The deubiquitinase Leon/USP5 regulates ubiquitin homeostasis during Drosophila development

Abstract

Ubiquitination and the reverse process deubiquitination regulate protein stability and function during animal development. The Drosophila USP5 homolog Leon functions as other family members of unconventional deubiquitinases, disassembling free, substrate-unconjugated polyubiquitin chains to replenish the pool of mono-ubiquitin, and maintaining cellular ubiquitin homeostasis. However, the significance of Leon/USP5 in animal development is still unexplored. In this study, we generated leon mutants to show that Leon is essential for animal viability and tissue integrity during development. Both free and substrate-conjugated polyubiquitin chains accumulate in leon mutants, suggesting that abnormal ubiquitin homeostasis caused tissue disorder and lethality in leon mutants. Further analysis of protein expression profiles in leon mutants shows that the levels of all proteasomal subunits were elevated. Also, proteasomal enzymatic activities were elevated in leon mutants. However, proteasomal degradation of ubiquitinated substrates was impaired. Thus, aberrant ubiquitin homeostasis in leon mutants disrupts normal proteasomal degradation, which is compensated by elevating the levels of proteasomal subunits and activities. Ultimately, the failure to fully compensate the dysfunctional proteasome in leon mutants leads to animal lethality and tissue disorder.

USP5 Is Dispensable for Monoubiquitin Maintenance in Drosophila

“…2A). Death at third instar larval stage is the same as observed with DmUSP5 null mutation (20). The level of knockdown obtained by the ubiquitous sqh-Gal4 driver was determined through qRT-PCR and is shown in Fig.…”

“…DmUSP5 is expressed throughout the fly (20); therefore, we targeted this protease in the whole organism by using the ubiquitous drivers sqh-Gal4, actin-Gal4, and da-Gal4 with similar results. We used three different RNAi lines specific for DmUSP5 that target different sites of its mRNA.…”

“…Our group and others previously reported that RNAi-dependent knockdown of DmUSP5 and mutations in its gene lead to developmental lethality in the fruit fly (16,19,20). To examine whether this DUB is also important in adults, we employed the power of the Gal4-UAS system (33).…”

“…Here, we tested this possibility in the fruit fly Drosophila melanogaster, whose USP5 is necessary during development (16,19,20). Our biochemical and genetic experiments indicate that Drosophila USP5 is not necessary for maintaining a ready pool of monoubiquitin in vivo.…”

Developmental- and tissue-specific changes of ubiquitin forms in Drosophila melanogaster.

“…It has been shown that the loss-of-function mutation in the Rpn10/p54 polyubiquitin receptor subunit of the proteasome [16,17] and in one of the DUB genes in Drosophila, Usp5 [18,19], leads to disruption of ubiquitin equilibrium and an excessive accumulation of free polyubiquitins and polyubiquitylated proteins. To quantitate these changes, we applied the immunoblot-based ubiquitin quantification assay to determine the steady-state distribution of ubiquitin forms in animals with impaired Rpn10/p54 and Usp5 functions and compared them to wild type data.…”