Inducible Dimerization and Inducible Cleavage Reveal a Requirement for Both Processes in Caspase-8 Activation

Oberst, Andrew; Pop, Cristina; Tremblay, Alexandre G.; Blais, Véronique; Denault, Jean Bernard; Salvesen, Guy S.; Green, Douglas R.

doi:10.1074/jbc.m109.095083

Cited by 190 publications

(192 citation statements)

References 40 publications

(54 reference statements)

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“…26,39,40 Here we have shown that dimerization of RIPK1 or RIPK3 can induce kinase-dependent necroptosis, provided that active RIPK3 and MLKL are present. Importantly, at least in the case of dimerized RIPK3, the kinase activity can be supplied by bystander endogenous RIPK3 monomers that are presumably recruited to a kinase-inactive dimer.…”

Section: Discussionmentioning

confidence: 88%

RIPK1- and RIPK3-induced cell death mode is determined by target availability

et al. 2014

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Both receptor-interacting protein kinase 1 (RIPK1) and RIPK3 can signal cell death following death receptor ligation. To study the requirements for RIPK-triggered cell death in the absence of death receptor signaling, we engineered inducible versions of RIPK1 and RIPK3 that can be activated by dimerization with the antibiotic coumermycin. In the absence of TNF or other death ligands, expression and dimerization of RIPK1 was sufficient to cause cell death by caspase-or RIPK3-dependent mechanisms. Dimerized RIPK3 induced cell death by an MLKL-dependent mechanism but, surprisingly, also induced death mediated by FADD, caspase 8 and RIPK1. Catalytically active RIPK3 kinase domains were essential for MLKL-dependent but not for caspase 8-dependent death. When RIPK1 or RIPK3 proteins were dimerized, the mode of cell death was determined by the availability of downstream molecules such as FADD, caspase 8 and MLKL. These observations imply that rather than a 'switch' operating between the two modes of cell death, the final mechanism depends on levels of the respective signaling and effector proteins. Mammalian cells can use a number of mechanisms to kill themselves. The best characterized depends on the Bcl-2 family members Bax and Bak that work via mitochondria to activate caspases. 1 Some caspases, notably caspase 8, can be activated independently of Bcl-2 family members, for example, after stimulation of members of the TNF receptor superfamily. 2 Recently, it has become apparent that some of these receptors, including TNFR1, can activate a third suicide mechanism that does not require caspases, and in which the morphology of the dying cell differs from classical apoptosis. This form of cell death, termed 'necroptosis', can often be blocked by necrostatin-1 (nec-1), an inhibitor of the kinase activity of receptor-interacting protein kinase 1 (RIPK1). 3,4 Accordingly, observations from several groups have shown that in some cell types, expression of RIPK1 can signal cell death by caspase-independent necroptosis. 5 It has previously been revealed that RIPK1 could function downstream of death receptors, but in those cases, cell death was usually blocked by coexpression of the viral inhibitor of caspases 1 and 8, CrmA, 6 and typically exhibited a classical 'apoptotic' morphology. It was revealed that RIPK1 engages FADD via homotypic binding of their death domains (DDs), and FADD in turn activates caspase 8. 6,7 RIPK3, like RIPK1, bears a kinase domain and RIP homology interaction motif (RHIM), but unlike RIPK1 does not have a DD. 8-11 RIPK3 is required for necroptosis. 12,13 Furthermore, RIPK1 appears to activate RIPK3 in this pathway, as cell death could be blocked by nec-1. 14 RIPK3 activates, by phosphorylation, MLKL, a pseudokinase essential for this death pathway. [15][16][17] Once activated, MLKL forms multimers that trigger breaches of the plasma membrane. [18][19][20] Although RIPK3 is necessary for necroptosis, it is unclear whether activation of RIPK3 is sufficient for cell death, because TNF activates signaling ...

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Section: Discussionmentioning

confidence: 88%

RIPK1- and RIPK3-induced cell death mode is determined by target availability

et al. 2014

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“…Self-processing occurs between the large and the small subunit of caspase-1, and appears to be mandatory for caspase-1-directed maturation of the inflammasome target cytokines, IL-1β and IL-18. This cleavage event may be associated with stabilisation of the active site, as reported for initiator apoptotic caspases [15]. In contrast, self-processing between the large and the small subunits appeared dispensable for the initiation of pyroptosis, as wild-type and uncleavable mutants of caspase-1 were equally able to trigger pyroptosis in murine macrophages [14].…”

Section: Introductionmentioning

confidence: 79%

Burn the house, save the day: pyroptosis in pathogen restriction

Boucher¹,

Chen²,

Schroder³

2016

Inflammasome

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Many programmed cell death pathways are essential for organogenesis, development, immunity and the maintenance of homeostasis in multicellular organisms. Pyroptosis, a highly proinflammatory form of cell death, is a critical innate immune response to prevent intracellular infection. Pyroptosis is induced upon the activation of proinflammatory caspases within macromolecular signalling platforms called inflammasomes. This article reviews our understanding of pyroptosis induction, the function of inflammatory caspases in pyroptosis execution, and the importance of pyroptosis for pathogen clearance. It also highlights the situations in which extensive pyroptosis may in fact be detrimental to the host, leading to immune cell depletion or cytokine storm. Current efforts to understand the beneficial and pathological roles of pyroptosis bring the promise of new approaches to fight infectious diseases.

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“…The data further support the notion that dimerization is sufficient for activity in procaspase-8. 15,28 Stabilizing the dimer, as observed for the two quad mutants, appears to be as efficient in forming the active procaspase as cleavage of the IL in the wildtype caspase-8 [ Fig. 2(C)], because the IL is not cleaved in these mutants.…”

Section: Procaspase-8 Variants With Mutated Dimer Interface Demonstramentioning

confidence: 99%

“…The direct relationship between dimerization and activation of caspase-8 has been well-established, 11,16,28 so the enzyme activity of procaspase-8 is proportional to the population of dimer in the sample. Salvesen and coworkers also have shown that procaspase-8 dimerization is facilitated by sodium citrate, a kosmotrope, resulting in activation.…”

Section: Procaspase-8 Variants With Mutated Dimer Interface Demonstramentioning

confidence: 99%

“…It was shown previously that the dimer of caspase-8 is stabilized upon cleavage of the IL. 15,16,28 In order to eliminate the influence of IL cleavage on the kinetics assay, we performed the studies on the D374A, D384A uncleavable mutants [called DDED,D 2 A, Fig. 2(A)], which removes the cleavage sites in the linker.…”

Section: The Redesigned Dimer Interface Accelerates the Dimerization mentioning

confidence: 99%

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Redesigning the procaspase‐8 dimer interface for improved dimerization

MacKenzie

Clark

2014

Protein Science

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Caspase-8 is a cysteine directed aspartate-specific protease that is activated at the cytosolic face of the cell membrane upon receptor ligation. A key step in the activation of caspase-8 depends on adaptor-induced dimerization of procaspase-8 monomers. Dimerization is followed by limited autoproteolysis within the intersubunit linker (IL), which separates the large and small subunits of the catalytic domain. Although cleavage of the IL stabilizes the dimer, the uncleaved procaspase-8 dimer is sufficiently active to initiate apoptosis, so dimerization of the zymogen is an important mechanism to control apoptosis. In contrast, the effector caspase-3 is a stable dimer under physiological conditions but exhibits little enzymatic activity. The catalytic domains of caspases are structurally similar, but it is not known why procaspase-8 is a monomer while procaspase-3 is a dimer. To define the role of the dimer interface in assembly and activation of procaspase-8, we generated mutants that mimic the dimer interface of effector caspases. We show that procaspase-8 with a mutated dimer interface more readily forms dimers. Time course studies of refolding also show that the mutations accelerate dimerization. Transfection of HEK293A cells with the procaspase-8 variants, however, did not result in a significant increase in apoptosis, indicating that other factors are required in vivo. Overall, we show that redesigning the interface of procaspase-8 to remove negative design elements results in increased dimerization and activity in vitro, but increased dimerization, by itself, is not sufficient for robust activation of apoptosis.

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Inducible Dimerization and Inducible Cleavage Reveal a Requirement for Both Processes in Caspase-8 Activation

Cited by 190 publications

References 40 publications

RIPK1- and RIPK3-induced cell death mode is determined by target availability

RIPK1- and RIPK3-induced cell death mode is determined by target availability

Burn the house, save the day: pyroptosis in pathogen restriction

Redesigning the procaspase‐8 dimer interface for improved dimerization

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