Deubiquitination of phosphoribosyl-ubiquitin conjugates by PDE domain-containingLegionellaeffectors

Abstract

24Posttranslational protein modification by ubiquitin (Ub) is a central eukaryotic mechanism 25 that regulates a plethora of physiological processes. Recent studies unveiled an unconventional 26 type of ubiquitination mediated by the SidE family of Legionella pneumophila effectors, such as 27 SdeA, that catalyzes the conjugation of Ub to a serine residue of target proteins via a 28 phosphoribosyl linker (hence named PR-ubiquitination). Comparable to the deubiquitinases 29 (DUBs) in the canonical ubiquitination pathway, here we show that two Legionella effectors, 30 named DupA (deubiquitinase for PR-ubiquitination) and DupB, reverse PR-ubiquitination by 31 specific removal of phosphoribosyl-Ub (PR-Ub) from substrates. Both DupA and DupB are fully 32 capable of rescuing the Golgi fragmentation phenotype caused by exogenous expression of SdeA 33 in mammalian cells. We further show that deletion of these two genes results in significant 34 accumulation of PR-ubiquitinated species in host cells infected with Legionella. In addition, we 35 have identified a list of specific PR-ubiquitinated host targets and show that DupA and DupB play 36 a role in modulating the association of PR-ubiquitinated host targets with Legionella containing 37 vacuoles (LCV). Together, our data establish a complete PR-ubiquitination and deubiquitination 38 cycle and demonstrate the intricate control that Legionella has over this unusual Ub-dependent 39 posttranslational modification.40 3 Statement of significance 41 Ubiquitination is a vital posttranslational modification in eukaryotes. A variety of 42 microbial pathogens exploit this pathway during their infection. Legionella pneumophila, the 43 causative bacterial pathogen of Legionnaires' disease, has been show to hijack host ubiquitination 44 pathway via a large number of effectors. Recent studies revealed a family of effectors catalyzing 45 a novel type of Ub-dependent posttranslational modification, namely PR-ubiquitination. Here we 46 report two new players, DupA and DupB, involved in this unconventional pathway. We found that 47 DupA and DupB function as PR-Ub specific DUBs and play a role in regulating the PR-48 ubiquitination levels of host targets. Our results not only provide an expanding view of the PR-49 ubiquitination pathway, but may also facilitate the future identification of PR-ubiquitination 50 pathways in eukaryotes. 51 4 \body 52 Introduction 53Ubiquitin (Ub), a 76 amino acid protein, is attached to specific proteins as a potent 54 posttranslational mechanism. Ubiquitination plays an essential role in a broad aspect of cellular 55 processes, including protein homeostasis [1], cell signaling [2], and membrane trafficking [3, 4]. 56 Following the conventional scheme of ubiquitination, Ub is covalently coupled to lysine residues 57 on target proteins via the sequential activities of a collection of enzymes known as E1, E2, and 58 E3s [5]. The C-terminal glycine residue of Ub is first activated and covalently linked to the 59 catalytic cysteine residue of the Ub act...

Protein polyglutamylation catalyzed by the bacterial calmodulin-dependent pseudokinase SidJ

“…A previous publication assigned SidJ as a deubiquitinase that deconjugates phosphoribosyl-linked protein ubiquitination (Qiu et al, 2017). However, this unusual deubiquitination activity was not repeatable in another study (Black et al, 2019), or in our recently published studies (Wan et al, 2019). The definitive biochemical function of SidJ is now revealed in this study, and in recent reports (Bhogaraju et al, 2019; Black et al, 2019; Gan et al, 2019), as a polyglutamylase that adds glutamates to a specific catalytic residue E860 of SdeA and subsequently inhibits the PR-ubiquitination activity of SdeA.…”

“…A recent study reported that SidJ functions as a unique deubiquitinase that counteracts the SidE-mediated phosphoribosyl-ubiquitination by deconjugating phosphoribosyl-ubiquitin from modified proteins (Qiu et al, 2017). However, our recent results do not support this SidJ-mediated deubiquitinase activity (Wan et al, 2019) and the exact function of SidJ remains elusive.…”

Ubiquitin‐targeted bacterial effectors: rule breakers of the ubiquitin system

“…In the absence of suitable substrate, the SidE PDE domain will hydrolyze ADPr-Ub to Pr-Ub. Reversal of SidE-catalyzed ubiquitination by DupA and DupB also produces Pr-Ub as a byproduct (Wan et al, 2019a;Shin et al, 2020b). Pr-Ub exhibits a defect in passing through conventional E1/E2/E3 conjugation enzymes, and if levels accumulate becomes toxic in cells (Bhogaraju et al, 2016).…”

“…When expressed on their own in yeast or human cell culture, SidE proteins are highly toxic (Havey & Roy, 2015;Jeong et al, 2015;Bhogaraju et al, 2016). Interestingly, it is specifically mART function and not DUB activity or PDE activity that inhibits yeast growth, suggesting ADPr-Ub-dependent stress on the ubiquitin system (Havey & Roy, 2015;Wan et al, 2019a). While neither DupA nor DupB can rescue yeast cells that co-express SdeA, expression of another Legionella effector called SidJ can (Havey & Roy, 2015;Jeong et al, 2015;Wan et al, 2019a).…”

Modulation of phagosome phosphoinositide dynamics by a Legionella phosphoinositide 3‐kinase

“…By controlling the activation cycle of Rab1, SidM/DrrA facilitates the fusion of ER-derived vesicles to the LCV (Arasaki et al, 2012). The hijacking of the activity of Rab1 and other Rab small GTPases is further regulated by several effector-induced reversible modifications, including AMPylation by SidM/DrrA (Muller et al, 2010) and SidD (Neunuebel et al, 2011;, phosphorylcholination by AnkX (Mukherjee et al, 2011; and Lem3 , phosphoribosyl-linked serine ubiquitination by members of SidEs (Bhogaraju et al, 2016;Qiu et al, 2016;Kotewicz et al, 2017), SidJ (Bhogaraju et al, 2019;Black et al, 2019;Gan et al, 2019;Sulpizio et al, 2019), DupA and DupB (Wan et al, 2019;Shin et al, 2020). These different layers of regulation appear to occur at distinct cellular locations and times during the intracellular life cycle of L. pneumophila (Qiu & Luo, 2017).…”