Cellular & Molecular Immunology (2016) 13, 560–576 & 2016 CSI and USTC All rights reserved 2042-0226/16 www.nature.com/cmi

REVIEW

The ubiquitin system: a critical regulator of innate immunity and pathogen–host interactions

Jie Li, Qi-Yao Chai and Cui Hua Liu

The ubiquitin system comprises enzymes that are responsible for ubiquitination and deubiquitination, as well as ubiquitin receptors that are capable of recognizing and deciphering the ubiquitin code, which act in coordination to regulate almost all host cellular processes, including host–pathogen interactions. In response to pathogen infection, the host innate immune system launches an array of distinct antimicrobial activities encompassing inflammatory signaling, phagosomal maturation, autophagy and apoptosis, all of which are fine-tuned by the ubiquitin system to eradicate the invading pathogens and to reduce concomitant host damage. By contrast, pathogens have evolved a cohort of exquisite strategies to evade host innate immunity by usurping the ubiquitin system for their own benefits. Here, we present recent advances regarding the ubiquitin system-mediated modulation of host–pathogen interplay, with a specific focus on host innate immune defenses and bacterial pathogen immune evasion. Cellular & Molecular Immunology (2016) 13, 560–576; doi:10.1038/cmi.2016.40; published online 15 August 2016

Keywords: apoptosis; autophagy; bacterial pathogen; innate immune signaling; phagosomal maturation; ubiquitin system

INTRODUCTION majority of E3s contain a signature RING domain coordinating The highly conserved ubiquitin (Ub) protein consists of 76 two Zn2+ in a cross-brace arrangement that functions as a amino acids and can be covalently attached to other proteins docking site for Ub-loaded E2s. Some RING proteins harbor via an isopeptide bond formed between its C-terminal glycine substrate-recognizing modules themselves and directly act as residue and the ε-amino radical of lysine residues on substrates. E3s (for example, tumor necrosis factor (TNF) receptor- Ubiquitination is carried out by the sequential actions of three associated factors (TRAFs) and inhibitor of apoptosis proteins enzymes. Initially, a Ub-activating enzyme (E1) catalyzes the (IAPs)), whereas others act in multisubunit E3 complexes that formation of an energy-rich thioester bond between its active-site employ additional subunits for substrate binding (for example, cysteine and the C-terminal glycine residue of Ub in an ATP- anaphase-promoting complex/cyclosome and Skp1-cullin-F dependent manner. The activated Ub is then delivered to a box protein (SCF)/cullin-RING-ligase (CRL)). In contrast to similar cysteine residue in the active site of a Ub-conjugating HECT and RBR E3s, E3s characterized by a RING domain enzyme (E2). Finally, a Ub ligase (E3) binds to both the possess no active-site cysteine and thus do not form a thioester Ub-charged E2 and the substrate protein, leading to the linkage intermediate, which means that the RING E3s function of Ub to the target protein (Figure 1).1 In addition to mono- more as scaffolds to facilitate the transfer of a donor Ub to ubiquitination, a protein can be modified with polymeric Ub an acceptor lysine by aligning the catalytic factors for nucleo- chains that are composed of several Ub moieties. The presence of philic attack. Consequently, linkage specificity is largely con- seven internal lysine residues (K6, K11, K27, K29, K33, K48 and ferred by the E2s rather than the RING E3s.3 K63) and the N-terminal methionine residue on Ub allows the HECT E3s feature the C-terminal HECT domain, which assembly of eight homotypic and multiple-mixed conjugates.2 assumes a bilobal architecture, with the N-terminal lobe The human genome encodes 2 E1s, ~ 40 E2s and ~ 600 E3s. containing the E2-binding domain and the C-terminal lobe The E3 ligases comprise three major classes: Really Interesting harboring the catalytic cysteine residue. Unlike RING E3s, the New Gene (RING) E3s, Homologous to E6AP C-Terminus HECT E3s modify target proteins through a two-step reaction (HECT) E3s and RING-In-Between-RING (RBR) E3s.3–5 The pathway in which the E2s first transfer the donor Ub to

CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China Correspondence: Professor CH Liu, PhD, Professor, Principal Investigator, CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China. E-mail: [email protected] Received: 8 May 2016; Revised: 30 May 2016; Accepted: 30 May 2016 The ubiquitin system in infection and immunity JLiet al

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Figure 1 The ubiquitination cascade and functional consequences of polyubiquitin chains with different linkage types. An E1 enzyme catalyzes the formation of a thioester bond between its active-site Cys residue and the C terminus of ubiquitin in an ATP-dependent manner. Activated ubiquitin is then transferred to the active-site Cys residue of an E2. The final step is mediated by an E3 ligase that either facilitates the direct transfer of the ubiquitin from E2 onto its substrate (RING E3 ligase) or forms a thioester intermediate with ubiquitin before transfer (HECT or RBR E3 ligase). The RING E3 can be a single chain or a large multiprotein complex that provides specificity and regulatory complexity. The substrate fate changes with different types of polyubiquitination. The K48- and K11-linked ubiquitin chains typically destine target proteins for degradation in the 26S proteasome, whereas the K63- and M1-linked chains are mostly implicated in nonproteolytic events such as signaling cascades. Notably, under autophagic conditions, the K63 linkage can act as a direct substrate for lysosomal destruction by binding to the adaptor p62. HECT, Homologous to E6AP C-Terminus; RBR, RING-In-Between- RING; RING, Really Interesting New Gene. the catalytic cysteine residue on the HECT domain through For instance, the K48- and K11-linked polyubiquitin chains a transthiolation reaction, retaining the high-energy thioester exhibit compact conformations and are usually direct bond. HECT E3s then facilitate optimal orientation of the substrates for 26S proteasome-mediated degradation, which acceptor lysine toward the Ub-E3 thioester bond to enable comprises the canonical Ub–proteasome system. By contrast, nucleophilic attack. Consequently, linkage type is determined when joined through M1 or K63, these conjugates adopt by the HECT E3s.4 extended conformations and enable the reversible assembly The RBR E3s harbor a conservative catalytic region embedded of multiprotein complexes, which are largely implicated with a RING1, an in-between RING and a RING2 domain. The in various nonproteolytic events such as immune signaling RBR E3s catalyze ubiquitination via a RING/HECT-hybrid cascades. In addition, homogeneous Ub chains linked via four mechanism in which the recruitment of a Ub-charged E2 other unconventional lysines have also been observed in cells. by RING1 facilitates the transfer of Ub to form a HECT-like Several of these atypical linkages mediate the proteasomal thioester intermediate with a cysteine on RING2 before coupling degradation of substrates in a similar manner to K11 and Ub to its substrate.5 As with HECT E3s, RBR E3s control K48 linkages; however, the reason for such redundancy linkage specificity as exemplified by the linear Ub chain remains enigmatic.8 Although previous research has primarily assembly complex (LUBAC), which exhibits a unique capacity focused on the homogenously linked Ub chains, the mixed and to synthesize linear Ub chains.6 branched Ub chains are emerging as crucial modifications As a versatile post-translational protein modification, ubi- bearing distinct physiological functions. For example, Meyer quitination triggers diverse biological outcomes. The addition and Rape9 recently reported that the anaphase-promoting of a single Ub typically alters the activity, interaction or complex/cyclosome E3 ligase and its cognate E2s, Ube2c and localization of the labeled protein, whereas the conjugation of Ube2s, collaboratively augment the rate of proteasomal polyubiquitin chains with different linkage types confers destruction by decorating their substrates with branched distinct functions because of dissimilar topology structures.7 K11- and K48-linked Ub chains.

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The Ub status is decoded and translated into specificcellular MODULATION OF INNATE IMMUNE SIGNALING BY THE outputs by various Ub receptors embedded with one or more UBIQUITIN SYSTEM Ub-binding domains (UBDs). UBDs differ in their structures, Accumulating data suggest that the ubiquitin system has a which encompass alpha-helical motifs (for example, ubiquitin- pivotal role in orchestrating signaling networks emanating from interacting motif (UIM)), zinc fingers (for example, Npl4 zinc the pattern-recognition receptors.12,13 Pattern-recognition finger (NZF)), pleckstrin-homology (PH) domains (for exam- receptors, such as Toll-like receptors (TLRs), RIG-I-like ple, GRAM-like ubiquitin-binding in EAP45 (GLUE)), receptors (RLR) and NOD-like receptors (NLRs), provoke Ub-conjugating (Ubc)-related structures (for example, ubiqui- various host countermeasures in response to pathogen- tin E2 variant (Uev)) and several other emerging Ub-binding associated molecular patterns (PAMPs) or pathogen-induced structures such as the Src homology 3 and phospholipase perturbation of cellular physiology. Upon engagement, most A2-activating protein (PLAA) family Ub-binding (PFU) pattern-recognition receptors transduce signaling to promote domains.10 Most UBDs, although structurally dissimilar, recog- the expression of pro-inflammatory cytokines, type I interferons nize the hydrophobic Ile44 patch present on the Ub surface, (IFNs; IFN-α and IFN-β) and other innate immune genes by which is composed of Leu8, Ile44, Val70 and His68. Beyond activating several immune signaling cascades including the this hotspot, several other motifs on the Ub molecule, such nuclear factor-κB(NF-κB), mitogen-activated protein kinases as the Phe4 patch, Ile36 patch, Asp58 patch and the TEK-box, (MAPK) and interferon regulatory factor 3 (IRF3) cascades. By can mediate either hydrophobic or hydrogen-bond interactions contrast, certain ligated cytosolic pattern-recognition receptors with UBDs. Remarkably, in addition to contacting a single initiate the formation of inflammasomes, which in turn activate Ub, UBDs have the capacity to distinguish between different the inflammatory caspases (caspase-1/4/5/11) to proteolytically polyUb linkage types by detecting the distance between process pro-IL-1β and pro-IL-18 and to evoke pyroptosis. successive Ub moieties, the sequence context surrounding the Following secretion, these cytokines prime other immune cells isopeptide bond and the free C termini of unanchored chains. to prepare them for defense against microbial infections. Given Akin to reversible phosphorylation, ubiquitination can be that the Ub system-mediated regulation of the TLR, RLR and removed by deubiquitinases (DUBs), leading to the enhance- TNF-α signaling pathways has been well documented in several ment of protein stability or attenuation of Ub signaling previous reviews,12,14 we will focus our discussion on NLR and contributing to Ub homeostasis. DUBs are classified as signaling. five groups owing to their distinct signature catalytic domains, which include Ub-specific protease (USP) domains, ovarian NOD1/2 signaling tumor protease (OTU) domains, Ub C-terminal hydrolase NOD1 and NOD2, two NLR family members, evoke NF-κB domains, JAMM4 domains and Josephin domains.11 All DUBs, and MAPK activation upon sensing peptidoglycan-derived except for the JAMM4 domain-containing DUBs, which constituents.15 NOD1 contains an N-terminal caspase activa- are zinc-dependent metalloproteases, belong to the cysteine tion and recruitment domain (CARD) belonging to the death proteases. Despite folding into divergent architectures, the domain (DD) superfamily, a centrally located NOD followed by cysteine DUBs possess either a catalytic dyad (Cys and His) C-terminal leucine-rich repeats (LRRs). NOD2 shares identical or triad (Cys, His, Asn or Asp) in their catalytic domains. It is domains with NOD1, except that it contains two N-terminal noteworthy that DUBs exhibit specificity at various layers to CARD modules. discriminate between different linkage types or substrates, When unstimulated, the LRRs occlude the NOD module including between Ub and many Ub-like tags (for example, to render NOD1/2 in a monomeric and autoinhibited state. NEDD8 and SUMO), and to cleave the Ub chains from the Once ligated, NOD1/2 switches to an open conformation, terminus or from within the target protein. Strikingly, such undergoes self-oligomerization and binds the proximal specificity is not necessarily confined to particular DUB adaptor receptor-interacting protein 2 through a homotypic families. In fact, DUBs from the same family can exhibit CARD–CARD interaction, which in turn recruits cellular dissimilar specificities. For instance, the 26S proteasome- inhibitor of apoptosis protein 1/2 (cIAP1/2) to build a NOD1- localized USP domain-containing USP14 exhibits exo-activity, /2-associated signaling complex. Within this signaling hub, exclusively disassembling K48-linked chains from the distal cIAP1/2 is activated to conjugate K63-linked chains to RIP2.16 end, yielding monoubiquitin, whereas the USP domain- The polyubiquitin chains then act like scaffolds to recruit containing CYLD possesses endo-activity, preferentially hydro- two kinase complexes, specifically the TGFβ-activated kinase lyzing K63-linked and linear chains to generate longer Ub 1 (TAK1) complex and the canonical IκB kinase (IKK) chains through internal cleavage. complexes, through their respective ubiquitin-binding subu- Here, we highlight the latest advances in the modulation nits, TAB2–TAB3 (TAK1-associated binding protein) and of many aspects of host innate immune responses by the Ub NEMO (NF-κB essential modulator, also termed IKKγ), system during infection, including inflammatory signaling, which in turn activate the NF-κB and, putatively, the MAPK phagosomal maturation, autophagy and apoptosis. In addition, signaling cascades. Likewise, ITCH, a HECT E3 ligase, was also we will summarize the strategies adopted by pathogens to found to mark RIP2 with K63-linked chains following NOD2 interfere with or to co-opt the Ub system to favor their own activation.17 However, unlike cIAP1/2, ITCH-conjugated intracellular survival and propagation. chains are required for optimal JNK and p38 activation but

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563 blunt NF-κB activation. Such a discrepancy might be attributed Recently, the ubiquitin system has emerged as a critical to the possibility that ITCH competes with cIAP1/2 to govern regulatory mechanism underlying the pyrin domain-containing whether MAPK or NF-κB is the predominant cascade down- inflammasomes (Figure 2).26 The NLRP3 is unique among the stream of NOD2 activation. In addition, NOD2 activation leads inflammasome sensors because, instead of one or two specific to NEMO ubiquitination, which was shown to amplify NF-κB ligands, it responds to a wide range of stimulatory signals such signaling and is partially accomplished by NOD2-activated as ATP, crystalline reagents and the microbial toxin nigericin. TRAF6.18 Finally, a recent study suggests that the ubiquitin Two-step signaling, priming and activation is required for molecule might compete with RIP2 to associate with CARD of the NLRP3 inflammasome to be competent in processing NOD1/2, thereby negatively affecting NOD1/2 signaling.19 pro-IL-1β and pro-IL-18 in macrophages. By contrast, the Intriguingly, XIAP (X-linked Inhibitor of Apoptosis) was priming signaling alone can induce IL-1β maturation 27 also recently observed to be present in the NOD2 signaling in monocytes where caspase-1 is constitutively activated. complex.20 Unlike cIAP1/2, XIAP primarily synthesizes Lipopolysaccharide (LPS) is best known to prime the NLRP3 fl κ K63- and K48-independent atypical polyubiquitin chains in ammasome by inducing NF- B activation and thus β 28 on RIP2, which enable the incorporation of LUBAC into the upregulating NLRP3 and pro-IL-1 protein levels. In addi- NOD2 signaling complex. RIP2 was later uncovered to be tion, the F-box protein FBXL2, a subunit of the SCF E3 ligase the prime substrate of LUBAC in NOD2 signaling through complex, serves as a sentinel inhibitor of NLRP3 by mediating quantitative proteomics analysis of the components obtained its K48-linked ubiquitination and proteasomal degradation fl from M1-Ub-affinity purification.21 Although the UBD in resting human in ammatory cells. LPS priming prolongs fi the lifespan of NLRP3 by inducing and activating the FBXL2 of TAB2/3, speci cally the NZF domain, preferentially binds 29 to K63-type chains, the UBD in NEMO, referred to as UBAN inhibitor FBXO3. Of note, apart from its protein levels, the activity of NLRP3 is also subject to post-translational (ubiquitin binding in ABIN and NEMO), binds significantly regulation by ubiquitination. It has recently been shown that more strongly to M1 chains compared with K63 chains. the DUB BRCC3 profoundly promotes inflammasome activa- Thus, the synchronous addition of K63- and M1-linked chains tion by deubiquitinating NLRP3, whereas the level of BRCC3 on RIP2 may provide an optimal platform to facilitate the does not correlate with the abundance of NLRP3 after LPS TAK1-catalyzed activation of the IKK complex. exposure, hinting that non-degradative K63-linked ubiquitina- For controlled and beneficial pro-inflammatory responses, tion might obstruct NLRP3 inflammasome activation in resting ubiquitination must be counterbalanced by DUBs that are as macrophages.30 critical as E3 ligases in regulating immune signaling. The linear The ubiquitin system is also implicated in modulating ubiquitin chain-specific DUB OTULIN binds to the catalytic other subunits of the NLRP3 inflammasome. The ASC can LUBAC subunit HOIP and selectively trims the M1-linked 21 be linearly ubiquitinated by LUBAC, which enhances activation chains from RIP2, thereby attenuating NOD2 signaling. of the NLRP3 inflammasome independent of its positive role in In addition, HOIP also interacts with CYLD, which limits the NF-κB signaling.31 Nonetheless, it remains to be determined extension of both K63- and M1-linked chains on RIP2 what signal triggers LUBAC-catalyzed modification of the ASC 22 to restrict NOD2 signaling. upon NLRP3 stimulation. Furthermore, the E3 ligases cIAP1/2 fl in association with the adaptor TRAF2 interact with and In ammasome signaling deposit K63-linked chains on caspase-1, thereby universally In response to microbial infection, certain NLR family mem- potentiating caspase-1 activation stimulated by various bers, including NLRP3 and NLRC4, and the cytosolic DNA inflammasome agonists, such as ATP and cytosolic flagellin.32 fl sensor AIM2 assemble into a multimeric canonical in amma- Finally, A20 acts to limit spontaneous or exogenously stimu- fl some complex to activate in ammatory caspase-1, which in lated NLRP3 inflammasome activation.33 Mechanistically, fl turn mediates the maturation of the pro-in ammatory inter- in LPS-primed bone marrow-derived macrophages, pro-IL-1β β leukin (IL)-1 and IL-18 as well as the induction of pyroptosis. was conjugated with polyubiquitin chains, which likely support Pyroptosis, distinct both morphologically and molecularly from the proteolytic cleavage of pro-IL-1β by encouraging higher- fl apoptosis, occurs following the activation of in ammatory order oligomerization of inflammasome subunits. Ablation caspases and cleavage of gasdermin D, which induces mem- of A20 in macrophages sharply increased the ubiquitination brane rupture and the leakage of cytosolic contents, thereby level of pro-IL-1β in response to LPS alone. damaging the replication niche of intracellular pathogens and Undoubtedly, the failure to obliterate unwanted inflamma- rendering them vulnerable to neutrophils.23,24 Pyrin domain- somes would likely bring about excessive inflammation and cell harboring NLRP3 and AIM2 inflammasomes signal exclusively death, which necessitates the ubiquitin system-mediated nega- through the ASC (apoptosis-associated speck-like protein tive regulation of inflammasomes. A recent study showed that containing a CARD) adaptor to recruit and activate caspase-1. AIM2 or NLRP3 inflammasome activation is accompanied by By contrast, the CARD domain-harboring NLRC4 inflamma- autophagosome formation in macrophages, which functions to some can signal to caspase-1 directly to elicit pyroptosis and temper inflammation by clearing active inflammasomes. indirectly through the adaptor ASC to augment IL-1β and Specifically, the ASC might undergo K63-linked ubiquitination IL-18 secretion.25 within the assembled inflammasomes, and the autophagic

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Figure 2 Modulation of the NLRP3 inflammasome by the ubiquitin system. The NLRP3 inflammasome relies on both priming and activating signals to be competent in processing pro-IL-1β and pro-IL-18 as well as inducing pyroptosis in macrophages. LPS priming upregulates the production of NLRP3, pro-IL-1β and FBXO3 through activation of the NF-κB pathway. FBXO3 functions to decrease FBXL2 protein levels, which targets NLRP3 for K48-linked ubiquitination and proteasomal degradation. BRCC3 enhances inflammasome activation by removing the K63-linked ubiquitin chains from NLRP3. A20 deubiquitinates and thereby limits the maturation of pro-IL-1β. In response to various agonists such as ATP and crystals, NLRP3, ASC and pro-caspase-1 assemble into a multisubunit inflammasome complex in the cytoplasm. LUBAC and cIAP1/2-TRAF2 assist in NLRP3 inflammasome activation through linear and K63-linked ubiquitination of ASC and pro-caspase-1, respectively. By contrast, the K63-linked ubiquitination of ASC directs the NLRP3 inflammasome for degradation in the autophagosome by binding to the autophagic adaptor p62, ultimately resulting in the termination of NLRP3 inflammasome activation. ASC, apoptosis-associated speck-like protein containing a CARD; CARD, caspase activation and recruitment domain; cIAP1/2, cellular inhibitor of apoptosis protein 1/2; IL, interleukin; LRR, leucine-rich repeat; LUBAC, linear Ub chain assembly complex; NBD, nucleotide-binding domain; NF-κB; nuclear factor-κB; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor 6.

adaptor p62 would then assist in the delivery of inflammasome exchange factors), GTPase-activating proteins and guanine- aggregates to autophagosomes for destruction.34 nucleotide dissociation inhibitors. Rab5 modulates early phagosomal morphogenesis by FUNCTION OF THE UBIQUITIN SYSTEM IN promoting the fusion of nascent phagosomes with early PHAGOSOMAL MATURATION DURING PATHOGEN endosomes. Initially, the recruitment of Rabex-5, the cognate INFECTION GEF of Rab5, to early phagosomes promotes Rab5 activation. Phagocytosis, the uptake and clearance of microbial pathogens, The activated Rab5 then recruits its effector protein Rabaptin- exerts a crucial role in innate immune defense against infection. 5, which is capable of boosting the activity of Rabex-5, thereby The nascent phagocytic vacuole is innocuous. These eventual constituting a positive feedback loop.37 The association of antiseptic properties are gained following phagosomal matura- Rabex-5 with early phagosomes is governed by cycling between tion, which entails a succession of precisely choreographed ubiquitin binding and monoubiquitination.38 Rabex-5 contains membrane fusion and fission events to remodel the phagoso- an A20-like ZnF that confers E3 activity and interacts with mal membrane and contents, culminating in the biogenesis an Asp58-centered polar region on ubiquitin, an inverted of phagolysosomes.35 As with the endocytic pathway, the UIM (IUIM) that binds to the Ile44 patch on ubiquitin, phagosome undergoes early, late and lysosome-fusion stages a central GEF catalytic core and a C-terminal coiled-coil (CC) of maturation. The conversion between phagosomal stages is domain comprising the Rabaptin-5-docking region.39 Rabex-5 delicately coordinated by the Rab family GTPases, which is recruited from the cytosol to the early phagosomes through comprise over 60 members in mammals.36 Akin to other its ZnF and IUIM domain-mediated interactions with the GTPases, the activity and localization of Rabs is tightly ubiquitinated membrane protein, as well as its CC domain- modulated by their cognate GEFs (guanine-nucleotide mediated association with other as yet undetermined factors.

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Following Rab5 activation and endosomal fusion or convey- Although ubiquitination provides a sorting signal for the ance of cargo, Rabex-5 undergoes Ub-binding-stimulated influx of cargo into the MVB compartment, the cargo must be monoubiquitination, leading to the migration of Rabex-5 from deubiquitinated before packaging into ILV to maintain stable the phagosomes to the cytoplasm, where the covalently linked cellular ubiquitin pools. In mammals, AMSH and UBPY are Ub moiety may bind intramolecularly or intermolecularly to its recruited to the endosome or phagosome not merely by the ZnF/IUIM motif. Rabex-5 deubiquitination, presumably car- ESCRT-0 subunit STAM but also by the ESCRT-III complex. ried out by the two endosomal DUBs, associated molecule with In contrast to AMSH, which exclusively removes K63-type the SH3 domain of STAM (AMSH) and ubiquitin-specific chains, UBPY disassembles both K63- and K48-type chains. In processing protease Y (UBPY), abrogates this binding and addition to recycling Ub or cargo from the MVB pathway, allows the resumption of this cycle.40 Together, ubiquitin these endosomal DUBs can reverse the autoinhibitory mono- system-mediated Rabex-5 regulation integrates cargo detection ubiquitination of ESCRT proteins. For example, AMSH is with endosomal fusion. capable of cleaving the ubiquitin moiety appended on Hrs.48 A hallmark of early-to-late phagosomal transition is the Beyond ESCRT complexes, several other endocytic adaptors acquisition of Rab7 and concomitant loss of Rab5. The active participate in MVB formation. Their stability and activity Rab7, through its effectors, mediates further maturation of the are similarly sensitive to modulation by the ubiquitin system. phagosomes and centripetally directs late phagosomes along For example, CRL3SPOPL mediates the degradative ubiquitina- the microtubules to facilitate fusion with lysosomes. Parkin tion of the endocytic adaptor EPS15, which is needed for was observed to regulate the endolysosomal pathway (and efficient ILV formation during endosomal/phagosomal matura- putatively the phagolysosomal pathway) by targeting Rab7 for tion. CRL3SPOPL may be activated at endosomes/phagosomes ubiquitination, thereby affecting the activity and protein level by the E3 ligase DCNL3, which is known to associate with of Rab7. Specifically, parkin-mediated ubiquitination might membranes by virtue of a covalent lipid modification that increase the activity and membrane association of Rab7. conjugates NEDD8 to the CUL3 subunit.49 Activated Rab7 demonstrates potentiated capacity for effector binding, which might in turn stabilize Rab7.41 INVOLVEMENT OF THE UBIQUITIN SYSTEM IN Of note, the phagosomal membrane proteins can be targeted ANTIMICROBIAL AUTOPHAGY to intraluminal vesicles (ILVs), similar to the multivesicular Autophagy is an evolutionarily conserved homeostatic process body (MVB) formed in the early or late endosomal system, in which cells eliminate protein aggregates, damaged or super- for degradation, which are generated after invagination and fluous organelles, and microbial pathogens from their cytosol scission of the limiting membrane.42 Importantly, perturbation or utilize bulky cytosolic contents for metabolism.50 A marked of this process adversely affects phagosomal maturation morphological feature of autophagy is the genesis of double- and pathogen eradication.43 The ESCRT (endosomal sorting membrane autophagosomes, which sequester and transport the complex required for transport) machinery consists of entrapped cargoes to lysosomes for decomposition. The process four complexes, ESCRT-0, -I, -II and -III, which operate of de novo autophagosomal formation is primarily regulated by sequentially to mediate MVB formation via three distinct four complexes: the ULK1 protein kinase complex, the VPS34 but connected steps: capturing the ubiquitin-tagged cargo, lipid kinase complex, two transmembrane proteins, ATG9 deforming the phagosomal/endosomal membrane and mem- and VMP1, and two ubiquitin-like protein conjugation machi- brane abscission from the inside.44 The ubiquitin system has a neries operating on ATG12 and LC3 (mammalian homolog of central role in ESCRT-mediated MVB biogenesis.45 Specifically, yeast ATG8). The first ubiquitin-like conjugation reaction ubiquitination specifies which membrane cargo should be facilitates the connection of ATG12 with ATG5, catalyzed by delivered to the lysosomal lumen. Then, the ESCRT machinery the E1-like ATG7 and E2-like ATG10. In complex with sorts ubiquitinated cargo into ILVs. ESCRT-0, -I and -II ATG16L1, ATG5–ATG12 in turn function as an E3-like ligase complexes employ several distinct UBDs, including UIM and collaborate with an E2-like ATG3 to facilitate the linkage (STAM1/2), double-sided UIM (Hrs), VHS (STAM1/2, Hrs), of LC3 to phosphatidylethanolamine at the phagophore. catalytically inactive Uev (Tsg101) and GLUE (EAP45), to Substantial data indicate that autophagic initiation and capture and gather the ubiquitinated cargo into a specific progression is tightly modulated by the ULK1 and VPS34 membrane compartment. Finally, ESCRT-III with no UBDs complexes. The Ser/Thr protein kinase ULK1 assembles a primarily promotes vesicle budding and scission. In addition to stable complex with FIP200 (ATG17 in yeast) and ATG13, and recognizing ubiquitinated cargo, certain ESCRT proteins can its activity is controlled by the nutrient-sensitive kinase themselves be ubiquitinated, leading to alterations in their complex, mTORC1 (mammalian target of rapamycin complex stability, activity or localization. The ESCRT-0 subunit Hrs is 1). The Ser/Thr protein kinase mTOR exists in two distinct monoubiquitinated, which maintains Hrs in an inactive con- multisubunit complexes known as mTORC1 and mTORC2 in formation and precludes its recognition of the Ub-labeled mammalian cells, with mTORC1 suppressing autophagy membrane cargo owing to the intramolecular Ub–UIM directly via the phosphorylation and deactivation of the interaction.46 Similarly, disturbance of the E3 Mahogunin- ULK1, whereas mTORC2 promotes survival and suppresses mediated ubiquitination of the ESCRT-I subunit Tsg101 autophagy indirectly via the phosphorylation and activation of compromises endosome/phagosome-to-lysosome traffic.47 AKT, which in turn activates mTORC1 along the PI3K–AKT–

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mTOR cascade.51 VPS34 is the sole class III phosphoinositide pathogens, such as Mycobacterium tuberculosis (Mtb) and 3-kinase in mammals that functions in a stable complex Salmonella enterica serovar typhimurium. The ubiquitin system containing p150 (VPS15 ortholog) and Beclin-1 to produce is widely implicated in the initiation of the autophagic pathway phosphatidylinositol 3-phosphate. Beclin-1 forms a binding as well as the subsequent coupling of autophagosomes to platform for several other regulatory proteins with the capacity bacterial targets (Figure 3).55 The kinase activities of both to either enhance (UVRAG, ATG14L, Bif1 and AMBRA1) or mTOR and AKT are subject to ubiquitin system-dependent suppress (Bcl-2, TAB2/3 and Rubicon) the autophagic activity modulation. In response to nutrients, p62 brings TRAF6 to of VPS34.52,53 Following mTOR suppression, activated ULK1 mTORC1 via its binding affinity for both raptor and TRAF6.56 boosts the kinase activity of the ATG14L-harboring VPS34 Then, the recruited TRAF6 marks mTOR with K63-linked complex through the phosphorylation of Beclin-1 on Ser14.54 ubiquitin chains, which are instrumental for the activation Pathogen infection-triggered selective autophagy is also of mTOR. In addition, K48-linked ubiquitination has been termed xenophagy and functions to restrict a range of documented to directly affect the stability or indirectly affect

Figure 3 The ubiquitin system regulates the xenophagy of bacterial pathogens. Both initiation of the autophagic pathway and the subsequent capture of bacterial targets is modulated by the ubiquitin system. On one hand, the E3 ligase TRAF6 supports K63-linked polyubiquitination and activation of mTORC1, ULK1 and Beclin-1-VPS34 complexes with the help of p62, AMBRA1 and TLR4, respectively, leading to the inhibition or activation of autophagy. On the other hand, cytosol invasion triggers the E3 ligase (for example, Parkin and LRSAM1)-mediated ubiquitination of the bacterial pathogen itself or the associated host components. Then, autophagic receptors such as p62, NDP52 and optineurin (OPTN) that bind to both ubiquitin and the autophagic marker LC3 direct ubiquitin-tagged bacteria to the autophagosome for elimination. The NDP52 is unique among the autophagic receptors because of its ability to bind both ubiquitin and galectin-8, another 'eat-me' signal that recognizes the exposed host glycans on the damaged phagosome. mTORC1, mammalian target of rapamycin complex 1; NDP52, nuclear domain 10 protein 52; TLR, Toll-like receptor; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor.

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567 the kinase activity of mTOR. For instance, DEPTOR, an cytosol-invasive bacteria with ubiquitin chains by virtue of its endogenous inhibitor for both mTORC1 and mTORC2, LRR and RING domains, respectively, in the absence of other becomes phosphorylated by mTOR in cooperation with casein assistant proteins. Unlike parkin, LRSAM1 predominantly kinase I upon stimulation by growth signals, which create a synthesizes K6- and K27-linked ubiquitin chains. Furthermore, phosphodegron for SCFβTrCP binding, ultimately resulting in in addition to Sa. typhimurium,LRSAM1wasfoundtolocalize DEPTOR degradation and mTOR activation.57 Finally, AKT to multiple Gram-negative and -positive bacteria. Thus, undergoes TRAF6-catalyzed K63-type ubiquitination at the PH LRSAM1 might represent a unique pathogen-recognizing E3 domain upon exposure to growth factor, which promotes AKT ligase that is active against a broad range of intracellular membrane adherence, phosphorylation and activation without bacteria in the autophagic pathway. influencing its phosphatidylinositol (3,4,5)-trisphosphate lipid- The ubiquitinated coat deposited on bacteria or the binding capability.58 surrounding vacuole are bound by three autophagic receptors, Recent data showed that the activity of ULK1 is also specifically, p62 (SQSTM1), NDP52 (nuclear domain 10 regulated by ubiquitination. In cells undergoing autophagy, protein 52) and optineurin through their C-terminal UBA, AMBRA1, a binding partner for both Beclin-1 and ULK1, in ZnF or UBAN domains, respectively, resulting in the isolation complex with TRAF6 supports K63-linked polyubiquitination of bacteria by autophagosomes.69 In addition, NDP52 detects and consequent self-association, stabilization and activation of galectin-8 signaling, which interacts with the host glycans ULK1.59 Notably, under normal conditions, mTORC1 phos- exposed on the impaired phagosomal membrane. It was phorylates AMBRA1 and precludes its recruitment of TRAF6. proposed that NDP52 is transiently recruited to the impaired Because ULK1 activates AMBRA1 via phosphorylation, such phagosome by galectin-8, whereas the persistent localization of AMBRA1-mediated modification of ULK1 likely operates as a NDP52 still requires ubiquitination.70 Notably, these three positive feedback regulatory loop. Furthermore, the ubiquitin autophagic receptors might perform non-redundant functions system targets Beclin-1 to modulate autophagic induction. in xenophagy. A possible mechanism is that these receptors Specifically, upon engagement by LPS, TLR4 recruits Beclin-1 bind to different ubiquitin chains with dissimilar affinities. For to the receptor-bound signaling node where the activated example, p62 displays a preference for K63 chains over K48 TRAF6 decorates Beclin-1 with K63-linked chains, thereby chains, whereas Optineurin preferentially binds linear chains. disrupting the interaction between Bcl-2 and Beclin-1 and Alternatively, there might be beneficial interactions among leading to the oligomerization of Beclin-1 and activation of them. For example, NDP52 recruits TBK1 to the cytosolic Sa. VPS34.60 In addition to its activity, the stability of Beclin-1 is typhimurium, which in turn enhances the affinity of Optineurin also under the control of ubiquitination. For example, RNF216 for LC3B through phosphorylation.71 binds to and destabilizes Beclin-1 through K48-linked ubiqui- tination, whereas USP19 maintains Beclin-1 protein levels APOPTOSIS REGULATION BY THE UBIQUITIN SYSTEM through K11-specific deubiquitination.61,62 Apoptosis is a vital cell suicide modality in innate immunity Upon induction, xenophagy, reminiscent of other selective that can be triggered either along the extrinsic pathway by the autophagic routes, such as mitophagy, utilizes autophagic pro-apoptotic TNF superfamily ligands or along the intrinsic receptors that bind both a molecular tag attached to the pathway by several internal apoptotic insults, such as DNA bacterial cargo and the autophagic modifier LC3 to selectively damage and endoplasmic reticulum (ER) stress, leading to the bridge bacterial cargo to the autophagosomal membrane.63 clearance of the colonization of intracellular pathogens.72 The Ubiquitin appears to represent a common 'eat-me' signal in cell-surface death receptors (DRs), belonging to the TNFR various selective autophagic pathways including xenophagy.64 superfamily and featuring the presence of a cytosolic DD, can For example, both K48- and K63-linked ubiquitin chains were be divided into two categories: TNFR1, DR3 and possibly DR6- previously observed to accumulate around the vacuolar patho- binding TNFR1-associated death domain-containing protein gen Mtb during infection in macrophages. Recent research and exerting pro-inflammatory and pro-survival functions, or partially sheds light on the underlying mechanism: the bacterial Fas/CD-95, DR4 and DR5 engaging FADD (Fas-associated ESX-1 T7SS-mediated phagosomal permeabilization allows the protein with DD) and exerting primarily pro-apoptotic func- cGAS-STING-dependent cytosolic DNA sensor pathway to tions. Furthermore, crosstalk might occur between these two access extracellular bacterial DNA and then triggers parkin- opposing processes.73 mediated K63-linked ubiquitination surrounding the Mtb- TNF-α-induced apoptosis can proceed via either a RIP1- containing phagosome, which is required for the delivery of dependent or a RIP-independent pathway.74 The RIP1- bacilli to autophagosomes.65–67 Despite these observations, it is dependent pathway is controlled by its ubiquitination status. still poorly understood how parkin is recruited and activated at Following the second mitochondrial activator of caspases the bacterial niche and which bona fide protein(s) are targeted (Smac)-induced cIAP autodegradation and CYLD/USP7/ by parkin, as well as the identity of other E3s responsible for USP2a-mediated K63 deubiquitination of RIP1, RIP1 is liber- K48-linked ubiquitination. Another well-defined example ated from TNFR1 signaling complex I and in turn recruits the involves the LRR-harboring RING family E3 ligase LRSAM1, FADD adaptor and the apical caspase-8 to build a cytosolic which targets Sa. typhimurium to the autophagic pathway in complex II, also dubbed the Death-Inducing Signaling Com- epithelial cells.68 Notably, LRSAM1 directly senses and tags the plex (DISC), which mediates apoptotic cell demise.75,76 It is

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noteworthy that Smac is discharged from mitochondria via compared with other pro-survival Bcl-2 proteins because of mitochondrial outer membrane permeabilization (MOMP), its rapid turnover. Several E3 ligases, including MULE, which suggests that TNF-α-induced RIP-dependent apoptosis SCFβTRCP and SCFFBW7,havebeenrevealedtomarkMcl-1 might occur downstream of the intrinsic mitochondrial path- for degradation in a cellular context-dependent manner.86–88 way. In addition, recent studies have indicated that the lack of Conversely, USP9X binds and cleaves the K48-type chains from linear ubiquitination in complex I accelerates complex II Mcl-1 to hamper the degradation of Mcl-1 by the 26S formation in TNF signaling.77 Whereas CYLD directly binds proteasome.89 LUBAC and removes linear chains, thereby sensitizing cells to Beyond Bcl-2 family proteins, the ubiquitin system governs TNF-provoked apoptosis, A20 binding to the linear chains via the stability, activity or localization of certain regulators of Bcl- its ZnF7 domain antagonizes their removal, consequently 2, such as p53, which induces apoptosis through transcription- blocking cell death.78 Dissimilarly, the RIP1-independent dependent and -independent activation of Bcl-2 family apoptotic branch is controlled by the level of cellular FLICE members.90 A critical negative modulator of p53 is MDM2 Inhibitory Protein (c-FLIPL), which effectively blocks caspase-8 and its homolog MDMX.91 Despite bearing a RING domain, activation by competitively binding to FADD. The E3 ligase MDMX exhibits no intrinsic E3 activity. Rather, MDMX drives Itch activated by JNK1 phosphorylation acts to provoke p53 degradation by forming a heterodimer with MDM2, the apoptosis by directing c-FLIPL for proteasomal destruction.79 bona fide RING E3 ligase, which is mediated by a homotypic In contrast to TNFR1, DR4, DR5 and Fas, upon stimulation RING–RING interaction. Conversely, in response to genotoxic by their cognate ligands, directly recruit the adaptor FADD and pressure, USP10 undergoes ATM-mediated phosphorylation the initiator caspase-8 to establish a membrane-bound DISC. A and then migrates to the nucleus, where it stabilizes p53 recent report proposed that this assembled DISC recruits a through deubiquitination.92 Furthermore, basal levels of Cul3-Rbx1 E3 ligase that deposits K63-linked chains on the MDM2 can function alone or in homodimers to predomi- small p10 catalytic domain of caspase-8.80 The p62 adaptor nantly monoubiquitinate p53 in resting cells, leading to nuclear then boosts caspase-8 aggregation within the intracellular export of p53. Under apoptotic conditions, such cytoplasmic ubiquitin-rich foci, thereby augmenting enzyme processing monoubiquitinated p53 translocates to the mitochondria, and leading to robust caspase-3 and caspase-7 activation. By where it undergoes rapid deubiquitination and activation by contrast, TRAF2 mediates K48-linked ubiquitination of the mitochondrial herpesvirus-associated ubiquitin-specific caspase-8 on its large p18 catalytic domain downstream of protease (HAUSP, also known as USP7).93 Cul3-Rbx1 at the DISC, thereby destining the activated Downstream of MOMP, the stability and activity of caspases caspase-8 for rapid degradation following autoprocessing and is regulated by IAPs, which primarily include XIAP and cIAPs. membrane separation.81 Thus, TRAF2 appears to place a Unlike cIAPs, XIAP acquires the ability to directly bind and stringent barrier that impedes apoptotic commitment upon restrain the initiator caspase-9 and the executor caspase-3/7.94 DR4/5 or Fas ligation. In addition, XIAP promotes the K48-linked polyubiquitination Mitochondria act as signaling hubs that integrate and of the processed caspase-3 instead of pro-caspase-3.95 Analo- translate diverse pro-survival and pro-apoptotic signals released gous to XIAP, cIAP1 interacts with and ubiquitinates the fully from the cell nucleus or compartments during intrinsic mature caspase-7 and partially processed caspase-3, thereby apoptosis. The Bcl-2 family proteins, featuring one or more facilitating their turnover.96 Furthermore, IAPs regulate their Bcl-2 homology (BH) domains, fine-tune this intrinsic apop- abundance reciprocally, thereby ensuring optimal levels to totic pathway by governing the mitochondrial outer membrane allow cells to respond quickly to the ever-changing environ- integrity.82 Substantial data have highlighted the key role of the ment. For example, in a homogeneous interaction mediated by ubiquitin system in controlling the dynamic balance between their respective RING domains, cIAP1 reduces the protein the protein levels of pro- and anti-apoptotic Bcl-2 proteins, levels of XIAP by inducing its degradative ubiquitination.97 which acts as a pivotal checkpoint for apoptosis. Upon stimulation by survival signals, BimEL, a pro-apoptotic BH3- INTERVENTION OF THE UBIQUITIN SYSTEM IN only protein, undergoes Rsk1/2-executed phosphorylation in a IMMNUE EVASION BY BACTERIAL PATHOGENS conserved degron, which is enhanced by the Erk1/2-catalyzed Because the ubiquitin system is indispensable for eukaryotic phosphorylation of BimEL, thus facilitating SCFβTRCP binding cells to regulate diverse immune responses, many pathogens and its subsequent proteasomal degradation.83 Conversely, the have developed multiple mechanisms to cunningly subvert this DUB USP27X binds BimEL upon its Erk1/2-dependent phos- well-balanced program to favor their own survival. Bacterial phorylation, removes the ubiquitin chains and stabilizes the pathogens inject diversified effector proteins into the host phosphorylated BimEL.84 Another example involves Bok (Bcl-2 cellular environment through special secretion systems, includ- ovarian killer), which acts as an unconventional effector of ing the type III secretion system (T3SS), T4SS, T6SS and T7SS, MOMP and triggers apoptotic cell death when the ER- during infection.98,99 After entry into the host cells, those associated degradation (ERAD) system is impaired during ER effectors recompose various cellular processes to destroy host stress. Strikingly, unlike Bax and Bak, Bok harbors constitutive immune defenses and create an adaptive niche for bacterial activity that is restricted by ERAD E3 ligase gp78-mediated intruders to survive and proliferate.100,101 Notably, the ubiqui- degradative ubiquitination.85 Finally, Mcl-1 is unique tin pathways are sensitive targets of many bacterial effectors

Cellular & Molecular Immunology Table 1 List of bacterial effector proteins and their interactions with host cells

Bacterial Organism Biochemical characteristics Host targets Cellular function Ref effector

Mimicry of E3 ubiquitin ligase SopA Salmonella typhimurium HECT-type E3 ligase Unclear Intervention in host inflammation 116 NleL EHEC HECT-type E3 ligase Unclear Modulation of actin-pedestal formation 134 NleG EHEC, EPEC and RING/U-box-type E3 ligase Unclear Unclear 135 Salmonella LubX Legionella pneumophila RING/U-box-type E3 ligase CLK1 Promotion of bacterial intracellular survival 118 AnkB L. pneumophila F-box protein K48-linked polyubiquitinated proteins, Assimilation of host amino acids 125 Trim21 LegU1 L. pneumophila F-box protein BAT3 Unclear 127 LegAU13 L. pneumophila F-box protein Unclear Unclear 127 IpaH9.8 Shigella flexneri NEL-type E3 ligase NEMO Inhibition of the NF-κBpathway 103 IpaH0722 S. flexneri NEL-type E3 ligase TRAF2 Inhibition of the NF-κBpathway 104 IpaH4.5 S. flexneri NEL-type E3 ligase p65 Inhibition of the NF-κBpathway 105 IpaH7.8 S. flexneri NEL-type E3 ligase GLMN Promotion of inflammasome activation and cell 106 death SlrP Salmonella typhimurium NEL-type E3 ligase TRX Intervention in host cell death 113 SspH1 Sa. typhimurium NEL-type E3 ligase PKN1 Inhibition of the NF-κBpathway 114 SspH2 Sa. typhimurium NEL-type E3 ligase NOD1 Intervention in host inflammation 115 h bqii ytmi neto n immunity and infection JLi in system ubiquitin The SidC/SdcA L. pneumophila C-H-D triad-containing E3 ligase Unclear Recruitment of host ER vesicles 128,129 tal et SdeA L. pneumophila E1/E2-independent E3 ligase; ADP- Multiple ER-associated Rabs such as Rab33b, Promotion of bacterial intracellular replication 131 ribosyltransferase Rab1

Mimicry of deubiquitination enzyme AvrA Sa. typhimurium DUB IκB-α, β-catenin Intervention in host inflammation 112 SseL Sal. typhimurium DUB Ubiquitin aggregates around the SCV Evasion of host autophagy 111 TssM Burkholderia DUB TRAF3, TRAF6 and IκBα Inhibition of type I IFN and NF-κBpathways 140 pseudomallei YopJ Yersinia DUB TRAF2, TRAF6, TRAF3, IκBα and TAK1 Inhibition of NF-κB and MAPK pathways 142,145 pseudotuberculosis YopP Y. enterocolitica DUB TRAF6, NEMO, TAK1 and TAB1 Inhibition of NF-κB and MAPK pathways 141,144

Other effector-mediated interference of the host ubiquitin system 108 ellr&MlclrImmunology Molecular & Cellular OspI S. flexneri Glutamine deamidase UBC13 Inhibition of NF-κBpathway OspG S. flexneri Serine/threonine kinase Ubiquitinated E2 such as UbcH5, UbcH7 Inhibition of NF-κBpathway 107 NleE EHEC and EPEC Methyltransferase TAB2, TAB3 Inhibition of NF-κBpathway 137 NleB Citrobacter rodentium O-GlcNAc transferase GAPDH Inhibition of NF-κBpathway 146 Tir EHEC and EPEC Unclear SHP-1 and SHP-2 Inhibition of NF-κBpathway 136 InlC Listeria monocytogenes Unclear IKKα subunit of IκB Inhibition of NF-κBpathway 150 LLO Li. monocytogenes Bacteria pore-forming toxin UBC9 Inhibition of TGFβ signaling 152 569 The ubiquitin system in infection and immunity JLiet al

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(Table 1). Here, we primarily focus on the detailed mechanisms 157 130 151 121 119 148 149 138,139 138,139 118,120 B; employed by bacterial pathogens to manipulate the host κ ubiquitin system.

Shigella flexneri EPEC, As a prevalent pathogenic Gram-negative bacterium, S. flexneri B; nuclear factor-

κ utilizes its T3SS to invade and colonize human intestinal epithelial cells. Notably, in addition to S. flexneri, the majority of other Gram-negative bacterial pathogens are also equipped B kinase; INl, internalin; LLO, κ with T3SS to release a vast array of bacterial proteins into host cells. The core device of T3SS, termed the injectisome, is a B and MAPK pathways on; IKK, I 3.5-MDa syringe-like protein complex composed of stacked κ rings embedded in the bacterial membrane and a needle-like

B essential modulator; NF- fi κ lament that extends into the extracellular space from the bacterial surface.99 To some extent, the injectisome acts as a pipeline to transport pathogenic cargo from the bacterial cell to

replication the host cell. Several of the T3SS effectors delivered by S. flexneri have been identified as Novel E3 Ligases (NELs), which are structurally distinct from the eukaryotic E3 ligases.102

l E3 Ligase; NEMO, NF- Certain NEL family members, including IpaH9.8, IpaH0722 ; Ena/VASP, enabled/vasodilator-stimulated phosphoprotein; and IpaH4.5, have been shown to tamper with the host NF-κB cascade by ubiquitinating NEMO, TRAF2 and p65 for protea- somal degradation, respectively.103–105 Another NEL effector, IpaH7.8, possesses E3 ligase activity in vitro and targets an Escherichia coli inhibitor of Cullin-based RING E3 ligase named GLMN for degradation, which augments inflammasome activation.106 In addition, OspG and OspI, belonging to a different subsets -activated kinase 1; TAB, TAK1-associated binding protein; TRX, thioredoxin; UIML, ubiquitin-interacting

β of S. flexneri effectors, have recently been identified to κ D8, ubiquitin Inactivation of host CRLs suppress NF- B activation by disrupting the ubiquitin system. Mammalian serine/threonine kinase imitator OspG binds to NSF, Syk Promotion of bacterial invasion and intracellular JNK, p38, TAB3 Inhibition of NF- many ubiquitinated E2 proteins, including UbcH5 and Ubch7, in host cells, thereby enhancing its own kinase activity and preventing the ubiquitination and degradation of phosphory- lated IκBα.107 OspI inactivates Ubc13 by functioning as a glutamine deamidase, thus causing the inhibition of TRAF6 108 -containing vacuole; TAK1, TGF polyubiquitination.

Sa. typhimurium

Salmonella Sa. typhimurium is an intracellular Gram-negative pathogenic agent that survives in a special vacuole called the Salmonella- containing vacuole (SCV) after internalization into the macro- phages or epithelial cells.109 Two different T3SSs, T3SS1 and T3SS2, encoded by pathogenicity island-1 (SPI-1) and SPI-2, Cysteine proteaseUnclearGlutamine deamidaseInositol phosphate phosphataseGEFProtein tyrosine phosphatase UnclearUnclear Lipid-conjugated Unclear Atg8 NEDD8, ubiquitin Arp2/3 complex and Ena/VASP Evasion of host autophagy Cdc42, Rac1 Evasion of host autophagy Inactivation E-cadherin of host CRLs c-Met Promotion of bacterial invasion and survival Promotion of bacterial invasion and survival Promotion of bacterial internalization Promotion of bacterial invasion Protein tyrosine phosphatase with UIML domain respectively, are employed by Sa. typhimurium to secrete effector proteins. T3SS1 is favorable during the invasion stage, whereas T3SS2 is conducive to the development of SCV after bacterial entry into the host cell. Several of these effectors function to disturb the ubiquitin system.110 For example, the effector SseL is identified as a DUB and deubiquitinates the

; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GEF, guanine-nucleotide exchange factor; HECT, homologous to E6-AP C terminus; IFN, interfer ubiquitin aggregates surrounding the SCV to help bacteria escape from autophagy.111 Another DUB discovered in L. pneumophila Li. monocytogenes B. pseudomallei Sa. typhimurium Sa. typhimurium Sa. typhimurium Li. monocytogenes Li. monocytogenes OrganismMycobacterium tuberculosis Biochemical characteristics Host targets Cellular function Ref E. coli Sa. typhimurium is AvrA, which inhibits host inflammatory responses by deubiquitinating IκBα and β-catenin.112 Similar to the IpaH family effectors in S. flexneri, Sa. typhimurium encodes three NELs: SlrP, SspH1 and SspH2. In particular, RavZ ActA CifCHBP SopB SopE EHEC and EPECSptP InlA Glutamine deamidaseInlB NED PtpA PKN1, protein kinase N1; RING, Really Interesting New Gene; SCV, listeriolysin O; MAPK, mitogen-activated protein kinase; NEDD8, neural-precursor-cell-expressed developmentally down-regulatedmotif-like. 8; NEL, Nove enteropathogenic Table 1 (Continued ) Bacterial effector Abbreviations: Arp2/3, actin-related protein 2/3; CLK1, CDC2-like kinase 1; DUB, deubiquitinase; EHEC, enterohemorrhagic SlrP has an impact on host cell death by targeting mammalian

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571 thioredoxin.113 SspH1 decreases NF-κB activation by ubiquitinating ribosyltransferase motif.131 Of note, this report not only the protein kinase N1,114 whereas SspH2 was shown to target demonstrates for the first time that ubiquitination can be NOD1 for monoubiquitination, thereby increasing the NOD1- catalyzed by bacterial effectors in the absence of E1 and E2 dependent secretion of IL-8.115 Another E3 effector of enzymes but also hints that ubiquitin can be activated by ADP Sa. typhimurium, SopA, is structurally similar to the eukaryotic ribosylation. homologous to E6-AP C terminus (HECT) E3 ligase and interacts with the host E2 enzyme UbcH7.116 Although the Enterohemorrhagic Escherichia coli and enteropathogenic substrate in the host cells has not yet been demonstrated, it is Escherichia coli believed that SopA-mediated ubiquitination is involved in the Both enterohemorrhagic Escherichia coli (EHEC) and enter- regulation of host inflammation.117 Intriguingly, Sa. typhimur- opathogenic E. coli (EPEC) are notable agents of the pathogenic ium also utilizes the host ubiquitin system to process and E. coli family, whose adhesion to intestinal epithelial cells or regulate its own effector proteins. SopE and SptP harbor invasion into host cells in some circumstances can induce dissimilar functions, and their degradation is temporally multiple human bowel diseases.132 Two types of E3 effectors, differentially regulated by the ubiquitin system to ensure NleL and NleG, are identified in HEHC. NleL shares a – efficient bacterial invasion.118 120 Another example involves homologous C-terminal sequence with Sa. typhimurium SopB, which is modified by monoubiquitination during a SopA and is approved as a HECT E3 effector because of its special invasion period or within the context of proper catalytic activity to form polyubiquitin chains in vitro.133 NleL subcellular localization such that it systematically performs modulates EHEC-induced actin-pedestal formation, which 121 multiple functions. contributes to EHEC adherence to intestinal epithelial cells; however, the molecular target of NleG in host cells remains Legionella pneumophila unclear.134 As mimics of the eukaryotic RING/U-box E3 Reminiscent of Sa. typhimurium, the causative agent of a severe ligases, NleG family effectors are also identified in other human pneumonia, L. pneumophila also establishes a special pathogenic E. coli such as EPEC and Salmonella.135 Both EHEC vacuole named the Legionella-containing vacuole (LCV) after and EPEC deliver the effectors NleE and Tir to subvert the intruding into host cells.122 L. pneumophila secrets various E3 NF-κB pathway. NleE directly methylates the cysteine residue ligase-mimic effectors via the Dot/Icm T4SS to maintain in the ZnF of TAB2/3 to hinder their ubiquitination, whereas its intracellular survival.123 T4SS, despite sharing structural Tir facilitates the binding of host protein tyrosine phosphatases similarity with T3SS, is extensively distributed in both Gram- SHP-1 and SHP-2 to TRAF6, which prevents the ubiquitina- negative and -positive bacteria and uniquely manages the tion of TRAF6.136,137 Finally, EPEC and EHEC also target host translocation of not only effector proteins but also toxins and neddylation via the Cif effector to favor their survival. Similarly, plasmids into host cells.99 For example, the effector LubX contains two U-box domains that ubiquitinate the host CDC2- Burkholderia pseudomallei encodes a Cif homolog effector like kinase 1.124 In addition, AnkB, LegU1 and LegAU13 named CHBP. These Cif family effectors function as novel exhibit structural similarity to the eukaryotic F-box-containing glutamine deamidases and prefer to deamidate NEDD8 rather E3 ligase. AnkB is crucial for L. pneumophila to acquire than ubiquitin and thus impair its conjugation to Cullin, nutrition as it locates to the LCV membrane and recruits which is an essential subunit of the host CRL E3 ligase 138,139 K48-linked polyubiquitinated proteins, the degradation of complexes. which yields amino acids for the bacteria.125 Another study showed that AnkB is also modified by Trim21-mediated Other Gram-negative bacterial pathogens K11-linked ubiquitination; however, this fails to change its Three bacterial DUBs called TssM, YopJ and YopP have fi stability.126 LegU1 and LegAU13 are integrated into the host recently been identi ed from B. pseudomallei, Yersinia pseudo- SCF E3 complex, with the former directly ubiquitinating BAT3, tuberculosis and Y. enterocolitica, respectively. These DUBs fl a host co-chaperone of Hsp70 that is implicated in ER analogously have a part in suppressing host in ammatory κ α stress modulation.127 SidC and its paralog SdcA, identified in signaling pathways. TssM targets TRAF3, TRAF6 and I B to L. pneumophila, are capable of recruiting the ER-derived cleave both K48- and K63-type ubiquitin chains, thereby κ 140 vesicles to build LCV.128 A new study showed that SidC/SdcA limiting the IRF3 and NF- B cascades. YopJ and YopP are is a novel type of bacterial E3 ligase with a special C46-H444- homologous to each other and both exert multiple functions D446 catalytic triad that can form high-molecular-weight during bacterial infection. YopJ targets host TRAF2, TRAF6, polyubiquitin chains.129 In addition, the effector RavZ specifi- TRAF3, IκBα and TAK1, whereas YopP targets TRAF6, cally deconjugates the lipid-attached LC3 marks and thereby NEMO, TAK1 and TAB1 to cleave ubiquitin moieties, resulting – prevents the autophagic pathway by functioning as a cysteine in interference of the NF-κB and MAPK cascades.141 145 In protease.130 Finally, a peculiar yet interesting study recently addition, the mouse pathogen Citrobacter rodentium,which reported that SdeA, belonging to the SidE family effectors of imitates EPEC, delivers the effector NleB to O-GlcNAcylate, the L. pneumophila, is a novel all-in-one ubiquitin conjugation host glyceraldehyde 3-phosphate dehydrogenase, thus disrupt- enzyme, which solely mediates the ubiquitination of many ing its interaction with TRAF2 and arresting the NF-κB host Rab GTPases by virtue of a putative mono-ADP- pathway.146

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572 Listeria monocytogenes CONCLUSIONS Li. monocytogenes is an important Gram-positive intracellular Mounting evidence has highlighted the essential regulatory role bacterial pathogen that causes the human disease listeriosis. of the ubiquitin system in host–pathogen interactions. We are, Typically, certain internalin family proteins on the surface of however, far from fully appreciating both the molecular Li. monocytogenes, such as internalin A (InlA), InlB and InlC, mechanisms and the physiological functions of these processes. function to disorder the host ubiquitin system for efficient As more members of the host immune defense pathways are bacterial immune evasion and survival.147 Specifically, InlA identified as being regulated by the ubiquitin system, we are promotes the internalization of Li. monocytogenes by targeting faced with a challenging mission to dissect the underlying E-cadherin on epithelial cells for tyrosine phosphorylation and molecular details. Which bona fide protein targets, E3s or subsequent ubiquitination mediated by Src kinase and Hakai DUBs are responsible for the observed phenotypes? How are E3 ligase, respectively.148 InlB triggers the autophosphorylation these E3s and DUBs regulated under both normal and infected and activation of the host receptor tyrosine kinase, c-Met, conditions? Considering that a specific protein is, more often resulting in the ubiquitin ligase c-Cbl-dependent ubiquitination than not, modulated by several different E3s and/or DUBs, of c-Met and facilitating Li. monocytogenes invasion.149 Briefly, attention should be paid to clarifying whether these different InlC directly interacts with IKKα to impair phosphorylation enzymes function redundantly or whether they function and thus delay the destruction of IκB to suppress the NF-κB distinctly in a cell type and context-dependent manner. Even pathway.150 Another surface protein of Li. monocytogenes,ActA, after thorough characterization of these biochemical events, functions as a recruiter for the actin-related protein 2/3 the importance of evaluating the physiological relevance fi fi complex and Ena/VASP, thereby promoting bacterial evasion of in vitro ndings before drawing de nitive conclusions about of the autophagic pathway.151 Notably, Li. monocytogenes also a mechanism cannot be overemphasized. Similarly, there are hijacks the host ubiquitin system by secreting a pore-forming still many gaps in our comprehension of ubiquitin system- toxin, listeriolysin O, which degrades the host SUMO E2 relevant pathogen immune-evasion strategies. How are these enzyme Ubc9, thereby decreasing SUMOylation and impairing pathogen-derived E3s and DUBs regulated after injection downstream TGFβ signaling.152 into the host environment? How do those multifunctional effector proteins orchestrate their diverse activities in physiological situations? Significantly, such knowledge will Mycobacterium tuberculosis provide informative clues for the development of effective Mtb is an excellent example of a human bacterial pathogen that therapeutic approaches as well as vaccine strains to curb successfully survives in phagosomes and prevents their normal infectious diseases. maturation after engulfment by macrophage cells.153 Although Mtb encodes a structurally ubiquitin-like protein called RpfB ACKNOWLEDGEMENTS and even possesses a prokaryotic ubiquitin-like protein system We acknowledge research funding from the National Basic Research (Pup system) that is functionally analogous to the prototypical Programs of China (Grant Nos 2012CB518700 and 2014CB744400), the National Natural Science Foundation of China (Grant Nos ubiquitin system, thus far no direct evidence has demonstrated 81371769 and 81571954) and the Youth Innovation Promotion the capacity of Mtb to resist host immune elimination by Association CAS (Grant No. Y12A027BB2). utilizing its ubiquitin-like degradation system.154,155 This is likely because the highly unstructured Pup is unable to act as a post-translational modification or to mediate signaling path- ways. Therefore, Mtb inevitably resorts to other tactics to 1 Kerscher O, Felberbaum R, Hochstrasser M. Modification of proteins disturb the host immune system. Recently, our research group by ubiquitin and ubiquitin-like proteins. Annu Rev Cell Dev Biol 2006; 22:159–180. first reported that PtpA, a secreted Mtb tyrosine phosphatase, 2 Komander D, Rape M. The ubiquitin code. Annu Rev Biochem 2012; subtly co-opts the host ubiquitin system to facilitate Mtb 81:203–229. 156 3 Deshaies RJ, Joazeiro CA. RING domain E3 ubiquitin ligases. Annu survival, which was methodically summarized by Chen. Rev Biochem 2009; 78:399–434. Our data showed that Mtb PtpA is activated to directly 4 Rotin D, Kumar S. Physiological functions of the HECT family of – dephosphorylate p-JNK and p-p38 by binding to the host ubiquitin ligases. Nat Rev Mol Cell Biol 2009; 10:398 409. 5 Smit JJ, Sixma TK. RBR E3-ligases at work. EMBO Rep 2014; 15: ubiquitin via a brand new UIM-like region, resulting in the 142–154. repression of innate immunity.157 Furthermore, we observed 6 Smit JJ, Monteferrario D, Noordermeer SM, van Dijk WJ, van der Reijden BA, Sixma TK. The E3 ligase HOIP specifies linear that PtpA interferes with the association between TAB3 and ubiquitin chain assembly through its RING-IBR-RING domain and the K63-linked ubiquitin chains by competitively interacting with unique LDD extension. EMBO J 2012; 31:3833–3844. the NZF domain of TAB3, thus subverting NF-κB activation. 7 Chen ZJ, Sun LJ. Nonproteolytic functions of ubiquitin in cell signaling. Mol Cell 2009; 33:275–286. Our discovery that Mtb commandeers the ubiquitin system 8 Xu P, Duong DM, Seyfried NT, Cheng D, Xie Y, Robert J et al. provides novel insight into the Mtb–macrophage interaction Quantitative proteomics reveals the function of unconventional ubiqui- – tin chains in proteasomal degradation. Cell 2009; 137: 133–145. and reveals potential pathogen host interface-based targets for 9 Meyer HJ, Rape M. Enhanced protein degradation by branched the treatment of tuberculosis.158 ubiquitin chains. Cell 2014; 157:910–921.

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