A unique deubiquitinase that deconjugates phosphoribosyl-linked protein ubiquitination

Qiu, Jiazhang; Yu, Kaiwen; Fei, Xiaowen; Liu, Yao; Nakayasu, Ernesto; Piehowski, Paul; Shaw, Jared; Puvar, Kedar; Das, Chittaranjan; Liu, Xiaoyun; Luo, Zhao‐Qing

doi:10.1038/cr.2017.66

Cited by 74 publications

(72 citation statements)

References 44 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2 A , lane 7 to 9). Furthermore, the L. pneumophila effector SidJ, which was previously reported as a PR-Ub specific DUB [28], showed no detectable changes of the PR-ubiquitinated signals when the PR-ubiquitinated substrate mixtures were incubated with purified recombinant SidJ and Calmodulin (Fig. 2 A , lane 10).…”

Section: Resultsmentioning

confidence: 78%

See 1 more Smart Citation

Deubiquitination of phosphoribosyl-ubiquitin conjugates by PDE domain-containingLegionellaeffectors

Wan

Sulpizio

Akturk

et al. 2019

Preprint

View full text Add to dashboard Cite

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...

show abstract

Section: Resultsmentioning

confidence: 78%

“…Expression of the Legionella effector SidJ, which is a negative regulator of SdeA activity, was able to suppress SdeA toxicity when the proteins were co-expressed ( SI Appendix , Fig. S9 D ) [28, 29]. As a result, we inquired whether DupA or DupB could similarly alleviate the toxicity of SdeA in yeast.…”

Section: Resultsmentioning

confidence: 99%

Deubiquitination of phosphoribosyl-ubiquitin conjugates by PDE domain-containingLegionellaeffectors

Wan

Sulpizio

Akturk

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…SidJ was first identified as a metaeffector that neutralizes the toxicity of the SidE family phosphoribosyl ubiquitin ligases in yeast (Havey and Roy, 2015; Jeong et al, 2015). 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), as well as in our unpublished studies.…”

Section: Discussionmentioning

confidence: 99%

Protein polyglutamylation catalyzed by the bacterial Calmodulin-dependent pseudokinase SidJ

Sulpizio

Minelli

Wan

et al. 2019

Preprint

View full text Add to dashboard Cite

23Pseudokinases are considered to be the inactive counterparts of conventional protein 24 kinases and comprise approximately 10% of the human and mouse kinomes. Here we report the 25 crystal structure of the Legionella pneumophila effector protein, SidJ, in complex with the 26 eukaryotic Ca 2+ -binding regulator, Calmodulin (CaM). The structure reveals that SidJ contains a 27 protein kinase-like fold domain, which retains a majority of the characteristic kinase catalytic 28 motifs. However, SidJ fails to demonstrate kinase activity. Instead, mass spectrometry and in vitro 29 biochemical analysis demonstrate that SidJ modifies another Legionella effector SdeA, an 30 unconventional phosphoribosyl ubiquitin ligase, by adding glutamate molecules to a specific 31 residue of SdeA in a CaM-dependent manner. Furthermore, we show that SidJ-mediated 32 polyglutamylation suppresses the ADP-ribosylation activity. Our work further implies that some 33 pseudokinases may possess ATP-dependent activities other than conventional phosphorylation. 34 35 36 KEYWORDS 37 SidJ; polyglutamylation; Legionella pneumophila; SdeA; phosphoribosyl ubiquitination; 38 ubiquitin 39 40 5effectors (Havey and Roy, 2015; Jeong et al., 2015; Urbanus et al., 2016). Furthermore, SidJ has 87 been shown to act on SidE proteins and releases these effectors from the LCV (Jeong et al., 2015). 88A recent study reported that SidJ functions as a unique deubiquitinase that counteracts the SidE-89 mediated phosphoribosyl-ubiquitination by deconjugating phosphoribosyl-ubiquitin from 90 modified proteins (Qiu et al., 2017). However, our recent results do not support this SidJ-mediated 91 deubiquitinase activity (Wan et al., 2019) and the exact function of SidJ remains elusive. 92The goal of the present study is to elucidate the molecular function of SidJ and to 93 investigate the mechanism that underlies how SidJ antagonizes the PR-ubiquitination activity of 94 SidEs. Here we report the crystal structure of SidJ in complex with human Calmodulin 2 (CaM) 95 and reveal that SidJ adopts a protein kinase-like fold. A structural comparison allowed us to 96 identify all the catalytic motifs conserved in protein kinases. However, SidJ failed to demonstrate 97 protein kinase activity. Using SILAC (Stable Isotope Labeling by Amino acids in Cell culture) 98 based mass spectrometry approach, we discovered that SidJ modifies SdeA by attaching the amino 99 acid glutamate to a key catalytic residue on SdeA. Moreover, we found that this glutamylation 100 activity by SidJ is CaM dependent and the glutamylation of SdeA suppresses its PR-ubiquitination 101 activity. Thus our work provides molecular insights of a key PR-ubiquitination regulator in 102 Legionella infection. We anticipate that our work will also have impact on the studies of 103 pseudokinases and CaM-regulated cellular processes. 104 6 Results 105 SidJ Binds CaM through its C-terminal IQ Motif 106To elucidate the biological function of SidJ, we performed sequence analyses and found 107 that the C-terminus of SidJ c...

show abstract

“…SidJ encoded by L. pneumophila counteracts SidE toxicity [246,253]. As prolonged activation of SdeA might be also unfavourable for bacterial growth inducing host cell death [254], Legionella may have evolved SidJ as a deubiquitinase enzyme described for its ability to hydrolyse the phosphodiester linkage of phosphoribosyl-ubiquitinated substrates [255].…”

Section: Adp-ribosylation-dependent Ubiquitination As Mechanism Of Pamentioning

confidence: 99%

Targeting ADP-ribosylation as an antimicrobial strategy

Catara

Corteggio

Valente

et al. 2019

Biochemical Pharmacology

View full text Add to dashboard Cite

A B S T R A C T ADP-ribosylation (ADPr) is an ancient reversible modification of cellular macromolecules controlling major biological processes as diverse as DNA damage repair, transcriptional regulation, intracellular transport, immune and stress responses, cell survival and proliferation. Furthermore, enzymatic reactions of ADPr are central in the pathogenesis of many human diseases, including infectious conditions. By providing a review of ADPr signalling in bacterial systems, we highlight the relevance of this chemical modification in the pathogenesis of human diseases depending on host-pathogen interactions. The post-antibiotic era has raised the need to find alternative approaches to antibiotic administration, as major pathogens becoming resistant to antibiotics. An in-depth understanding of ADPr reactions provides the rationale for designing novel antimicrobial strategies for treatment of infectious diseases. In addition, the understanding of mechanisms of ADPr by bacterial virulence factors offers important hints to improve our knowledge on cellular processes regulated by eukaryotic homologous enzymes, which are often involved in the pathogenesis of human diseases.T large number of worldwide spread diseases, affecting humans, insects and plants [31,[56][57][58], with a great impact on human health. In addition, infectious diseases strongly affect the world economy in terms of costs for the human health as well as food and agricultural economic losses [59]. The use of antibiotics to counteract the pathogen infections has represented the therapeutic strategy of choice in the last century, however the emergence of antibiotic resistance strains represents the major challenge of the 21st century [60][61][62]. Targeting bacterial pathogenic mechanisms by disarming pathogens of virulence factors, for instance by inhibiting the enzymatic activity of bART, represents a valid alternative intervention strategy to overcome antibiotic resistance [63][64][65][66]. In addition, because of the conservation of ADPr systems throughout the evolution, the understanding of bacterial pathogenic mechanisms may contribute to unveil novel and conserved cellular processes regulated by ADPr in high organisms [34,45,67,68]. The dysregulation of such processes can be either cause of human diseases or be target of therapeutic intervention [39,[69][70][71].Herein, we will provide a summary of bacterial ADPr systems describing their pathogenic mechanisms and highlighting the similarities with ADPr systems in mammals as well. Further, we discuss the potential of targeting ADPr for therapeutic strategies of infection diseases. ADP-ribosylation in pathophysiologyADPr was originally described in the 60s; two independent studies reported the identification of a new polymer, poly(ADP-ribose) (PAR) synthesised from NAD + in vertebrate cells [72] and, simultaneously, the finding that bacterial DTX activity from C. diphteriae is dependent on NAD + content [73]. In the following decades, research in the field has expanded the involvement of ADPr ...

show abstract

A unique deubiquitinase that deconjugates phosphoribosyl-linked protein ubiquitination

Cited by 74 publications

References 44 publications

Deubiquitination of phosphoribosyl-ubiquitin conjugates by PDE domain-containingLegionellaeffectors

Deubiquitination of phosphoribosyl-ubiquitin conjugates by PDE domain-containingLegionellaeffectors

Protein polyglutamylation catalyzed by the bacterial Calmodulin-dependent pseudokinase SidJ

Targeting ADP-ribosylation as an antimicrobial strategy

Contact Info

Product

Resources

About