A biochemical analysis of the activation of the Drosophila caspase DRONC

Dorstyn, Loretta; Kumar, Sharad

doi:10.1038/sj.cdd.4402288

Cited by 53 publications

(55 citation statements)

References 41 publications

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“…Caspase-1, however, apparently differs from these two initiator caspases as cleavage of the enzyme leads to large increases in enzyme activity and dimerization. Caspase-9 and DRONC activation have been shown to be enhanced much more by oligomerization than by proteolysis (39,40,46,47). An interpretation of our combined structural, kinetic, and biochemical data can help explain what is different about caspase-1 and why both dimerization and proteolysis are important for activity.…”

Section: Procaspase-1 Zymogen Domain Structurementioning

confidence: 78%

Crystal Structure of Procaspase-1 Zymogen Domain Reveals Insight into Inflammatory Caspase Autoactivation

Elliott¹,

Rougé²,

Wiesmann³

et al. 2009

Journal of Biological Chemistry

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One key event in inflammatory signaling is the activation of the initiator caspase, procaspase-1. Presented here is the crystal structure of the procaspase-1 zymogen without its caspase recruitment domain solved to 2.05 Å . Although the isolated domain is monomeric in solution, the protein appeared dimeric in crystals. The loop arrangements in the dimer provide insight into the first autoproteolytic events that occur during activation by oligomerization. Additionally, in contrast to other caspases, we demonstrate that autoproteolysis at the second cleavage site, Asp 316 , is necessary for conversion to a stable dimer in solution. Critical elements of secondary structure are revealed in the crystal structure that explain why a dimeric protein is favored after proteolysis at this aspartic acid. Dimer stabilization is concurrent with a 130-fold increase in k cat , the sole contributing kinetic factor to an activated and efficient mediator of inflammation.Caspases are a family of aspartic acid-specific cysteine proteases that are involved in inflammation and apoptosis. This enzyme family is generally categorized into three groups as follows: inflammatory initiators (caspases-1, -4, and -5), apoptotic initiators (caspases-2, -8, -9, and -10), and apoptotic effectors (caspases-3, -6, and -7). Because of its role as the protease responsible for processing prointerleukin-1␤ (pro-IL 2 -1␤) to an active 17-kDa form, caspase-1 is a major driver of inflammation and innate immunity (1-3). The enzyme also participates in the activation of prointerleukin-18 and prointerleukin-33 (4, 5). Caspase-1 activation and the subsequent release of IL-1␤, IL-18, and IL-33 are one of the first lines of defense against invading microbes and viruses. Excessive caspase-1 activity, however, can lead to pathologies associated with several autoimmune and inflammatory diseases such as septic shock, inflammatory bowel disease, familial cold autoinflammatory syndrome, rheumatoid arthritis, osteoarthritis, and gout (6 -8).Caspase-1 knock-out mice have strongly supported this assertion, demonstrating an absence of processed IL-1␤ and IL-18 and subsequent decreases in production of cytokines IL-1␣, IL-6, interferon-␥, and tumor necrosis factor-␣ when challenged with lipopolysaccharide or Listeria monocytogenes (6, 9, 10). More recent studies have established the involvement of caspase-1 in neurodegenerative disorders such as trauma and ischemic brain injury, Huntington disease, and amyotrophic lateral sclerosis (7).Pathways leading to the activation of the enzyme are triggered by diverse pathogen and damage-associated molecular patterns such as lipopolysaccharide, flagellin, DNA, RNA, uric acid crystals, bacterial toxins, and UVB damage (11). These patterns are often recognized by Toll-like receptors on the cell surface or by nod-like receptors inside the cell. After pattern recognition, intracellular events result in the conversion of the inactive procaspase-1 zymogen to a fully processed, active enzyme.Procaspase-1 conversion is induced by macromole...

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Section: Procaspase-1 Zymogen Domain Structurementioning

confidence: 78%

Crystal Structure of Procaspase-1 Zymogen Domain Reveals Insight into Inflammatory Caspase Autoactivation

Elliott¹,

Rougé²,

Wiesmann³

et al. 2009

Journal of Biological Chemistry

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“…Our data support recent work questioning the role of autoproteolysis in DRONC activation. 15 Cleavage in the inter-chain linker clearly has a role to play in the regulation of DRONC activity. In caspase buffer, singlechain DRONC has almost no activity on synthetic substrates when compared with the two-chain enzyme, and it was this observation that led to the suggestion that cleavage in the inter-chain linker was the mechanism of activation.…”

Section: Discussionmentioning

confidence: 99%

“…Assembling a Drosophila apoptosome that efficiently cleaves peptide substrates is not trivial. Indeed, in a recent paper, 15 the only assay possible for ARK activated DRONC was to amplify the signal and measure downstream DEVD-ase activity. Unfortunately this cannot tell us about DRONC specificity on synthetic substrates because the signal needed executioner caspase activation.…”

Section: Discussionmentioning

confidence: 99%

“…[10][11][12] Moreover, unlike human caspase-9, which is activated by dimerization within the apoptosome, 13 DRONC is reported to require proteolytic autoprocessing for activation, 14 although this has recently been disputed. 15 Finally, in normal living S2 cells, DRONC is processed between the large and small subunits at Glu352, implying that DRONC can cleave C-terminally to a Glu residue. 14,16 The requirement for autoprocessing and specificity for Glu residues thus places DRONC in a separate category mechanistically from its human ortholog caspase-9, and thereby violates the principle of conservation of mechanism.…”

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confidence: 99%

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Activation mechanism and substrate specificity of the Drosophila initiator caspase DRONC

et al. 2008

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Drosophila Nedd2-like caspase (DRONC), an initiator caspase in Drosophila melanogaster and ortholog of human caspase-9, is cleaved during its activation in vitro and in vivo. We show that, in contrast to conclusions from previous studies, cleavage is neither necessary nor sufficient for DRONC activation. Instead, our data suggest that DRONC is activated by dimerization, a mechanism used by its counterparts in humans. Subsequent cleavage at Glu352 stabilizes the active dimer. Since cleavage is at a Glu residue, it has been proposed that DRONC is a dual Asp-and Glu-specific caspase. We used positional-scanning peptide libraries to define the P1-P4 peptide sequence preferences of DRONC, and show that it is indeed equally active on optimized tetrapeptides containing either Asp or Glu in P1. Furthermore, mutagenesis reveals that Asp and Glu residues are equally tolerated at the primary autoprocessing site of DRONC itself. However, when its specificity is tested on a natural substrate, the Drosophila executioner caspase DRICE, a clear preference for Asp emerges. The formerly proposed Glu preference is thus incorrect. DRONC does not differentiate between Asp and Glu in poor substrates, but prefers Asp when tested on a good substrate.

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“…First, the 20E-EcR-USP complex and the 20E primary response genes [including Br-C (broad -FlyBase) E74 (Eip74EF), E75 (Eip75B) and E93 (Eip93F)] induce expression of several 20E secondary response genes that account for PCD, including the caspases Dronc (Nc) and Drice (Ice) (Cakouros et al, 2004;Kilpatrick et al, 2005) and the death activators reaper and Hid (Wrinkled) (Yin and Thummel, 2005). Second, Reaper and HID prevent Dronc and Drice from ubiquitin-regulated protein degradation, and Dronc and Drice activate each other by protein cleavage Dorstyn and Kumar, 2008). Third, Reaper, HID, Dronc and Drice promote IAP1 (Thread) to undergo ubiquitin-regulated protein degradation, and vice versa .…”

Section: Introductionmentioning

confidence: 99%

Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death inDrosophila

et al. 2009

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Juvenile hormone (JH) regulates many developmental and physiological events in insects, but its molecular mechanism remains conjectural. Here we report that genetic ablation of the corpus allatum cells of the Drosophila ring gland (the JH source) resulted in JH deficiency, pupal lethality and precocious and enhanced programmed cell death (PCD) of the larval fat body. In the fat body of the JH-deficient animals, Dronc and Drice, two caspase genes that are crucial for PCD induced by the molting hormone 20-hydroxyecdysone (20E), were significantly upregulated. These results demonstrated that JH antagonizes 20E-induced PCD by restricting the mRNA levels of Dronc and Drice. The antagonizing effect of JH on 20E-induced PCD in the fat body was further confirmed in the JH-deficient animals by 20E treatment and RNA interference of the 20E receptor EcR. Moreover, MET and GCE, the bHLH-PAS transcription factors involved in JH action, were shown to induce PCD by upregulating Dronc and Drice. In the Met-and gce-deficient animals, Dronc and Drice were downregulated, whereas in the Met-overexpression fat body, Dronc and Drice were significantly upregulated leading to precocious and enhanced PCD, and this upregulation could be suppressed by application of the JH agonist methoprene. For the first time, we demonstrate that JH counteracts MET and GCE to prevent caspase-dependent PCD in controlling fat body remodeling and larval-pupal metamorphosis in Drosophila.

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A biochemical analysis of the activation of the Drosophila caspase DRONC

Cited by 53 publications

References 41 publications

Crystal Structure of Procaspase-1 Zymogen Domain Reveals Insight into Inflammatory Caspase Autoactivation

Crystal Structure of Procaspase-1 Zymogen Domain Reveals Insight into Inflammatory Caspase Autoactivation

Activation mechanism and substrate specificity of the Drosophila initiator caspase DRONC

Juvenile hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent caspase-dependent programmed cell death inDrosophila

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