Abstract
Death receptors are transmembrane proteins that can induce the activation of caspase-8 upon ligand binding, initiating apoptosis. Recent work has highlighted the great molecular complexity of death receptor signalling, in particular through ubiquitination/deubiquitination. We have earlier defined the deubiquitinase Ubiquitin-Specific Protease 27x (Usp27x) as an enzyme capable of stabilizing the pro-apoptotic Bcl-2 family member Bim. Here, we report that enhanced expression of Usp27x in human melanoma cells leads to the loss of cellular FLICE-like inhibitory protein (cFLIP) and sensitizes to Tumor necrosis factor receptor 1 (TNF-R1) or Toll-like receptor 3 (TLR3)-induced extrinsic apoptosis through enabling enhanced processing of caspase-8. The loss of cFLIPL upon overexpression of Usp27x was not due to reduced transcription, could be partially counteracted by blocking the ubiquitin proteasome system and was independent of the known cFLIPL destabilizing ubiquitin E3-ligases Itch and DTX1. Instead, Usp27x interacted with the E3-ligase TRIM28 and reduced ubiquitination of TRIM28. Reduction of cFLIPL protein levels by Usp27x-induction depended on TRIM28, which was also required for polyI:C-induced cell death. This work defines Usp27x as a novel regulator of cFLIPL protein expression and a deubiquitinase in fine tuning death receptor signalling pathways to execute apoptosis.
Highlights
One of the two major apoptosis pathways starts at the cell surface with signals from death receptors such as TNFR1 and Fas/CD95; Toll-like receptor 3 (TLR3), a pattern recognition receptor that recognizes double-stranded RNA and is expressed by many tumour cells, can assemble a similar death-inducing signalling complex (DISC) and trigger similar death-inducing events [1].The DISC consists of the assembly of a number of proteins and orchestrates the activation of caspase-8
Using custom-made Ubiquitin-Specific Protease 27x (Usp27x) antibodies, Atanassov et al reported a size of human and mouse Usp27x of ~72 kDa, which differs from the estimated size of 50 kDa of annotated Usp27x [24]
This band was less pronounced in cells genomically deficient for Usp27x (Fig. 1B, C), and this protein was not immunoprecipitated under native conditions from Usp27x-deficient Caco2 cells (Fig. 1C)
Summary
One of the two major apoptosis pathways starts at the cell surface with signals from death receptors such as TNFR1 and Fas/CD95; Toll-like receptor 3 (TLR3), a pattern recognition receptor that recognizes double-stranded RNA (dsRNA) and is expressed by many tumour cells, can assemble a similar death-inducing signalling complex (DISC) and trigger similar death-inducing events [1].The DISC consists of the assembly of a number of proteins and orchestrates the activation of caspase-8. One of the two major apoptosis pathways starts at the cell surface with signals from death receptors such as TNFR1 and Fas/CD95; Toll-like receptor 3 (TLR3), a pattern recognition receptor that recognizes double-stranded RNA (dsRNA) and is expressed by many tumour cells, can assemble a similar death-inducing signalling complex (DISC) and trigger similar death-inducing events [1]. Ubiquitin itself is targeted for ubiquitination at various lysine (K) or at the N-terminal methionine (M), resulting in polyubiquitination of different linkage types that have different outcomes due to the recruitment of proteins with different ubiquitin binding domains (UBDs) [5, 6]. K63-linked ubiquitination of RIPK1 at the DISC of TNFR1 by cIAP1/2 leads to the recruitment TAK1, formation of the linear ubiquitin chain assembly complex (LUBAC) and subsequent M1-ubiquitination of RIPK1 by LUBAC, resulting in the activation of the pro-survival NF-κΒ [7].
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