Abstract

During apoptosis induction by TNF, the extrinsic and intrinsic apoptosis pathways converge at the lysosomal-mitochondrial interface. Earlier studies showed that the lysosomal aspartic protease Cathepsin D (CtsD) cleaves Bid to tBid, resulting in the amplification of the initial apoptotic cascade via mitochondrial outer membrane permeabilization (MOMP).The goal of this study was to identify further targets for CtsD that might be involved in activation upon death receptor ligation. Using a proteomics screen, we identified the heat shock protein 90 (HSP90) to be cleaved by CtsD after stimulation of U937 or other cell lines with TNF, FasL and TRAIL. HSP90 cleavage corresponded to apoptosis sensitivity of the cell lines to the different stimuli. After mutation of the cleavage site, HSP90 partially prevented apoptosis induction in U937 and Jurkat cells. Overexpression of the cleavage fragments in U937 and Jurkat cells showed no effect on apoptosis, excluding a direct pro-apoptotic function of these fragments. Pharmacological inhibition of HSP90 with 17AAG boosted ligand mediated apoptosis by enhancing Bid cleavage and caspase-9 activation. Together, we demonstrated that HSP90 plays an anti-apoptotic role in death receptor signalling and that CtsD-mediated cleavage of HSP90 sensitizes cells for apoptosis. These findings identify HSP90 as a potential target for cancer therapy in combination with death ligands (e.g. TNF or TRAIL).

Highlights

  • Tumor necrosis factor alpha (TNF-α) is a pleiotropic cytokine involved in regulating various cellular processes such as inflammation, differentiation, proliferation and apoptosis induction

  • Which is critical for lysosomal membrane permeabilization (LMP), which often serves to amplify the cell death signalling, but can be an initial trigger [7, 14]

  • We demonstrated a role for the aspartic protease Cathepsin D (CtsD) in TNF induced apoptosis signalling

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Summary

Introduction

Tumor necrosis factor alpha (TNF-α) is a pleiotropic cytokine involved in regulating various cellular processes such as inflammation, differentiation, proliferation and apoptosis induction. TNF can trigger signal transduction via two different members of the TNF-receptor superfamily: TNF-receptor 1 (TNF-R1) and TNFreceptor 2 (TNF-R2). TNF-R1 is a member of the deathreceptor subgroup of this protein family sharing the so called “death domain” (DD) in their C-terminal domain, which enables them to trigger apoptosis induction [1]. The switch between non-apoptotic and apoptotic signal transduction induced by TNF-R1 depends on the selective recruitment of adaptor proteins to its intracellular part. We have shown that different adaptor proteins are recruited depending on the subcellular localization and ubiquitination state of the receptor. Internalized, activated TNF-R1 recruits FADD and caspase-8 to the so called “complex II” and initiaties apoptosis induction [2,3,4,5,6]. Caspase-8 is activated by autoproteolysis and subsequently activates caspase-3 directly or via a lysosomal-mitochondrial amplification loop that involves the release of cytochrome C and APAF-1, and formation of the apoptosome with caspase-9 [7]

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