The deubiquitinating enzyme DUBAI stabilizes DIAP1 to suppress Drosophila apoptosis

Abstract

Deubiquitinating enzymes (DUBs) counteract ubiquitin ligases to modulate the ubiquitination and stability of target signaling molecules. In Drosophila, the ubiquitin-proteasome system has a key role in the regulation of apoptosis, most notably, by controlling the abundance of the central apoptotic regulator DIAP1. Although the mechanism underlying DIAP1 ubiquitination has been extensively studied, the precise role of DUB(s) in controlling DIAP1 activity has not been fully investigated. Here we report the identification of a DIAP1-directed DUB using two complementary approaches. First, a panel of putative Drosophila DUBs was expressed in S2 cells to determine whether DIAP1 could be stabilized, despite treatment with death-inducing stimuli that would induce DIAP1 degradation. In addition, RNAi fly lines were used to detect modifiers of DIAP1 antagonist-induced cell death in the developing eye. Together, these approaches identified a previously uncharacterized protein encoded by CG8830, which we named DeUBiquitinating-Apoptotic-Inhibitor (DUBAI), as a novel DUB capable of preserving DIAP1 to dampen Drosophila apoptosis. DUBAI interacts with DIAP1 in S2 cells, and the putative active site of its DUB domain (C367) is required to rescue DIAP1 levels following apoptotic stimuli. DUBAI, therefore, represents a novel locus of apoptotic regulation in Drosophila, antagonizing cell death signals that would otherwise result in DIAP1 degradation.

Expansion of DUB functionality generated by alternative isoforms – USP35, a case study

“…Hence, USP35 is a hitherto unstudied DUB that regulates programmed cell death. This is consistent with the role of DUBAI, the D. melanogaster orthologue of both mammalian USP35 and the related DUB USP38 (Yang et al, 2014). DUBAI protects cells from apoptosis by stabilising DIAP1, a major IAP in fly.…”

“…The D. melanogaster orthologue of USP35 and USP38, DUBAI, has previously been shown to be an anti-apoptotic protein (Yang et al, 2014). To test whether USP35 iso1 has the same function in mammalian cells, we monitored apoptosis in HEK293 cells overexpressing USP35 iso1 following treatment with the protein TRAIL, an apoptotic stimulus, by monitoring cleavage of caspase-8, the main initiator caspase of the extrinsic apoptotic pathway.…”

The deubiquitinase Leon/USP5 regulates ubiquitin homeostasis during Drosophila development

“…There are 41 DUBs in the Drosophila genome with only a handful of them being revealed for their developmental roles [8]. Nonstop is required for photoreceptor axon growth regulated by glial cells [9], Fat facets regulates the ubiquitination status of Liquid facets in retina patterning [10], and DUBAI stabilizes DIAP1 to suppress apoptosis [11]. However, the functions for most of Drosophila DUBs are still unknown.…”

Screening for E3-Ubiquitin ligase inhibitors: challenges and opportunities

“…Ubiquitination is a dynamic process that is negatively regulated by deubiquitinases (DUBs). These enzymes catalyse the deconjugation of ubiquitin from substrates or ubiquitin chains, acting as important regulators of the ubiquitin machinery [26-28]. …”