Cell death regulation in Drosophila: Conservation of mechanism and unique insights

Programmed cell death, or apoptosis, is a genetically encoded form of cell suicide that results in the orderly death and phagocytic removal of excess, damaged, or dangerous cells during normal development and in the adult. The cellular machinery required to carry out apoptosis is present in most, if not all cells, but is only activated in cells instructed to die (for review see Jacobson et al. 1997). Here, we review cell death regulation in the fly in the context of a first pass look at the complete Drosophila genome and what is known about death regulation in other organisms, particularly worms and vertebrates

Reaper is regulated by IAP-mediated ubiquitination

In most cases, apoptotic cell death culminates in the activation of the caspase family of cysteine proteases, leading to the orderly dismantling and elimination of the cell. The IAPs (inhibitors of apoptosis) comprise a family of proteins that oppose caspases and thus act to raise the apoptotic threshold. Disruption of IAP-mediated caspase inhibition has been shown to be an important activity for pro-apoptotic proteins in Drosophila (Reaper, HID, and Grim) and in mammalian cells (Smac/DIABLO and Omi/HtrA2). In addition, in the case of the fly, these proteins are able to stimulate the ubiquitination and degradation of IAPs by a mechanism involving the ubiquitin ligase activity of the IAP itself. In this report, we show that the Drosophila RHG proteins (Reaper, HID, and Grim) are themselves substrates for IAP-mediated ubiquitination. This ubiquitination of Reaper requires IAP ubiquitin-ligase activity and a stable interaction between Reaper and the IAP. Additionally, degradation of Reaper can be blocked by mutating its potential ubiquitination sites. Most importantly, we also show that regulation of Reaper by ubiquitination is a significant factor in determining its biological activity. These data demonstrate a novel function for IAPs and suggest that IAPs and Reaper-like proteins mutually control each other's abundance

A pathway of signals regulating effector and initiator caspases in the developing Drosophila eye

Regulated cell death and survival play important roles in neural development. Extracellular signals are presumed to regulate seven apparent caspases to determine the final structure of the nervous system. In the eye, the EGF receptor, Notch, and intact primary pigment and cone cells have been implicated in survival or death signals. An antibody raised against a peptide from human caspase 3 was used to investigate how extracellular signals controlled spatial patterning of cell death. The antibody crossreacted specifically with dying Drosophila cells and labelled the activated effector caspase Drice. It was found that the initiator caspase Dronc and the proapoptotic gene head involution defective were important for activation in vivo. Dronc may play roles in dying cells in addition to activating downstream effector caspases. Epistasis experiments ordered EGF receptor, Notch, and primary pigment and cone cells into a single pathway that affected caspase activity in pupal retina through hid and Inhibitor of Apoptosis Proteins. None of these extracellular signals appeared to act by initiating caspase activation independently of hid. Taken together, these findings indicate that in eye development spatial regulation of cell death and survival is integrated through a single intracellular pathway

The Drosophila Caspase DRONC Cleaves following Glutamate or Aspartate and Is Regulated by DIAP1, HID, and GRIM

The caspase family of cysteine proteases plays important roles in bringing about apoptotic cell death. All caspases studied to date cleave substrates COOH-terminal to an aspartate. Here we show that the Drosophila caspase DRONC cleaves COOH-terminal to glutamate as well as aspartate. DRONC autoprocesses itself following a glutamate residue, but processes a second caspase, drICE, following an aspartate. DRONC prefers tetrapeptide substrates in which aliphatic amino acids are present at the P2 position, and the P1 residue can be either aspartate or glutamate. Expression of a dominant negative form of DRONC blocks cell death induced by the Drosophila cell death activators reaper, hid, and grim, and DRONC overexpression in flies promotes cell death. Furthermore, the Drosophila cell death inhibitor DIAP1 inhibits DRONC activity in yeast, and DIAP1's ability to inhibit DRONC-dependent yeast cell death is suppressed by HID and GRIM. These observations suggest that DRONC acts to promote cell death. However, DRONC activity is not suppressed by the caspase inhibitor and cell death suppressor baculovirus p35. We discuss possible models for DRONC function as a cell death inhibitor

Stratifying the early radiologic trajectory in dyspneic patients with COVID-19 pneumonia

OBJECTIVE: This study aimed to stratify the early pneumonia trajectory on chest radiographs and compare patient characteristics in dyspneic patients with coronavirus disease 2019 (COVID-19). MATERIALS AND METHODS: We retrospectively included 139 COVID-19 patients with dyspnea (87 men, 62.7+/-16.3 years) and serial chest radiographs from January to September 2020. Radiographic pneumonia extent was quantified as a percentage using a previously-developed deep learning algorithm. A group-based trajectory model was used to categorize the pneumonia trajectory after symptom onset during hospitalization. Clinical findings, and outcomes were compared, and Cox regression was performed for survival analysis. RESULTS: Radiographic pneumonia trajectories were categorized into four groups. Group 1 (n = 83, 59.7%) had negligible pneumonia, and group 2 (n = 29, 20.9%) had mild pneumonia. Group 3 (n = 13, 9.4%) and group 4 (n = 14, 10.1%) showed similar considerable pneumonia extents at baseline, but group 3 had decreasing pneumonia extent at 1-2 weeks, while group 4 had increasing pneumonia extent. Intensive care unit admission and mortality were significantly more frequent in groups 3 and 4 than in groups 1 and 2 (P \u3c .05). Groups 3 and 4 shared similar clinical and laboratory findings, but thrombocytopenia ( \u3c 150x103/muL) was exclusively observed in group 4 (P = .016). When compared to groups 1 and 2, group 4 (hazard ratio, 63.3; 95% confidence interval, 7.9-504.9) had a two-fold higher risk for mortality than group 3 (hazard ratio, 31.2; 95% confidence interval, 3.5-280.2), and this elevated risk was maintained after adjusting confounders. CONCLUSION: Monitoring the early radiologic trajectory beyond baseline further prognosticated at-risk COVID-19 patients, who potentially had thrombo-inflammatory responses

The Drosophila Inhibitor of Apoptosis (IAP) DIAP2 Is Dispensable for Cell Survival, Required for the Innate Immune Response to Gram-negative Bacterial Infection, and Can Be Negatively Regulated by the Reaper/Hid/Grim Family of IAP-binding Apoptosis Inducers

Many inhibitor of apoptosis (IAP) family proteins inhibit apoptosis. IAPs contain N-terminal baculovirus IAP repeat domains and a C-terminal RING ubiquitin ligase domain. Drosophila IAP DIAP1 is essential for the survival of many cells, protecting them from apoptosis by inhibiting active caspases. Apoptosis initiates when proteins such as Reaper, Hid, and Grim bind a surface groove in DIAP1 baculovirus IAP repeat domains via an N-terminal IAP-binding motif. This evolutionarily conserved interaction disrupts DIAP1-caspase interactions, unleashing apoptosis-inducing caspase activity. A second Drosophila IAP, DIAP2, also binds Rpr and Hid and inhibits apoptosis in multiple contexts when overexpressed. However, due to a lack of mutants, little is known about the normal functions of DIAP2. We report the generation of diap2 null mutants. These flies are viable and show no defects in developmental or stress-induced apoptosis. Instead, DIAP2 is required for the innate immune response to Gram-negative bacterial infection. DIAP2 promotes cytoplasmic cleavage and nuclear translocation of the NF-{kappa}B homolog Relish, and this requires the DIAP2 RING domain. Increasing the genetic dose of diap2 results in an increased immune response, whereas expression of Rpr or Hid results in down-regulation of DIAP2 protein levels. Together these observations suggest that DIAP2 can regulate immune signaling in a dose-dependent manner, and this can be regulated by IBM-containing proteins. Therefore, diap2 may identify a point of convergence between apoptosis and immune signaling pathways

The Drosophila caspase Ice is important for many apoptotic cell deaths and for spermatid individualization, a nonapoptotic process

Caspase family proteases play important roles in the regulation of apoptotic cell death. Initiator caspases are activated in response to death stimuli, and they transduce and amplify these signals by cleaving and thereby activating effector caspases. In Drosophila, the initiator caspase Nc (previously Dronc) cleaves and activates two short-prodomain caspases, Dcp-1 and Ice (previously Drice), suggesting these as candidate effectors of Nc killing activity. dcp-1-null mutants are healthy and possess few defects in normally occurring cell death. To explore roles for Ice in cell death, we generated and characterized an Ice null mutant. Animals lacking Ice show a number of defects in cell death, including those that occur during embryonic development, as well as during formation of adult eyes, arista and wings. Ice mutants exhibit subtle defects in the destruction of larval tissues, and do not prevent destruction of salivary glands during metamorphosis. Cells from Ice animals are also markedly resistant to several stresses, including X-irradiation and inhibition of protein synthesis. Mutations in Ice also suppress cell death that is induced by expression of Rpr, Wrinkled (previously Hid) and Grim. These observations demonstrate that Ice plays an important non-redundant role as a cell death effector. Finally, we demonstrate that Ice participates in, but is not absolutely required for, the non-apoptotic process of spermatid differentiation

Funding structures for Build-to-Suit developments in Brazil: advantages and risks

Empreendimentos build-to-suit são aqueles em que o locador desenvolve um imóvel sob medida para o locatário, que o ocupará pelo prazo previsto em contrato. Dadas as peculiaridades desse tipo de contrato no contexto do real estate, o objetivo deste artigo é analisar as diferentes origens de recursos (fontes de funding) e a forma como eles são empregados (estruturas de funding) para desenvolver os empreendimentos, e discutir as vantagens e riscos dessas estruturas de funding do ponto de vista do empreendedor, que também é o locador. De forma a desenvolver este estudo e formatar as estruturas de funding apresentadas, parte-se de uma revisão das\ud práticas atuais do mercado imobiliário brasileiro (através de notícias veiculadas\ud na mídia e de prospectos de negócios realizados), da literatura brasileira sobre o tema e do conhecimento gerado no Grupo de Real Estate da Escola Politécnica da USP. De maneira a verificar a validade legal das soluções, é realizada uma checagem com\ud base na legislação brasileira e nas normas da Comissão de Valores Mobiliários.\ud Considera-se fontes de funding aquelas tratadas (1) como equity: capital próprio do empreendedor, capital de parceiros (e sócios) no empreendimento na forma de dinheiro ou imóveis (notadamente, o terreno onde será construído o empreendimento), ou investimento de Fundo de Investimento Imobiliário (FII); e (2) como dívida: financiamento bancário, securitização dos recebíveis de aluguéis com CRI ou debêntures. As estruturas de funding apresentadas serão combinações dessas fontes. A análise evidencia que estruturas com financiamento por securitização e emissão de CRI são as mais adequadas de forma geral para os negócios, assim como o investimento completo por FII para negócios de maior porte e nos quais o FII é proprietário direto do empreendimento. \ud Palavras-chave: real estate, build-to-suit, locação, funding, project financeBuild-to-suit real estate assets are tailor made developments for the tenant purposes, who occupies and operates the property for the duration agreed. Given the peculiarities of these contracts and the specificities of the property, this article aims at analyzing the sources of capital and how these funds are mixed and structured for the developments. The article discusses the risks and benefits of each of these funding\ud structures assuming the role of developer. In order to do this study and establish the funding structures shown, the research starts with a review of the current practices in Brazilian real estate market (based on press releases and prospects of deals), of local research papers, and will use the knowledge created at the Real Estate Research Group at Escola Politécnica at Universidade de São Paulo. Since it’s necessary to validate\ud the solutions proposed, Brazilian laws and Comissão de Valores Mobiliários (CVM) norms\ud are reviewed. Funding sources considered will be treated as (1) equity: developers own funds, partnership (via capital or real state – mainly land – investment), or Fundo de Investimento Imobiliário (Brazilian investment structure comparable to REITs); or as (2) debt: banks traditional credit lines, securitization of receivables with CRI emissions\ud , and debt bond emissions. The funding structures presented are mixes of these sources. The analysis shows that the structures best suited for this purpose are those with debt by securitization with CRI emissions, along with the complete investment by a FII but only with large emissions and having the FII as the sole owner of the real estate. \ud Keywords: real estate, build-to-suit, rent, funding, project financ