Deubiquitinating enzymes (DUBs) are proteases that control the post-translational modification of proteins by ubiquitin and in turn regulate diverse cellular pathways. Despite a growing understanding of DUB biology at the structural and molecular level, little is known about the physiological importance of most DUBs. Here, we systematically identify DUBs encoded by the genome of Drosophila melanogaster and examine their physiological importance in vivo. Through domain analyses we uncovered 41 Drosophila DUBs, most of which have human orthologues. Systematic knockdown of the vast majority of DUBs throughout the fly or in specific cell types had dramatic consequences for Drosophila development, adult motility or longevity. Specific DUB subclasses proved to be particularly necessary during development, while others were important in adults. Several DUBs were indispensable in neurons or glial cells during developmental stages; knockdown of others perturbed the homeostasis of ubiquitinated proteins in adult flies, or had adverse effects on wing positioning as a result of neuronal requirements. We demonstrate the physiological significance of the DUB family of enzymes in intact animals, find that there is little functional redundancy among members of this family of proteases, and provide insight for future investigations to understand DUB biology at the molecular, cellular and organismal levels.

OTUB1 is a deubiquitinating enzyme that cleaves Lys-48–linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2∼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1–E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1−/− knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.

A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild‐type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight‐binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30‐fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.

Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction for combined single-platform analysis, examining the viability of NLO microscopy as an alternative to the conventional X-ray raster scan for the purposes of sample centering. Second-harmonic generation microscopy and two-photon excited ultraviolet fluorescence microscopy were evaluated for crystal detection, and assessed by X-ray raster scanning.

Ubiquitination is countered by a group of enzymes collectively called deubiquitinases (DUBs) - about 100 of them can be found in the human genome. One of the most interesting aspects of these enzymes is the ability of some members to selectively recognize specific linkage types between ubiquitin in polyubiquitin chains and their endo and exo specificity. The structural basis of exo-specific deubiquitination catalyzed by a DUB is poorly understood. UCH37, a cysteine DUB conserved from fungi to humans, is a proteasome-associated factor that regulates the proteasome by sequentially cleaving polyubiquitin chains from their distal ends, i.e., by exo-specific deubiquitination. In addition to the catalytic domain, the DUB features a functionally uncharacterized UCH37-like domain (ULD), presumed to keep the enzyme in an inhibited state in its proteasome-free form. Herein we report the crystal structure of two constructs of UCH37 from Trichinella spiralis in complex with a ubiquitin-based suicide inhibitor, ubiquitin vinyl methyl ester (UbVME). These structures show that the ULD makes direct contact with ubiquitin stabilizing a highly unusual intra-molecular salt bridge between Lys48 and Glu51 of ubiquitin, an interaction that would be favored only with the distal ubiquitin but not with the internal ones in a Lys48-linked polyubiquitin chain. An inspection of 39 DUB-ubiquitin structures in the protein data bank reveals the uniqueness of the salt bridge in ubiquitin bound to UCH37, an interaction that disappears when the ULD is deleted, as revealed in the structure of the catalytic domain alone bound to UbVME. The structural data are consistent with previously reported mutational data on the mammalian enzyme, which, together with the fact that the ULD residues that bind to ubiquitin are conserved, points to a similar mechanism behind the exo specificity of the human enzyme. To the best of our knowledge, these data provide the only structural example so far of how the exo specificity of a DUB can be determined by its non-catalytic domain. Thus, our data show that, contrary to its proposed inhibitory role, the ULD actually contributes to substrate recognition and could be a major determinant of proteasome-associated function of UCH37. Moreover, our structures show that the unproductively oriented catalytic cysteine in the free enzyme is aligned correctly when ubiquitin binds, suggesting a mechanism for ubiquitin selectivity.

OTUB1 is a highly expressed cysteine protease that specifically cleaves K48-linked polyubiquitin chains. This unique deubiquitinating enzyme (DUB) can bind to a subset of E2 ubiquitin conjugating enzymes, forming complexes in which the two enzymes can regulate one another’s activity. OTUB1 can noncatalytically suppress the ubiquitin conjugating activity of its E2 partners by sequestering the charged E2∼Ub thioester and preventing ubiquitin transfer. The same E2 enzymes, when uncharged, can stimulate the DUB activity of OTUB1 in vitro, although the importance of OTUB1 stimulation in vivo remains unclear. To assess the potential balance between these activities that might occur in cells, we characterized the kinetics and thermodynamics governing the formation and activity of OTUB1:E2 complexes. We show that both stimulation of OTUB1 by E2 enzymes and noncatalytic inhibition of E2 enzymes by OTUB1 occur at physiologically relevant concentrations of both partners. Whereas E2 partners differ in their ability to stimulate OTUB1 activity, we find that this variability is not correlated with the affinity of each E2 for OTUB1. In addition to UBE2N and the UBE2D isoforms, we find that OTUB1 inhibits the polyubiquitination activity of all three UBE2E enzymes, UBE2E1, UBE2E2, and UBE2E3. Interestingly, although OTUB1 also inhibits the auto-monoubiquitination and autopolyubiquitination activity of UBE2E1 and UBE2E2, it is unable to suppress autoubiquitination by UBE2E3. Our quantitative analysis provides a basis for further exploring the biological roles of OTUB1:E2 complexes in cells.

The development of the lesions of swine dysentery in surgically isolated colonic loops of growing pigs was studied. Operated, uninoculated pigs served as controls, and test pigs received one of the following preparations: Vibrio coli in saline, V. coli in gastric mucin, crude minced colon from pigs with active dysentery, and filtered minced colon from pigs with active dysentery. A comparison was made of lesions of dysentery in isolated colonic loops with those that developed after oral inoculation of normal pigs. When inoculated into the surgical loops, filtered and unfiltered crude minced colon produced lesions of dysentery within 72 h after inoculation. Intact animals orally inoculated with the same material were unaffected up to 196 h. Use of the isolated loop may be a more sensitive system for the development of lesions of swine dysentery. Direct instillation of loops with V. coli preparations did not produce lesions.

A common strategy for studying the biological role of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8 C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.