Identification and characterization of diverse OTU deubiquitinases in bacteria

Abstract

25Manipulation of host ubiquitin signaling is becoming an increasingly apparent evolutionary 26 strategy among bacterial and viral pathogens. By removing host ubiquitin signals, for example, 27 invading pathogens can inactivate immune response pathways and evade detection. The Ovarian 28Tumor (OTU) family of deubiquitinases regulates diverse ubiquitin signals in humans. Viral 29 pathogens have also extensively co-opted the OTU fold to subvert host signaling, but the extent 30 to which bacteria utilize the OTU fold was unknown. We have predicted and validated a set of 31 OTU deubiquitinases encoded by several classes of pathogenic bacteria. Biochemical assays 32 highlight the ubiquitin and polyubiquitin linkage specificities of these bacterial deubiquitinases. 33By determining the ubiquitin-bound structures of two examples, we demonstrate the novel 34 strategies that have evolved to both thread an OTU fold and to recognize a ubiquitin substrate. 35With these new examples, we perform the first cross-kingdom structural analysis of the OTU 36 fold that highlights commonalities among distantly-related OTU deubiquitinases. 37 38 KEYWORDS 39

An evolutionary approach to systematic discovery of novel deubiquitinases, applied to Legionella

“…Structurally, all known DUBs more or less closely follow the papain fold, which already narrows down their evolutionary origins ( 34 ). The papain fold, which in MEROPS is typically represented by the CA-clan, is also found in the CE clan as a circularly permuted form; several recently identified OTU-type bacterial deubiquitinases also show circular rearrangement of the core structural elements ( 35 ). However, the papain fold is also seen in many non-DUB proteases and our HMM-to-HMM comparisons shown in Figs 1 and 3A reveal significant sequence relationships only between some DUB and UbL-protease families such as ATX3-UCH or ZUFSP-UFSP-ATG4, leaving other families unconnected.…”

Legionella Manipulates Non-canonical SNARE Pairing Using a Bacterial Deubiquitinase

“…The DlotB single deletion strain did not show any differences in intracellular growth from the wild-type strain, which indicates that intracellular replication of L. pneumophila is the result of multiple cascades of reactions mediated by many effector proteins with functional redundancy as described previously (Isberg et al, 2009). While this manuscript was under revision, studies on the biochemical characterization of LotB were reported (Ma et al, 2020;Schubert et al, 2020;Shin et al, 2020). In these studies, it was proven that LotB functions as a DUB with high specificity toward K63-linked ubiquitin chains.…”

An evolutionary approach to systematic discovery of novel deubiquitinases, applied to Legionella

“…Structurally, all known DUBs more or less closely follow the papain fold, which already narrows down their evolutionary origins [34]. The papain fold, which in MEROPS is typically represented by the CA-clan, is also found in the CE-clan as a circularly permuted form; several recently identified OTU-type bacterial deubiquitinases also show circular rearrangement of the core structural elements [35]. However, the papain fold is also seen in many non-DUB proteases, and our HMM-to-HMM comparisons shown in figures 1 and 3a reveal significant sequence relationships only between some DUB and UBL-protease families such as ATX3-UCH or ZUFSP-UFSP-ATG4, leaving other families unconnected.…”