Human Bcl-2 cannot directly inhibit the <i>Caenorhabditis elegans</i> Apaf-1 homologue CED-4, but can interact with EGL-1

Jabbour, Anissa; Puryer, Michelle A; Yu, Jinxin; Lithgow, Trevor; Riffkin, Christopher D.; Ashley, David M.; Vaux, David L.; Ekert, Paul G.; Hawkins, Christine J.

doi:10.1242/jcs.02985

Cited by 23 publications

(21 citation statements)

References 68 publications

(81 reference statements)

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“…Yeast cells can be rescued from this growth arrest by the overexpression of mammalian antiapoptotic proteins, such as Bcl-x L , which leads to robust colony growth (32,51). Thus, in this well-established functional assay (23,29,37,76), the expression of Bak blocks yeast growth, which can be rescued by the overexpression of Bcl-x L (Fig. 3A, "on").…”

Section: Resultsmentioning

confidence: 98%

Deerpox Virus Encodes an Inhibitor of Apoptosis That Regulates Bak and Bax

Banadyga

Lam

Okamoto

et al. 2011

J Virol

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Poxviruses encode numerous proteins that inhibit apoptosis, a form of cell death critical to the elimination of virally infected cells. Sequencing of the deerpox virus genome revealed DPV022, a protein that lacks obvious homology to cellular members of the Bcl-2 family but shares limited regions of amino acid identity with two unique poxviral inhibitors of apoptosis, M11L and F1L. Given the limited homology, we sought to determine whether DPV022 could inhibit apoptosis. Here we show that DPV022 localized to the mitochondria, where it inhibited apoptosis. We used a Saccharomyces cerevisiae model system to demonstrate that in the absence of all other Bcl-2 family proteins, DPV022 interacted directly with Bak and Bax. We confirmed the ability of DPV022 to interact with Bak and Bax by immunoprecipitation and showed that DPV022 prevented apoptosis induced by Bak and Bax overexpression. Moreover, we showed that DPV022 blocked apoptosis even when all the endogenous mammalian antiapoptotic proteins were neutralized by a combination of selective BH3 ligands. During virus infection, DPV022 interacted with endogenous Bak and Bax and prevented the conformational activation of both of them. Thus, we have characterized a novel poxviral inhibitor of apoptosis with intriguing amino acid differences from the well-studied proteins M11L and F1L.

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Section: Resultsmentioning

confidence: 98%

Deerpox Virus Encodes an Inhibitor of Apoptosis That Regulates Bak and Bax

Banadyga

Lam

Okamoto

et al. 2011

J Virol

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“…How, then, is Bcl-2, which acts by preventing MOMP, able to substitute for CED-9, whose antiapoptotic effect is exerted through sequestration of the nematode APAF-1 homolog CED-4? A more recent study 27 found that Bcl-2 does not interact with CED-4 in a yeast two hybrid screen; it does, however, interact with EGL-1. Furthermore, when the basic nematode apoptotic program is recapitulated in yeast, CED-9, but not Bcl-2, was able to prevent lethality caused by CED-3/CED-4 co-expression.…”

Section: Nematodes: Like Us Only Differentmentioning

confidence: 95%

“…The authors proposed that Bcl-2 prevents cell death in wild-type nematodes by binding EGL-1 through that protein's BH-3 domain, and that the ability of Bcl-2 to rescue the apoptotic phenotype of CED-9-deficient worms could be due to Bcl-2 preventing EGL-1 from neutralizing the small amount of maternally derived CED-9 present at the developmental stage at which the original experiments were performed. [26][27][28] Despite their frequent depiction as isolated, globular organelles, in healthy cells the mitochondria form a complex and interconnected network, subject to frequent fusion, fission, and remodeling events (reviewed in Hoppins et al 29 ). Imaging of mammalian cells undergoing apoptosis has revealed that mitochondria fragment during MOMP; subsequent studies have lent credence to the idea that mitochondrial dynamics and MOMP are intimately linked with increased mitochondrial fission leading to sensitivity to MOMP and increased fusion blocking or delaying it.…”

Section: Nematodes: Like Us Only Differentmentioning

confidence: 99%

Living with death: the evolution of the mitochondrial pathway of apoptosis in animals

2008

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The mitochondrial pathway of cell death, in which apoptosis proceeds following mitochondrial outer membrane permeabilization, release of cytochrome c, and APAF-1 apoptosome-mediated caspase activation, represents the major pathway of physiological apoptosis in vertebrates. However, the well-characterized apoptotic pathways of the invertebrates C. elegans and D. melanogaster indicate that this apoptotic pathway is not universally conserved among animals. This review will compare the role of the mitochondria in the apoptotic programs of mammals, nematodes, and flies, and will survey our knowledge of the apoptotic pathways of other, less familiar model organisms in an effort to explore the evolutionary origins of the mitochondrial pathway of apoptosis.

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“…2,19 The low affinity of EGL-1 for most mammalian pro-survival proteins, shown in the present study, is interesting in light of a recent report using a yeast model system. 20 Their data suggest Bcl-2 does not directly inhibit CED-4, as CED-9 does, but rather acts as a 'sink' to sequester EGL-1, preventing it from engaging CED-9. Our binding data would support this hypothesis provided the levels of Bcl-2 are sufficient to overcome its relatively low affinity (K D B10 mM) for EGL-1.…”

Section: Egl-1 Mutants Reveal Killing Thresholds Ef Lee Et Almentioning

confidence: 99%

“…2,19 More recently, studies employing yeast-based functional and other biochemical assays determined that in the nematode studies described above, Bcl-2 is probably functioning by binding to EGL-1 thereby preventing its association with CED-9. 20 However, although CED-9 appears able to prevent cellular processes such as mitochondrial fission in mammalian cells (suggesting potential interactions with mammalian proteins), 21 EGL-1 seems ineffective at inducing apoptosis. 21 We have also shown that mammalian BH3-only proteins such as Bim and Bad, unlike EGL-1, cannot dissociate CED-4:CED-9 complexes.…”

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confidence: 99%

EGL-1 BH3 mutants reveal the importance of protein levels and target affinity for cell-killing potency

et al. 2008

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Studies of the cell death pathway in the nematode Caenorhabditis elegans provided the first evidence of the evolutionary conservation of apoptosis signalling. Here we show that the worm Bcl-2 homology domain-3 (BH3)-only protein EGL-1 binds mammalian pro-survival proteins very poorly, but can be converted into a high-affinity ligand for Bcl-2 and Bcl-x L by subtle mutation of the cysteine residue at position 62 within the BH3 domain. A 100-fold increase in affinity was observed following a single atom change (cysteine to serine substitution), and a further 10-fold increase by replacement with glycine. The low affinity of wild-type EGL-1 for mammalian pro-survival proteins and its poor expression correlates with its weak killing activity in mammalian cells whereas the high-affinity C62G mutant is a very potent killer of cells lacking Mcl-1. Cell killing by the C62S mutant with intermediate affinity only occurs when this EGL-1 BH3 domain is placed in a more stable context, namely that of Bim S , which allows higher expression, though the kinetics of cell death now vary depending on whether Mcl-1 is neutralized by Noxa or genetically deleted. These results demonstrate how levels of BH3-only proteins, target affinity and the spectrum of neutralization of pro-survival proteins all contribute to killing activity. Landmark studies in Caenorhabditis elegans revealed that four genes, CED-9, CED-4, CED-3 and EGL-1, are essential for the programmed cell death (apoptosis) of 131 out of 1090 somatic cells during hermaphrodite nematode development. 1 Of these, EGL-1, CED-3 and CED-4 encode pro-apoptotic proteins whereas CED-9 encodes a pro-survival protein. CED-9 is the nematode homologue of mammalian B-cell lymphoma-2 (Bcl-2) pro-survival proteins (including Bcl-2 itself as well as Bcl-x L , Bcl-w, Mcl-1 and A1), 2 while CED-4 is similar to the mammalian adapter protein APAF-1 3 acting as a positive regulator of caspases, in particular, CED-3. 4 EGL-1 is the nematode Bcl-2 homology domain-3 (BH3)-only protein equivalent to mammalian BH3-only proteins such as Bim, Bad and Noxa. [5][6][7] In nematode cells, CED-9 functions by sequestering CED-4 at the mitochondrial membrane. 8-10 Following a developmental cue, EGL-1 is transcriptionally upregulated and the protein binds CED-9, 5-7 releasing CED-4, allowing its oligomerization. 11 These CED-4 oligomers then translocate to the perinuclear region of the cell 12 where they facilitate CED-3 activation by bringing these caspases into close proximity. 4,9 Biochemical and structural studies have revealed that binding of the EGL-1 BH3 domain into a hydrophobic groove on CED-9 causes a conformational change at the CED-4:CED-9 interface that results in CED-4 dissociation. [13][14][15] The BH3 domains of mammalian BH3-only proteins bind into similar grooves on mammalian pro-survival molecules, but a conformational change as seen in CED-9 has yet to be observed. [16][17][18] The functional equivalence, and hence evolutionary conservation, of CED-9 and mammalian Bcl-2 was demonstrated in st...

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Human Bcl-2 cannot directly inhibit the Caenorhabditis elegans Apaf-1 homologue CED-4, but can interact with EGL-1

Cited by 23 publications

References 68 publications

Deerpox Virus Encodes an Inhibitor of Apoptosis That Regulates Bak and Bax

Deerpox Virus Encodes an Inhibitor of Apoptosis That Regulates Bak and Bax

Living with death: the evolution of the mitochondrial pathway of apoptosis in animals

EGL-1 BH3 mutants reveal the importance of protein levels and target affinity for cell-killing potency

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