Characterisation of de-ubiquitylating enzymes in the regulation of apoptosis
Final Report Abstract
We have studied two previously uncharacterised Drosophila de-ubiquitylating enzymes of the OTU family, which had been identified as potential regulators of apoptosis in an in vivo RNAi screen. 1) CG6091: We have named this gene Duba according to its mammalian orthologue. Loss of Duba suppressed apoptosis induced by the IAP-antagonists Reaper and Hid in the fly eye. The initiator caspase Dronc, which is regulated by ubiquitylation, was identified as interaction partner of Duba and Duba was able to de-ubiquitylate Dronc in vitro, suggesting a potential mechanism how Duba is involved in regulation of IAP-antagonist-induced apoptosis. However, Duba nullmutant flies did not display defects in developmental apoptosis, suggesting that Duba might regulate caspases in non-apoptotic settings. Duba null mutant males were sterile and showed defects in spermatid individualisation, a process that depends on non-apoptotic caspase activity. We showed that fertility could be restored by genetic rescue with Duba but not with catalytically inactive Duba, confirming Duba’s role as de-ubiquitylating enzyme during spermatogenesis. Further experiments have shown that Duba preferentially cleaves ubiquitin chains with K11- or K63-linkage and that the enzyme needs to be activated by phosphorylation at Serine 183 in vitro and in vivo. 2) CG3251: Our experiments could not pinpoint a mechanism for the observed modulation of apoptotic eye phenotypes by CG3251 RNAi. We found that CG3251 is not an active de-ubiquitylating enzyme. The first reason for this seemed a replacement of a catalytic cysteine residue with serine. However, additional critical residues must have been lost because re-mutation to cysteine did not restore activity. This was also true for otu. otu and CG3251 are shared homologues of human OTUD4. These findings prompted a systematic bioinformatical analysis of genes of the OTUD4 family. In this family, replacement of cysteine with serine seems to have occurred in multiple species in an independent way, suggesting that catalytic activity is dispensable.
Publications
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Evolutionary Loss of Activity in De-Ubiquitylating Enzymes of the OTU Family. PLoS One. 2015 Nov 20;10(11)
Louis M, Hofmann K, Broemer M
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The de-ubiquitylating enzyme DUBA is essential for spermatogenesis in Drosophila. Cell Death Differentiation, 2016 Dec;23(12):2019-2030
Koerver L, Melzer J, Aguado Roca E, Teichert D, Glatter T, Arama E, Broemer M
