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Glutamine Deamidation and Dysfunction of Ubiquitin/NEDD8 Induced by a Bacterial Effector Family

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Science  03 Sep 2010:
Vol. 329, Issue 5996, pp. 1215-1218
DOI: 10.1126/science.1193844

Abstract

A family of bacterial effectors including Cif homolog from Burkholderia pseudomallei (CHBP) and Cif from Enteropathogenic Escherichia coli (EPEC) adopt a functionally important papain-like hydrolytic fold. We show here that CHBP was a potent inhibitor of the eukaryotic ubiquitination pathway. CHBP acted as a deamidase that specifically and efficiently deamidated Gln40 in ubiquitin and ubiquitin-like protein NEDD8 both in vitro and during Burkholderia infection. Deamidated ubiquitin was impaired in supporting ubiquitin-chain synthesis. Cif selectively deamidated NEDD8, which abolished the activity of neddylated Cullin-RING ubiquitin ligases (CRLs). Ubiquitination and ubiquitin-dependent degradation of multiple CRL substrates were impaired by Cif in EPEC-infected cells. Mutations of substrate-contacting residues in Cif abolished or attenuated EPEC-induced cytopathic phenotypes of cell cycle arrest and actin stress fiber formation.

Gram-negative bacterial pathogens use a type III secretion system (TTSS) to translocate effectors into eukaryotic host cells, serving as a key virulence mechanism (1, 2). Several effectors from different bacteria inhibit host cell cycle progression (36). Cif (cycle inhibiting factor) from Enteropathogenic E. coli (EPEC) arrests cell cycle at G2/M or G1/S transition (7). Cif homolog in Burkholderia pseudomallei (CHBP) also arrests cell cycle when delivered into eukaryotic cells (8). Cif and CHBP belong to a growing family of TTSS effectors that adopt a papain-like hydrolytic fold with a Cys-His-Asp/Asn/Glu/Gln catalytic triad (810). The host target and underlying mechanism for cell cycle arrest by the Cif/CHBP family are unknown.

Progression of eukaryotic cell cycle is driven by the ubiquitin-proteasome system (UPS) that mediates timed degradation of key cell cycle regulators. When delivered into HeLa cells that express green fluorescent protein (GFP) reporters of the UPS (11), purified CHBP, but not its catalytic mutant (C156S) (12), blocked degradation of UbG76V-GFP and Ub-R-GFP, but not the control Ub-M-GFP reporter (Fig. 1A). TNFα (tumor necrosis factor alpha) induces NF-κB (nuclear factor κB)–regulated gene transcription through UPS-dependent degradation of IκBα (inhibitor of NF-κB), which was also suppressed by CHBP (fig. S1). Ubiquitination is a sequential three-enzyme cascade composed of ubiquitin-activation enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. CHBP blocked free ubiquitin-chain synthesis catalyzed by different E3-E2 pairs, including a RING-domain E3 gp78c/Ube2g2 (13) (Fig. 1B and fig. S2, A to C). CHBP also blocked ubiquitination of RhoA mediated by a Cullin-based E3 complex (14) (Fig. 1C). Blocking ubiquitination required the catalytic cysteine in CHBP. CHBP is not a deubiquitinase, as it failed to disassemble polyubiquitin chains (fig. S2D). CHBP did not affect formation of E1~Ub and E2~Ub thioester intermediates (fig. S3). However, when CHBP-incubated E2~Ub thioester was reacted with E3, chain synthesis was largely inhibited (fig. S4). Thus, CHBP inactivates the E2~Ub thioester and is a potent and general inhibitor of the ubiquitination pathway.

Fig. 1

CHBP blocks ubiquitination by covalently modifying ubiquitin. (A) GFP reporter assays of effects of CHBP on the host ubiquitination pathway. Purified CHBP was directly delivered into HeLa cells expressing UbG76V-GFP, Ub-R-GFP, or Ub-M-GFP reporters. CHBPWT, wild-type CHBP; CHBPC/S, the catalytic cysteine mutant (C156S). (B and C) Effects of CHBP on in vitro ubiquitin-chain synthesis. In (B), gp78c and Ube2g2 were used as the E3 and E2, respectively, and reactions were stopped at indicated time points. Ubiquitination of RhoA by the Cul3/Roc1/BACURD complex was examined in (C). Ubn, ubiquitin chain; #, immunoglobulin G (IgG) heavy and light chains. (D) Native PAGE analysis of free ubiquitin treated with purified CHBP. WT, wild-type CHBP; C/S, C/A, H/A, and Q/A are mutations in the catalytic triad of CHBP. Coomassie blue–stained gel is shown.

None of E1, E2, ubiquitin, and E2~Ub thioester exhibited any changes when CHBP-incubated E2-charge reaction mixtures were subjected to reducing or nonreducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). When analyzed on a native PAGE gel, the E2~Ub thioester showed a faster migration toward the anode (fig. S5A). E2 recovered from the shifted E2~Ub thioester showed no mobility changes (fig. S5B). When free ubiquitin was treated with purified CHBP, but not its catalytic-triad mutants, a similar faster migration appeared (Fig. 1D). The mobility shift was greater than that of the E2~Ub thioester due to the smaller size of ubiquitin. Mass spectrometry was employed to reveal CHBP-induced modification on ubiquitin. Among the 15 tryptic peptides that cover the entire ubiquitin sequence, three overlapping ones (28AKIQDKEGIPPDQQR42, 30IQDKEGIPPDQQR42, and 30IQDKEGIPPDQQRLIFAGK48) from CHBP-treated ubiquitin showed a 1-dalton mass increase (fig. S6). Tandem mass spectrometry revealed that the 1-dalton increase occurred on Gln40 in ubiquitin (Fig. 2A), indicating a deamidation reaction. Substitution of Gln40 with Glu, but not Ala, caused a mobility change indistinguishable from that of CHBP-modified ubiquitin, and further CHBP treatment generated no additional shifts (Fig. 2B). Thus, CHBP specifically deamidates Gln40 in ubiquitin.

Fig. 2

CHBP deamidates Gln40 in ubiquitin and NEDD8 in vitro and during infection. (A) Electrospray ionization tandem mass spectrometry (MS/MS) spectrum of a Gln40-containing tryptic peptide from CHBP-treated ubiquitin. b and y ions are marked in the spectrum. The fragmentation patterns that generate the observed b and y ions are illustrated along the peptide sequence shown on top of the spectrum. The arrow marks the residue that shows a 1-dalton mass increase after CHBP treatment and is converted from glutamine into glutamate. (B) Native PAGE analysis of ubiquitin Gln40 mutants and effects of further CHBP treatment. Coomassie blue–stained gel is shown. (C) Chain formation activities of ubiquitin Q40E. Ubiquitination reaction was performed as that in Fig. 1B. (D) Native PAGE analysis of glutathione S-transferase (GST)–tagged ubiquitin and indicated UBLs after CHBP treatment. Coomassie blue–stained gel is shown. (E) Native PAGE assay of ubiquitin and NEDD8 deamidation by type III-secreted CHBP. Flag-UbΔGG and NEDD8ΔGG (deletion of the last two glycine residues) were immunopurified from 293T cells infected with indicated B. thailandensis strains and subjected to native gel electrophoresis followed by immunoblotting of antibodies by using Flag.

Similar to the effect of CHBP, ubiquitin Q40E was compromised in supporting in vitro ubiquitin-chain synthesis (Fig. 2C) without affecting E1 and E2 charging (fig. S3). CHBP-inactivated E2~Ub thioester also contained a deamidated Gln40 (fig. S7), suggesting that ubiquitin Q40E is defective in being transferred from E2 to the acceptor ubiquitin during chain synthesis. Consistently, Gln40 side chain is involved in interactions with E3 in the NEDD4L/E2~Ub oxyester complex (15). Ectopic expression of ubiquitin Q40E impaired TNFα-induced NF-κB luciferase reporter activation and led to the accumulation of several UPS substrates (fig. S8). Translocation of CHBP into HeLa cells by the EPEC TTSS also stabilized UPS substrates (fig. S9). Thus, deamidation of Gln40 in ubiquitin by CHBP attenuates ubiquitination both in vitro and in cells.

Ubiquitin-like proteins (UBLs) share ubiquitin’s three-dimensional fold (16). NEDD8 harbors ~80% sequence similarity with ubiquitin, whereas other UBLs are generally not similar to ubiquitin in primary sequence. Gln40 is conserved in NEDD8, SUMO2/3, and LC3, but only NEDD8 showed a mobility shift on the native gel upon CHBP treatment (Fig. 2D), resulting from deamidation of Gln40 (fig. S10). Thus, NEDD8 is another specific deamidation substrate of CHBP.

B. thailandensis serves as a model to study the virulence-associated TTSS system of B. pseudomallei (17). B. thailandensis harboring a CHBP expression plasmid was used to infect 293T cells that express UbΔGG or NEDD8ΔGG. Nearly 100% of NEDD8ΔGG and about 50% of UbΔGG were deamidated in a CHBP-dependent manner (Fig. 2E). This agrees with the potent in vitro activity of CHBP and its slight preference for NEDD8 over ubiquitin. Thus, CHBP deamidates both NEDD8 and ubiquitin during Burkholderia infection.

To compare enzymatic properties of CHBP with its EPEC homolog, 350 pmol of NEDD8 or ubiquitin in a 20-μl reaction (18 μM) was titrated with recombinant CHBP or Cif (fig. S11). After a 20-min reaction, 0.3 pmol and 0.03 pmol of CHBP were sufficient to deamidate nearly all 350 pmol of ubiquitin and NEDD8, respectively (Fig. 3A and fig. S11A). Although the activity of Cif on NEDD8 was comparable to that of CHBP on NEDD8, the activity of Cif on ubiquitin was lower by a factor of about 1000 than that on NEDD8 (Fig. 3A and fig. S11B). Consistently, NEDD8ΔGG was completely deamidated in HeLa cells infected with the Cif-bearing EPEC strain (E22), but not the Cif-deficient strain (E2348/49). In contrast, deamidation of UbΔGG was not detected (Fig. 3B). Complementation of E2348/49 with wild-type Cif, but not the catalytic mutant, restored NEDD8 deamidation (Fig. 3B).

Fig. 3

Cif selectively inactivates CRLs by deamidating NEDD8 in vitro and in vivo. (A) Enzyme-titration measurements of the deamidase activity of CHBP/Cif toward ubiquitin and NEDD8. Intensity of native ubiquitin/NEDD8 bands on native gels (fig. S11) was quantified and plotted versus the amount of CHBP/Cif used in each reaction. (B) Native PAGE assay of Cif deamidation of NEDD8 during EPEC infection of HeLa cells. EPEC E22 strain bears a functional Cif, whereas EPEC E2348/49 harbors a naturally truncated and nonfunctional Cif. Experiments were performed and data are presented similar to those in Fig. 2E. (C) Effects of Cif on steady levels of CRL and non-CRL substrates in EPEC-infected HeLa cells. GFP*, transfected Ub-R-GFP reporter. (D) Effects of Cif on ubiquitination of endogenous Nrf2 and p27 in EPEC-infected cells. #, IgG. (E and F) Effects of NEDD8 deamidation on neddylation-stimulated CRL activity of catalyzing substrate ubiquitination. Neddylation of Cul3 performed with NEDD8 (WT) or NEDD8 Q40E was shown in (E) as an immunoblot by using antibodies to Cul3. The Cul3/GST-Roc1 complex from the left three reactions in (E) was used to ubiquitinate Flag-Nrf2 (1–97) (F). Flag-Nrf2-Ub, Flag-Nrf2-Ub2, and Flag-Nrf2-Ubn denote mono-, di-, and poly-ubiquitinated Nrf2, respectively. #, IgG heavy and light chains; *, a nonspecific band.

NEDD8 is conjugated to Cullins that mediate the assembly of a large repertoire of Cullin-RING ubiquitin ligases (CRLs), and neddylation stimulates the activity of CRLs (18, 19). In EPEC-infected cells, substrates of CRLs (p27, Nrf2 and HIF-1α) accumulated in a Cif catalytic cysteine-dependent manner (Fig. 3C and fig. S12), whereas their mRNA levels were not affected (fig. S13). Consistently, ubiquitination of Nrf2 and p27 was markedly decreased (Fig. 3D). Non-CRL substrates—including p53, Mcl-1 (20), PINK1 (21), MOAP1 (22), and Ub-R-GFP reporter—were not stabilized (Fig. 3C and fig. S12). Thus, NEDD8 deamidation by Cif inactivates CRLs in infected cells.

Neddylation-stimulated CRL activation was reconstituted using Cul3/Roc1/Keap1 complex-catalyzed Nrf2 ubiquitination (Fig. 3, E and F). Neddylation of Cul3/Roc1 complex resulted in increased Nrf2 ubiquitination, and replacement of NEDD8 with NEDD8 Q40E abolished this stimulation effect (Fig. 3F) without disturbing autoneddylation of the Cul3/Roc1 complex (Fig. 3E and fig. S14). Similarly, deamidated ubiquitin did not affect Cul3 monoubiquitination (fig. S14). NEDD8 Q40E-conjugated Cul3 complex was even less active than the unneddylated counterpart (Fig. 3F), likely due to Glu-40 interfering with conformational changes of the Cullin/Roc1 complex (23). Thus, NEDD8 deamidation directly impairs the ubiquitination ligase activity of the neddylated CRL complex.

EPEC infection produces Cif-dependent actin stress fibers (6). Mutation of Cif deamidase catalytic residues abolished the development of stress fibers (fig. S15A) without affecting type III–dependent secretion (fig. S16). Actin stress fiber formation is controlled by RhoA, a Rho-family small guanosine triphosphatase (GTPase) and also a specific substrate of the Cul3/BACURD CRL complex (14). A panel of Rho GTPases was examined after EPEC infection, and only RhoA was stabilized by the deamidase activity of Cif (fig. S15B). Thus, Cif infection–induced formation of prominent actin stress fibers is probably due to dysfunction of CRLs as a result of NEDD8 deamidation.

Ectopic expression of NEDD8 Q40E resulted in accumulation of CRL substrates such as p27, Nrf2, and HIF-1α, but not non-CRL substrates (Fig. 4A and fig. S17). HeLa cells expressing NEDD8 Q40E showed decreased bromodeoxyuridine (BrdU) incorporation (Fig. 4B), indicating a defect in cell cycle progression. About 35% of transfected HeLa cells became enlarged with strong actin stress fibers, resembling the effect of EPEC infection. Thus, ectopic expression of NEDD8 Q40E partially recapitulates effects of Cif infection on impairing the CRL function.

Fig. 4

NEDD8 deamidation is linked to Cif-induced cytopathic effect during EPEC infection. (A) Effects of ectopic expression of NEDD8 Q40E on steady levels of CRL and non-CRL substrates. (B) Effects of ectopic expression of NEDD8 Q40E on cell cycle progression. Cell cycle of transfected HeLa cells was analyzed by BrdU incorporation. BrdU staining is in red and 4′,6′-diamidino-2-phenylindole (DAPI) is in blue. Scale bar, 300 μm. Statistics of BrdU-positive cells are presented in the graph as means ± SD of four independent countings of about 200 cells each. The experiment was repeated at least three times. (C and D) Effects of mutations in the substrate-contacting surface in Cif on stress fiber formation and cell cycle progression. Rhodamine-phalloidin–stained actin stress fibers and DAPI-stained nuclei in EPEC-infected HeLa cells are shown (C), and cell cycle profiles were determined by flow cytometry analysis of DNA contents (D). Scale bar, 50 μm.

Several point mutations in Cif were generated according to the crystal structure of CHBP/ubiquitin complex (Protein Data Bank ID code 3NZ5). D58A/D59A and N114A/N159A are mutations at the two enzyme-substrate contact interfaces; the former completely abolished Cif deamidase activity on NEDD8, and activity of the latter mutant is markedly attenuated (fig. S18). Both mutants failed to produce actin stress fibers during EPEC infection (Fig. 4C). The D58A/D59A mutant did not arrest cell cycle and the N114A/N159A mutant only marginally delayed cell cycle progression (Fig. 4D). Mutations of residues not involved in substrate binding (V111A, E139A, and K152A) had little effect on in vitro deamidation of NEDD8 (fig. S18), and these mutants behaved identically as wild-type Cif in producing stress fibers and arresting cell cycle (Fig. 4, C and D). All the mutants were competent for type III–dependent secretion (fig. S16). Thus, NEDD8 deamidation is closely linked to Cif-induced cytopathic effects of actin stress fiber formation and cell cycle arrest.

Here, we have shown that the CHBP/Cif family of TTSS effectors harbor a specific deamidase activity toward Gln40 in ubiquitin and NEDD8, rendering them inactive. Deamidated ubiquitin exhibits reduced ubiquitin ligase–catalyzed ubiquitin-chain synthesis, whereas deamidated NEDD8 suppresses the ubiquitin ligase activity of CRLs upon conjugation to Cullins. Selective deamidation of NEDD8 by Cif is linked to EPEC infection–induced cell cycle arrest and actin stress fiber formation. Given the universal role of ubiquitination and Cullin-mediated ubiquitination in many important cellular processes, our discovery further predicts a possible pleiotropic function of the Cif/CHBP family of effectors in bacterial pathogenesis.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1193844/DC1

Materials and Methods

Figs. S1 to S18

References

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Single-letter abbreviations for the amino acid residues are as follows: A, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; X, any amino acid; and Y, Tyr.
  3. We thank Y. Ye, C. Fathman, Z. Pan, D. Haas, V. Yu, E. Boedeker, and Z. Chen for providing reagents. We are grateful to F. Du and X. Wang for their assistance in obtaining reagents. We also thank members of the Shao laboratory for helpful discussions and technical assistance. This work was supported by Chinese Ministry of Science and Technology grant 2008AA022309 and National Basic Research Plan of China 973 grants to F.S. and National Institutes of Health grant R01 CA107134 to N.Z. and H.M. N.Z. is a Howard Hughes Medical Institute investigator.
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