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Cleavage of Atg3 protein by caspase-8 regulates autophagy during receptor-activated cell death

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Abstract

Autophagy is an evolutionarily conserved mechanism contributing to cell survival under stress conditions including nutrient and growth factor deprivation. Connections and cross-talk between cell death mechanisms and autophagy is under investigation. Here, we describe Atg3, an essential regulatory component of autophagosome biogenesis, as a new substrate of caspase-8 during receptor-mediated cell death. Both, tumor necrosis factor α and tumor necrosis factor-related apoptosis inducing ligand induced cell death was accompanied by Atg3 cleavage and this event was inhibited by a pan-caspase inhibitor (zVAD) or a caspase-8-specific inhibitor (zIETD). Indeed, caspase-8 overexpression led to Atg3 degradation and this event depended on caspase-8 enzymatic activity. Mutation of the caspase-8 cleavage site on Atg3 abolished its cleavage both in vitro and in vivo, demonstrating that Atg3 was a direct target of caspase-8. Autophagy was inactive during apoptosis and blockage of caspases or overexpression of a non-cleavable Atg3 protein reestablished autophagic activity upon death receptor stimulation. In this system, autophagy was important for cell survival since inhibition of autophagy increased cell death. Therefore, Atg3 provides a novel link between apoptosis and autophagy during receptor-activated cell death.

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Abbreviations

LC3:

Microtubule-associated protein 1 (MAP1) light chain 3

PI3K:

Phosphatidylinositol 3-kinase

zVAD-fmk:

Benzyloxycarbonyl-valyl-alanyl-aspartic-acid (O-methyl)-fluoromethylketone

CHX:

Cycloheximide

TNF:

Tumor necrosis factor

TRAIL:

Tumor necrosis factor-related apoptosis-inducing ligand

FADD:

Fas-associated protein with death domain

TRADD:

Tumor necrosis factor receptor type 1-associated death domain protein

PE:

Phosphatidylethanolamine

HEK293T:

Human embryonic kidney cells

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Acknowledgments

pcDNA-HA-caspase-8 and pcDNA3-caspase-8-C377S-HA (mutant caspase-8) plasmids were kindly provided by Nesrin Ozoren and Gabriel Nunez. This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) 1001 grant (Project No. 107T153), EMBO Strategical Development and Installations Grant (EMBO-SDIG, Project No. 1449) and Sabanci University. D.G. is a recipient of Turkish Academy of Sciences (TUBA) GEBIP Award. O. O. and D. O. A. are recipients of TUBITAK-2218 postdoctoral scholarship.

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Faculty of Engineering and Natural Sciences, Biological Sciences and Bioengineering Program, Sabanci University, Orhanli-Tuzla, 34956, Istanbul, Turkey

    Ozlem Oral, Devrim Oz-Arslan, Zeynep Itah & Devrim Gozuacik

  2. Faculty of Medicine, Department of Biophysics, Acibadem University, Maltepe, Istanbul, Turkey

    Devrim Oz-Arslan

  3. Faculty of Science, Department of Biology, Ege University, Bornova, Izmir, Turkey

    Atabak Naghavi, Remziye Deveci & Sabire Karacali

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  1. Ozlem Oral
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Correspondence to Devrim Gozuacik.

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Ozlem Oral and Devrim Oz-Arslan contributed equally to this work.

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Oral, O., Oz-Arslan, D., Itah, Z. et al. Cleavage of Atg3 protein by caspase-8 regulates autophagy during receptor-activated cell death. Apoptosis 17, 810–820 (2012). https://doi.org/10.1007/s10495-012-0735-0

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