Engineering a Dimeric Caspase-9: A Re-evaluation of the Induced Proximity Model for Caspase Activation

Chao, Yang; Shiozaki, Eric N.; Srinivasula, Srinivasa M.; Rigotti, Daniel J.; Fairman, Robert; Shi, Yigong

doi:10.1371/journal.pbio.0030183

Cited by 103 publications

(109 citation statements)

References 33 publications

(68 reference statements)

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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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“…Based on the molecular dimensions of APAF-1, this suggests a side-by-side arrangement of the CED-4 protomers in the heterotetramer, although no information is available on the details. In a recent paper by Chao et al, 32 it was elegantly demonstrated that dimerisation of caspase-9 alone is not sufficient for full activity (compared to APAF-1-activated caspase-9). Instead, an induced conformational Figure 7 Sequences of BH3 peptides used.…”

Section: Discussionmentioning

confidence: 99%

CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13

et al. 2005

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The pathway to cell death in Caenorhabditis elegans is well established. In cells undergoing apoptosis, the Bcl-2 homology domain 3 (BH3)-only protein EGL-1 binds to CED-9 at the mitochondrial membrane to cause the release of CED-4, which oligomerises and facilitates the activation of the caspase CED-3. However, despite many studies, the biophysical features of the CED-4/CED-9 complex have not been fully characterised. Here, we report the purification of a soluble and stable 2 : 2 heterotetrameric complex formed by recombinant CED-4 and CED-9 coexpressed in bacteria. Consistent with previous studies, synthetic peptides corresponding to the BH3 domains of worm BH3-only proteins (EGL-1, CED-13) dissociate CED-4 from CED-9, but not from the gain-offunction CED-9 (G169E) mutant. Surprisingly, the ability of worm BH3 domains to dissociate CED-4 was specific since mammalian BH3-only proteins could not do so.

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“…11,12,[14][15][16] Furthermore, this dimeric caspase-9 has significantly enhanced activity compared to its monomeric form. 17,18 Interestingly, cleavage at D315 is not essential for caspase-9 activation by the apoptosome or for cleavage of caspase-3. 16,[18][19][20] Active caspase-3 also cleaves caspase-9 at D330 in a feedback loop to remove an XIAP-binding IBM site, thereby alleviating XIAP inhibition of caspase-9 to allow sustained caspase activation.…”

mentioning

confidence: 99%

“…17,18 Interestingly, cleavage at D315 is not essential for caspase-9 activation by the apoptosome or for cleavage of caspase-3. 16,[18][19][20] Active caspase-3 also cleaves caspase-9 at D330 in a feedback loop to remove an XIAP-binding IBM site, thereby alleviating XIAP inhibition of caspase-9 to allow sustained caspase activation. 19 In Drosophila, the caspase-2/-9 homologue DRONC is required for specific developmental cell death pathways and stress-induced apoptosis [21][22][23][24][25] and is activated by the Drosophila Apaf-1 homologue, ARK.…”

mentioning

confidence: 99%

A biochemical analysis of the activation of the Drosophila caspase DRONC

Dorstyn

Kumar

2007

Cell Death Differ

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The activation of caspases is the principal event in the execution of apoptosis. Initiator caspases are activated through an autocatalytic mechanism often involving dimerisation or oligomerisation. In Drosophila, the only initiator caspase DRONC, is tightly inhibited by DIAP1 and removal of DIAP1 permits activation of DRONC by the Drosophila Apaf-1-related killer, ARK. ARK is proposed to facilitate DRONC oligomerisation and autoprocessing at residue E352. This study examines whether autoprocessing of DRONC is required for its activation and for DRONC-mediated cell death. Using purified recombinant proteins, we show here that while DRONC autocleaves at residue E352, mutation of this site did not abolish enzyme activation, DRICEinduced cleavage of DRONC or DRONC-mediated activation of DRICE. We performed a detailed mutational analysis of DRONC cleavage sites and show that overexpression of DRONC cleavage mutants in Drosophila cells retain pro-apoptotic activity. Using an in vitro cell-free assay, we found ARK alone did not activate DRONC and demonstrate a requirement for an additional cytosolic factor in ARK-mediated DRONC activation. These results suggest that, similar to mammalian caspase-2 and caspase-9, the initial cleavage of DRONC is not essential for its activation and suggest a mechanism of ARK-mediated DRONC activation different from that proposed previously.

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Engineering a Dimeric Caspase-9: A Re-evaluation of the Induced Proximity Model for Caspase Activation

Cited by 103 publications

References 33 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

CED-4 forms a 2 : 2 heterotetrameric complex with CED-9 until specifically displaced by EGL-1 or CED-13

A biochemical analysis of the activation of the Drosophila caspase DRONC

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