Abstract
The HOIP ubiquitin E3 ligase generates linear ubiquitin chains by forming a complex with HOIL‐1L and SHARPIN in mammals. Here, we provide the first evidence of linear ubiquitination induced by a HOIP orthologue in Drosophila. We identify Drosophila CG11321, which we named Linear Ubiquitin E3 ligase (LUBEL), and find that it catalyzes linear ubiquitination in vitro. We detect endogenous linear ubiquitin chain‐derived peptides by mass spectrometry in Drosophila Schneider 2 cells and adult flies. Furthermore, using CRISPR/Cas9 technology, we establish linear ubiquitination‐defective flies by mutating residues essential for the catalytic activity of LUBEL. Linear ubiquitination signals accumulate upon heat shock in flies. Interestingly, flies with LUBEL mutations display reduced survival and climbing defects upon heat shock, which is also observed upon specific LUBEL depletion in muscle. Thus, LUBEL is involved in the heat response by controlling linear ubiquitination in flies.
Highlights
Linear ubiquitin chains play an important role in the regulation of immune responses in mammals [1,2,3,4,5]
Linear Ubiquitin E3 ligase” (LUBEL) does not contain the N-terminal PNGase/ Ub-associated (PUB) domain found in HOIL-1-interacting protein” (HOIP), which forms a critical interaction with the deubiquitinase (DUB) OTULIN [19,20,21]
We found that OTULIN, but not a catalytically dead OTULIN mutant (Cys 129 Ala) or vOTU, cleaved the ubiquitin chains generated by LUBEL-RING in-between RING (RBR)-C in vitro (Fig 2B)
Summary
Linear ubiquitin chains play an important role in the regulation of immune responses in mammals [1,2,3,4,5]. HOIP was found to play an essential role during mouse embryonic development and cell death, based on phenotypes of HOIP-deficient mice and catalytically dead HOIP knockin mice [14,17]. Taken together, these findings suggest that HOIP-dependent linear ubiquitination plays a critical role in development and immune response signaling pathways.
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