Glutamine deamidation and dysfunction of ubiquitin/NEDD8 induced by a bacterial effector family

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.

Fig. 1. CHBP blocks the ubiquitination pathway by covalently modifying ubiquitin

(A) GFP reporter assays of effects of CHBP on the host ubiquitination pathway. HeLa cells were transfected with Ub^G76V-GFP, Ub-R-GFP or Ub-M-GFP reporter plasmid. Purified CHBP was delivered into transfected cells using the anthrax lethal factor system. Ub^G76V-GFP and Ub-R-GFP are rapidly degraded in cells, while Ub-M-GFP (a negative control) is not sensitive to the ubiquitin-proteasome pathway. CHBP^WT, wild-type CHBP; CHBP^C/S, the catalytic cysteine mutant (C156S). MG132 treatment serves as a positive control. Shown are anti-GFP and anti-actin immunoblots of lysates of cells harvested at indicated time points following CHBP delivery. (B and C) Effects of CHBP on in vitro ubiquitin-chain synthesis. Ubiquitination reaction, using bacterially purified ROC1/Cul1-CTD complex as the E3 and UbcH5c as the E2, was carried out in the presence of indicated amounts of recombinant CHBP (B). In (C), gp78c and Ube2g2 were used as the E3 and E2, respectively, and reactions carried out in the presence/absence of CHBP were stopped at indicated time points. Shown are anti-ubiquitin immunoblots to examine ubiquitin-chain (Ub_n) formation. (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 native gel is shown. (E) Chain formation activities of CHBP-modified ubiquitin. In vitro ubiquitination reaction was carried out as that in (C). WT, wild-type ubiquitin; MS, mobility-shifted ubiquitin generated by CHBP treatment and recovered from the native gel shown in (D) by electro-elution. Reactions were subjected to immunoblotting analysis for ubiquitin chain (Ub_n) formation.

Fig. 2. CHBP deamidates Gln-40 in ubiquitin/NEDD8 in vitro and during infection

(A) Electrospray ionization (ESI) tandem mass spectrometry (MS/MS) spectrum of a Gln-40-containing tryptic peptide from CHBP-treated ubiquitin (left) or the control untreated ubiquitin (right). 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 one-dalton mass increase after CHBP treatment and is converted from glutamine into glutamic acid. (B) Native PAGE analysis of ubiquitin Gln-40 mutants and effects of further CHBP treatment. Wild-type (WT) and indicated ubiquitin mutants were loaded onto the native gel either directly or after incubation with CHBP (wild-type or the catalytic cysteine mutant (C/A)). The gel was stained with Coomassie blue. (C) Chain formation activities of ubiquitin Q40E and comparison with effects of CHBP on wild-type ubiquitin. In vitro ubiquitination reaction was carried out as that in Fig. 1C. Reactions were subjected to immunoblotting analysis for ubiquitin chain (Ub_n) formation. (D) Effects of ectopic expression of ubiquitin Q40E on TNFα-induced IκBα degradation and NF-κB pathway activation. Intact HeLa cells (for the lower immunoblot panel) or HeLa cells expressing NF-κB dual-luciferase reporters were transfected with ubiquitin Q40E expression plasmid or a vector control and then stimulated with TNFα. Shown are mean relative luciferase activities from duplicate determinations with error bars indicating standard deviation. The lower panel shows anti-IκBα and anti-actin immunoblots of total cell lysates. (E) Native PAGE analysis of GST-tagged ubiquitin and indicated UBLs following incubation with CHBP. Coomassie blue stained native gel is shown. (F) Native PAGE assay of ubiquitin and NEDD8 deamidation by type III-secreted CHBP. 293T cells transfected with Flag-UbΔGG or Flag-NEDD8ΔGG expression plasmid were infected with B. thailandensis or B. thailandensis expressing CHBP. UbΔGG and NEDD8ΔGG immunopurified from infected cells were subjected to native gel electrophoresis followed by anti-Flag immunoblotting. UbΔGG and NEDD8ΔGG (deletion of the last two glycine residues) were used to avoid polyconjugation and conjugation to other cellular proteins.

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 towards ubiquitin and NEDD8. 350 pmol (3 μg) of ubiquitin or NEDD8 were subjected to 20-min incubation with increasing amounts of purified CHBP or Cif in a 20-μl reaction at 37°C. The whole reaction mixtures were analyzed by native gel electrophoresis followed by Coomassie blue staining as shown in fig. S12. Intensity of native ubiquitin/NEDD8 bands on native gels 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 while EPEC E2348/49 strain harbors a naturally truncated and nonfunctional Cif. Experiments were performed and data are presented similarly as those in Fig. 2F. (C) Effects of Cif on steady levels of CRL and non-CRL substrates in EPEC-infected cells. HeLa cells were infected with EPEC strains expressing wild-type Cif (Cif^WT) or the catalytic mutant (Cif^C/A) for indicated time durations. Endogenous levels of indicated proteins were shown by immunoblotting of total cell lysates using specific antibodies. p27, HIF-1α, and Nrf2 are ubiquitination substrates of Cul1, Cul2 and Cul3 CRL complex, respectively. p53, MOAP1, PINK1 and Mcl-1 are unstable proteins whose degradation is independent of CRLs in HeLa cells. GFP* indicates transfected Ub-R-GFP reporter. MG132 treatment was included as a control. (D) Effects of Cif on ubiquitination of Nrf2 and p27 in infected cells. Endogenous Nrf2 or p27 was immunoprecipitated under denaturing conditions from EPEC-infected and MG132-treated HeLa cells (expressing Flag-ubiquitin) using the specific or a control antibody. The immunoprecipitates (IP) were analyzed by anti-Flag (upper panels) and anti-Nrf2 (or p27) immunoblotting. Total cells lysates (Input) were also blotted with anti-Nrf2 (or anti-p27) and anti-actin antibodies as shown. # marks IgG. (E and F) Effects of NEDD8 deamidation on neddylation-stimulated CRL activity of catalyzing substrate ubiquitination. Purified Cul3/GST-Roc1 complex was first subjected to neddylation reaction with NEDD8 (WT) or NEDD8 Q40E and neddylated Cul3 is shown by anti-Cul3 immunoblot in (E). The Cul3/GST-Roc1 complex present in the left three reactions in (E) was immobilized onto glutathione beads and then used to ubiquitinate Flag-Nrf2 (1–97) in the presence of Keap1 as indicated in (F). Reaction mixtures were analyzed by anti-Flag immunoblotting (upper) or subjected to anti-Flag immunoprecipitation under denaturing conditions followed by anti-ubiquitin immunoblotting (lower). 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.

Fig. 4. NEDD8 deamidation is linked to Cif-induced cytopathic effect during infection

(A) Effects of ectopic expression of NEDD8 Q40E on steady levels of CRL and non-CRL substrates. HeLa cells were transfected with indicated Flag-NEDD8 expression plasmid. Endogenous levels of indicated proteins were shown by immunoblotting of total cell lysates using specific antibodies. (B) Effects of ectopic expression of NEDD8 Q40E on cell cycle progression. 10 μM of BrdU were added into medium of HeLa cells transfected with indicated Flag-NEDD8 expression plasmid for 1 h. Cells were stained with anti-BrdU antibody and DAPI. Statistics of BrdU-positive cells are presented in the graph as means ± SD of four independent counting of about 200 cells each. The experiment was repeated for at least three times. (C and D) Effects of mutations in the substrate-contacting surface in Cif on stress fibers formation and cell cycle progression. HeLa cells were infected with EPEC strains expressing wild-type Cif (WT) or indicated Cif mutants. Fluorescence images of actin stress fibers stained by Rhodamine-phalloidin and DAPI-stained nuclei are shown (C) and cell cycle profiles of infected cells were determined by flow cytometry analysis of DNA contents (D).