ATG5 Promotes Death Signaling in Response to the Cyclic Depsipeptides Coibamide A and Apratoxin A.

Xuemei Wan,Michelle Tan,Ian Ganley,Jeffrey Serrill,Ian Humphreys,Jane Ishmael,Kerry Mcphail

doi:10.3390/md16030077

Abstract

Our understanding of autophagy and lysosomal function has been greatly enhanced by the discovery of natural product structures that can serve as chemical probes to reveal new patterns of signal transduction in cells. Coibamide A is a cytotoxic marine natural product that induces mTOR-independent autophagy as an adaptive stress response that precedes cell death. Autophagy-related (ATG) protein 5 (ATG5) is required for coibamide-induced autophagy but not required for coibamide-induced apoptosis. Using wild-type and autophagy-deficient mouse embryonic fibroblasts (MEFs) we demonstrate that coibamide-induced toxicity is delayed in ATG5−/− cells relative to ATG5+/+ cells. Time-dependent changes in annexin V staining, membrane integrity, metabolic capacity and caspase activation indicated that MEFs with a functional autophagy pathway are more sensitive to coibamide A. This pattern could be distinguished from autophagy modulators that induce acute ER stress (thapsigargin, tunicamycin), ATP depletion (oligomycin A) or mTORC1 inhibition (rapamycin), but was shared with the Sec61 inhibitor apratoxin A. Coibamide- or apratoxin-induced cell stress was further distinguished from the action of thapsigargin by a pattern of early LC3-II accumulation in the absence of CHOP or BiP expression. Time-dependent changes in ATG5-ATG12, PARP1 and caspase-3 expression patterns were consistent with the conversion of ATG5 to a pro-death signal in response to both compounds.

Highlights

Macroautophagy and apoptosis are most conveniently studied as two distinct signal transduction pathways, the capacity for cross-talk between these processes has been appreciated for many years [1,2,3,4]
Given that coibamide A is capable of triggering apoptosis in the presence or absence of autophagy, we extended our analysis of wild-type and autophagy-deficient mouse embryonic fibroblasts (MEFs) to gain a better understanding of the role of coibamide-induced autophagy in cell fate
PARP1 and these results demonstrate that autophagy-competent cells are more vulnerable to coibamide Ainduced apoptosis than autophagy-deficient MEFs in a pattern that does not generalize to several other well characterized modulators of autophagy

Summary

Introduction

Macroautophagy (hereafter abbreviated to autophagy) and apoptosis are most conveniently studied as two distinct signal transduction pathways, the capacity for cross-talk between these processes has been appreciated for many years [1,2,3,4]. Autophagy can be induced beyond normal physiological levels as an early adaptive response to internal or external stress as cells attempt to maintain homeostasis through the “self-eating” of proteins and organelles [2]. In this survival mode, such as that triggered by starvation or exposure to an exogenous chemical, autophagy is favored and apoptosis signaling is inhibited [5]. If internal or environmental stress is sustained or cells fail to adapt, autophagy signaling can be suppressed or actively used by the cell to favor death signaling. The dynamic and context-specific nature of autophagy represents a current challenge to the development of autophagy modulators as therapeutic agents for

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