination is being compared with standard-of-care sorafenib in a phase 3 trial. F Hoffmann-La Roche/Genentech.

The prognosis of patients with advanced biliary tract cancer who have progressed on gemcitabine plus cisplatin is dismal. We aimed to investigate the efficacy and safety of second-line liposomal irinotecan plus fluorouracil and leucovorin in patients with metastatic biliary tract cancer that has progressed on gemcitabine plus cisplatin. This multicentre, open-label, randomised, phase 2b (NIFTY) study was done at five academic institutions in South Korea and included patients aged 19 years or older with histologically or cytologically confirmed metastatic biliary tract cancer that had progressed on first-line gemcitabine plus cisplatin and an Eastern Cooperative Oncology Group performance status of 0 or 1. By use of an interactive web-based response system integrated with an electronic data capture system, patients were randomly assigned (1:1) using permuted blocks (block size 4) to receive either intravenous liposomal irinotecan (70 mg/m2 for 90 min) plus intravenous leucovorin (400 mg/m2 for 30 min) and intravenous fluorouracil (2400 mg/m2 for 46 h) every 2 weeks or leucovorin and fluorouracil only every 2 weeks, and were stratified by primary tumour site, previous surgery with curative intent, and participating centre. Study treatment was continued until the patient had disease progression or unacceptable toxicities, or withdrew consent. The primary endpoint was blinded independent central review (BICR)-assessed progression-free survival. The primary endpoint and safety were assessed in the full analysis set and the safety analysis set, respectively, both of which comprised all randomly assigned patients who received at least one dose of the study treatment. This trial is registered with ClinicalTrials.gov, NCT03524508, and enrolment is complete. Between Sept 5, 2018, and Feb 18, 2020, 193 patients were screened for eligibility, of whom 174 (88 in the liposomal irinotecan plus fluorouracil and leucovorin group and 86 in the fluorouracil plus leucovorin group) were enrolled and included in the full analysis and safety analysis sets. At a median follow-up of 11·8 months (IQR 7·7–18·7), the median BICR-assessed progression-free survival was significantly longer in the liposomal irinotecan plus fluorouracil and leucovorin group (7·1 months, 95% CI 3·6–8·8) than in the fluorouracil and leucovorin group (1·4 months, 1·2–1·5; hazard ratio 0·56, 95% CI 0·39–0·81; p=0·0019). The most common grade 3–4 adverse events were neutropenia (21 [24%] of 88 in the liposomal irinotecan plus fluorouracil and leucovorin group vs one [1%] of 86 in the fluorouracil and leucovorin group) and fatigue or asthenia (11 [13%] vs three [3%]). Serious adverse events occurred in 37 (42%) patients receiving liposomal irinotecan plus fluorouracil and leucovorin and 21 (24%) patients receiving fluorouracil and leucovorin. There were no treatment-related deaths. Adding liposomal irinotecan to fluorouracil and leucovorin significantly improved BICR-assessed progression-free survival in patients with advanced biliary tract cancer. Liposomal irinotecan plus fluorouracil and leucovorin could be considered a standard-of-care second-line therapy for advanced biliary tract cancer. Servier and HK inno.N For the Korean translation of the abstract see Supplementary Materials section.