Structural and Biochemical Studies on Procaspase-8: New Insights on Initiator Caspase Activation

Keller, N.; Mareš, Jiřı́ J.; Zerbe, Oliver; Grütter, Markus G.

doi:10.1016/j.str.2008.12.019

Cited by 81 publications

(113 citation statements)

References 54 publications

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“…This assumption was motivated by structural considerations. Namely, procaspase-8 homodimers or procaspase-8/c-FLIP L heterodimers form hydrogen bonds between the catalytic subunits, 28,33 which are lacking in the short isoforms of c-FLIP as well as in the procaspase-8 prodomain.…”

Section: Resultsmentioning

confidence: 99%

Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain

et al. 2015

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The CD95/Fas/APO-1 death-inducing signaling complex (DISC), comprising CD95, FADD, procaspase-8, procaspase-10, and c-FLIP, has a key role in apoptosis induction. Recently, it was demonstrated that procaspase-8 activation is driven by death effector domain (DED) chains at the DISC. Here, we analyzed the molecular architecture of the chains and the role of the short DED proteins in regulating procaspase-8 activation in the chain model. We demonstrate that the DED chains are largely composed of procaspase-8 cleavage products and, in particular, of its prodomain. The DED chain also comprises c-FLIP and procaspase-10 that are present in 10 times lower amounts compared with procaspase-8. We show that short c-FLIP isoforms can inhibit CD95-induced cell death upon overexpression, likely by forming inactive heterodimers with procaspase-8. Furthermore, we have addressed mechanisms of the termination of chain elongation using experimental and mathematical modeling approaches. We show that neither c-FLIP nor procaspase-8 prodomain terminates the DED chain, but rather the dissociation/association rates of procaspase-8 define the stability of the chain and thereby its length. In addition, we provide evidence that procaspase-8 prodomain generated at the DISC constitutes a negative feedback loop in procaspase-8 activation. Overall, these findings provide new insights into caspase-8 activation in DED chains and apoptosis initiation.

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Section: Resultsmentioning

confidence: 99%

Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain

et al. 2015

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“…Since dimerization is a precondition for self-processing and caspase-8 activity [66], we speculated that HSV-2 R1 could interact with caspase-8 in a way that inhibits this event. Supporting our hypothesis, we found that HSV-2 R1 protects cells against apoptosis induced by over-expression of GFP-tagged caspase-8 that is known to be triggered by dimerization/activation of the over-expressed zymogen [51].…”

Section: Discussionmentioning

confidence: 99%

The ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 protect cells against TNFα- and FasL-induced apoptosis by interacting with caspase-8

et al. 2010

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We previously reported that HSV-2 R1, the R1 subunit (ICP10; UL39) of herpes simplex virus type-2 ribonucleotide reductase, protects cells against apoptosis induced by the death receptor (DR) ligands tumor necrosis factor-alpha-(TNFa) and Fas ligand (FasL) by interrupting DR-mediated signaling at, or upstream of, caspase-8 activation. Further investigation of the molecular mechanism underlying HSV-2 R1 protection showed that extracellularregulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3-K)/Akt, NF-jB and JNK survival pathways do not play a major role in this antiapoptotic function. Interaction studies revealed that HSV-2 R1 interacted constitutively with caspase-8. The HSV-2 R1 deletion mutant R1(1-834)-GFP and Epstein-Barr virus (EBV) R1, which did not protect against apoptosis induced by DR ligands, did not interact with caspase-8, indicating that interaction is required for protection. HSV-2 R1 impaired caspase-8 activation induced by caspase-8 over-expression, suggesting that interaction between the two proteins prevents caspase-8 dimerization/activation. HSV-2 R1 bound to caspase-8 directly through its prodomain but did not interact with either its caspase domain or Fas-associated death domain protein (FADD). Interaction between HSV-2 R1 and caspase-8 disrupted FADD-caspase-8 binding. We further demonstrated that individually expressed HSV-1 R1 (ICP6) shares, with HSV-2 R1, the ability to bind caspase-8 and to protect cells against DR-induced apoptosis. Finally, as the long-lived Fas protein remained stable during the early period of infection, experiments with the HSV-1 UL39 deletion mutant ICP6D showed that HSV-1 R1 could be essential for the protection of HSV-1-infected cells against FasL.Keywords FasL Á ICP6 Á ICP10 Á Viral inhibitor of apoptosis Á Caspase-8Electronic supplementary material The online version of this article

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“…36,37 In contrast, the IL of the procaspase-8 monomer interacts with active site loop 1 and prevents productive active site formation. 17 In addition to the structural features of the IL and pro-domain regions, the dimer interfaces are very different between caspases-3 and -8. While the interface of caspase-3 contains several backbone hydrogen bond interactions below 3 Å , caspase-8 contains few hydrogen bonds, and the interactions are at longer distances (>3 Å , Fig.…”

Section: Discussionmentioning

confidence: 99%

“…15 The structure of uncleaved procaspase-8 shows that activation occurs through the rearrangement of surface loops that form the substrate-binding cleft, which properly orients the catalytic residues. 17 In addition to homodimerization, procaspase-8 also forms heterodimers with the structurally similar protein, FLIP. [18][19][20] In the cell, the heterodimer inactivates RIPK1, preventing necroptosis and promoting cell survival, and it is thought that heterodimerization represents the default cellular pathway.…”

Section: Introductionmentioning

confidence: 99%

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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Structural and Biochemical Studies on Procaspase-8: New Insights on Initiator Caspase Activation

Cited by 81 publications

References 54 publications

Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain

Molecular architecture of the DED chains at the DISC: regulation of procaspase-8 activation by short DED proteins c-FLIP and procaspase-8 prodomain

The ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 protect cells against TNFα- and FasL-induced apoptosis by interacting with caspase-8

Redesigning the procaspase‐8 dimer interface for improved dimerization

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