EMBO/soda-nel huggingface dataset:
PLK4 deubiquitination by Spata2-CYLD suppresses NEK7-mediated NLRP3 inflammasome activation at the centrosome (2019)
Figure 2

Figure 2. Spata2 suppresses NLRP3 inflammasome activation in vitro and in vivo.

(A and B) ELISA of IL-1β secretion by WT or Spata2-KO BMDMs that were either untreated (-) or LPS-primed and then treated with the indicated NLRP3 inducers (A) or stimulated with the NLRC4 inducer (Salmonella infection) or AIM2 inducer ( Poly(dA:dT) transfection) (B). Bars and error bars represent the mean ± S.D. of triplicate experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Statistical analysis was performed using unpaired two-tailed Student's t-test.

(C) Immunoblot analysis of the mature IL-1β p17 and the active Caspase-1 p20 in cell supernatants (Sup) and the indicated proteins in the cell extracts (Cell ext) of WT or Spata2-KO BMDMs stimulated as in (A).

(D) WT and Spata2 CRISPR knock out (KO) iBMDMs stably expressing GFP-ASC were primed with LPS and stimulated with Nigericin for 1 h. GFP-ASC specks were imaged by confocal microscope, and percentage of ASC speck-positive cells was quantified. Scale bar, 10 µm. Bars and error bars represent the mean ± S.D. of triplicate experiments. ****P < 0.0001. Statistical analysis was performed using unpaired two-tailed Student's t-test.

(E) BMDMs from Spata2+/+ (WT) and Spata2-/- (KO) mice were pretreated with Caspase-1 inhibitor, Z-YVAD-FMK, for 30 min, primed with LPS, and then stimulated with Nigericin for 1 h. ASC specks were assessed by Immunofluorescence with antibody against ASC and imaged by confocal microscope, and percentage of ASC speck-positive cells was quantified. Scale bar, 10 µm. Bars and error bars represent the mean ± S.D. of triplicate experiments. *P < 0.05. Statistical analysis was performed using unpaired two-tailed Student's t-test.

(F) Immunoblot analysis of ASC in soluble cell lysate and DSS cross-linked insoluble fraction of WT or Spata2-KO BMDMs primed with LPS and stimulated without or with nigericin (Nig) for 1 h. Relative levels of ASC in cross-linked samples from LPS+ Nig treated cells were quantitated and shown below.

(G-I) Spata2+/+ (WT) and Spata2-/- (KO) mice were injected intraperitoneally (i.p.) with alum (20 mg/kg bodyweight) and sacrificed 6 h latter to collect peritoneal lavages. Absolute numbers of neutrophils recruited to the peritoneum were counted by FACS (G); levels of IL-1β (H) and IL-6 (I) in the peritoneal fluids were measured by ELISA. Circles and triangles represent individual mice. Horizontal bars indicate mean values. *P < 0.05. Statistical analysis was performed using unpaired two-tailed Student's t-test.

Data information: For all Immunoblotting data, molecular weights in kDa are indicated to the left.
PLK4 deubiquitination by Spata2-CYLD suppresses NEK7-mediated NLRP3 inflammasome activation at the centrosome (2019)
Figure 3

Figure 3. CYLD suppresses NLRP3 inflammasome activation in a manner dependent on Spata2-CYLD interaction.

(A) ELISA of IL-1β secretion by WT or CYLD KO BMDMs that were either untreated (-) or LPS-primed and then treated with the indicated NLRP3 inducers. Bars and error bars represent the mean ± S.D. of triplicate experiments. *P < 0.05; **P < 0.01; ***P < 0.001. Statistical analysis was performed using unpaired two-tailed Student's t-test.

(B) Immunoblot analysis of IL-1β and active caspase-1 p20 in cell supernatants (Sup) and the indicated proteins in cell extracts (Cell ext) from WT and CYLD KO BMDMs, stimulated as in (A).

(C) BMDMs from WT and CYLD KO mice were pretreated with Caspase-1 inhibitor, Z-YVAD-FMK, for 30 min, primed with LPS, and then stimulated with Nigericin for 30 min. ASC specks were assessed by Immunofluorescence with antibody against ASC and the percentage of ASC speck-positive cells was quantified. Bars and error bars represent the mean ± S.D. of triplicate experiments. *P < 0.05. Statistical analysis was performed using unpaired two-tailed Student's t-test.

(D) LDH release from WT and Cyld KO BMDMs primed with LPS and stimulated as indicated. Bars and error bars represent the mean ± S.D. of triplicate experiments. **P < 0.01. Statistical analysis was performed using unpaired two-tailed Student's t-test.

(E) Immunoblotting analysis of active caspase-1 p20 and IL-1β in cell supernatants (Sup) and the indicated proteins in cell extracts (Cell ext) of WT iBMDMs and Spata2 KO iBMDMs reconstituted with WT Spata2 or a Spata2 mutant (F108A) defective in CYLD binding that were primed with LPS and stimulated with Nigericin for 1 h.

Data information: For all Immunoblotting data, molecular weights in kDa are indicated to the left.
PLK4 deubiquitination by Spata2-CYLD suppresses NEK7-mediated NLRP3 inflammasome activation at the centrosome (2019)
Figure 4

Figure 4. Centrosomal localization of Spata2 is critical for recruiting CYLD to the centrosome and suppressing NLRP3 inflammasome activation.

(A) Confocal immunofluorescence analysis of Spata2 centrosome localization in HEK293 cells stably expressing control shRNA or shRNA against Spata2, stained with Spata2 and γ-tubulin antibodies and the nuclear dye DAPI. Scale bar, 10 µm.

(B) Confocal immunofluorescence analysis of Spata2 localization in RAW264.7 cells stably expressing GFP-Spata2, stained with γ-tubulin antibody and DAPI. Scale bar, 10 µm.

(C) Confocal microscopy of HEK293 cells stably expressing GFP-tagged Spata2 or Spata2 mutants (ΔPUB orΔ51-81) that were stained with γ-tubulin antibody and DAPI.

(D) Confocal microscopy of HEK293 cells stably expressing GFP-tagged CYLD along with an empty vector or vectors encoding Flag-tagged Spata2 or Spata2 mutants (ΔPUB orΔ51-81) stained as in (C). Scale bar, 10 µm.

(E) Co-immunoprecipitation analysis of CYLD physical interaction with Spata2 or Spata2 mutants (ΔPUB orΔ51-81) in HEK293 cells transfected with the indicated expression vectors. CYLD and Spata2 proteins were detected by immunoblot using anti-HA and anti-Flag antibodies, respectively.

(F) Immunoblotting analysis of the active caspase-1 p20 in cell supernatants (Sup) and pro-caspase-1 and Flag-Spata2 proteins in cell extracts (Cell ext) from WT iBMDMs and Spata2 KO iBMDMs reconstituted with WT Spata2 or Spata2 mutant defective in centrosome localization (ΔPUB orΔ51-81). Cells were primed with LPS and stimulated with Nigericin for 1 h.

Data information: For all Immunoblotting data, molecular weights in kDa are indicated to the left.
PLK4 deubiquitination by Spata2-CYLD suppresses NEK7-mediated NLRP3 inflammasome activation at the centrosome (2019)
Figure 5

Figure 5. Centrosomal kinase PLK4 suppresses NLRP3 inflammasome activation in a kinase activity-dependent manner.

(A) WT and Spata2 KO iBMDMs stably expressing an empty vector or a vector encoding Flag-NEK7 were untreated (NT), primed with LPS for 4 h, or primed with LPS for 4 h followed by stimulation with Nigericin for 30 min. Flag-NEK7 was immunoprecipitated and the association of endogenous NLRP3 was assessed by immunoblotting. Relative levels of NLRP3 in immunoprecipitates were normalized to Flag-NEK7 and shown below.

(B) Co-immunoprecipitation analysis of PLK4 interaction with endogenous Spata2 and CYLD in parental HEK293 cells and HEK293 cells stably expressing GFP-PLK4.

(C) iBMDMs stably expressing GFP-PLK4 and parental iBMDMs were unstimulated or stimulated with 0.5 ug/ml LPS for 4h. The PLK4 interaction with endogenous CYLD was analyzed by co-immunoprecipitation with anti-GFP antibody.

(D) Confocal microscopy analysis of ASC specks in iBMDMs stably expressing GFP-ASC, pretreated for 2 days with DMSO and two different PLK4 inhibitors, Centrinone (150 nM) and Centrinone-B(500 nM), primed with LPS for 4 h, and stimulated with Nigericin for 30 min. GFP-ASC specks were imaged and quantified (>500 cells counted). Scale bar, 10 µm. Bars and error bars represent the mean ± S.D. of triplicate experiments. *P < 0.05; ***P < 0.001. Statistical analysis was performed using unpaired two-tailed Student's t-test.

(E and F) Immunoblot analysis of the indicated proteins in cell supernatants (Sup) and cell extracts (Cell ext) of LPS-primed and Nigericin-stimulated BMDMs that were pretreated for 2 days with PLK4 inhibitors, Centrinone (150 nM) and Centrinone-B(500 nM) (E) or of LPS-primed and Nigericin-stimulated iBMDMs stably expressing a control shRNA or 3 different PLK4-specific shRNAs (F).

(G) Immunoblot analysis of active caspase-1 (p20) generation in HEK293 cells that were transfected with inflammasome components (Flag-tagged NLRP3, NEK7, ASC, and Caspase-1) along with EGFP, GFP-tagged PLK4, PLK4 kinase dead mutant (D154A), or PLK1 and stimulated with 10 µM Nigericin for 1h 24h post-transfection.

Data information: For all Immunoblotting data, molecular weights in kDa are indicated to the left.
PLK4 deubiquitination by Spata2-CYLD suppresses NEK7-mediated NLRP3 inflammasome activation at the centrosome (2019)
Figure 6

Figure 6. PLK4 inhibits NEK7-NLRP3 interaction by mediating NEK7 phosphorylation at Ser204.

(A) WT and Spata2 CRISPR knock out (KO) iBMDMs stably expressing GFP-ASC were pretreated with DMSO or PLK4 inhibitor Centrinone at a concentration of 150nM for 2 days, primed with LPS, and stimulated with Nigericin for 1 h. GFP-ASC specks were imaged by confocal microscope, and percentage of ASC speck-positive cells was quantified. Bars and error bars represent the mean ± S.D. of triplicate experiments. *P < 0.05; **P < 0.01. Statistical analysis was performed using unpaired two-tailed Student's t-test. See Appendix Fig S9 for images of ASC specks and levels of IL-1β and LDH.

(B) Co-immunoprecipitation analysis of NEK7/NLRP3 interaction in iBMDMs stably expressing Flag-NEK7 that were transduced with control or PLK4-specific shRNA. The cells were untreated, primed with LPS, or primed with LPS and stimulated with Nigericin. Flag-NEK7 was immunoprecipitated and the association of endogenous NLRP3 was assessed by immunoblot analysis. Relative levels of NLRP3 in immunoprecipitates were normalized to Flag-NEK7 and shown below.

(C) Co-immunoprecipitation analysis of NEK7/NLRP3 interaction in HEK293 cells transfected with HA-NEK7 and Flag-NLRP3 along with (+) or without (-) GFP-PLK4. The cells were either treated (+) or not treated (-) with the PLK4 inhibitor Centrinone.

(D) iBMDMs stably expressing Flag-tagged NEK7 or NEK7 mutant S204A were treated with LPS. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for the interaction between NEK7 and endogenous NLRP3.

(E) Immunoblot analysis of active caspase-1 (p20) in supernatants (Sup) and the indicated proteins in cell extracts (Cell ext) of iBMDMs stably expressing WT NEK7 or NEK7 mutant (S204A), primed with LPS and stimulated with nigericin.

(F) iBMDMs stably expressing Flag-tagged WT NEK7 or NEK7 mutant S204A were left untreated or stimulated with LPS for indicated time. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204 (P-NEK7).

(G) iBMDMs stably expressing Flag-tagged WT NEK7 were left untreated or pretreated with PLK4 inhibitor Centrinone, and stimulated without or with LPS for 4h. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204.

(H) iBMDMs stably expressing GFP-tagged PLK4 and parental iBMDMs were left untreated or stimulated with LPS for 4h. Anti-GFP immunoprecipitates were prepared for immunoblot analysis for the interaction between GFP-PLK4 and endogenous NEK7.

Data information: For all Immunoblotting data, molecular weights in kDa are indicated to the left.
PLK4 deubiquitination by Spata2-CYLD suppresses NEK7-mediated NLRP3 inflammasome activation at the centrosome (2019)
Figure 7

Figure 7. Spata2 and CYLD regulate PLK4 ubiquitination and function in NLRP3 inflammasome inhibition.

(A) Co-immunoprecipitation analysis of NEK7/NLRP3 interaction in HEK293 cells transfected with the indicated expression vectors, treated with either DMSO or 150 nM PLK4 inhibitor Centrinone.

(B) WT and Spata2 KO iBMDMs stably expressing Flag-tagged WT NEK7 were left untreated or pretreated with the PLK4 inhibitor Centrinone, and stimulated without or with LPS for 4h. Anti-Flag immunoprecipitates were prepared for immunoblot analysis for NEK7 phosphorylation at Ser 204.

(C) HEK293 cells stably expressing GFP-PLK4Δ24 were transfected with a control siRNA or siRNAs targeting Spata2 or CYLD. GFP-PLK4Δ24 was immunoprecipitated under denaturing conditions and immunoblotted for K63-, K48-linked and linear ubiquitination. Relative levels of PLK4 ubiquitination were normalized to GFP-PLK4 and shown below.

(D) Co-immunoprecipitation analysis of the PLK4-NEK7 interaction in HEK293 cells co-transfected with Flag-NEK7 and HA-PLK4 in the presence (+) or absence (-) of GFP, GFP-Spata2, GFP-CYLD, or GFP-CYLD C601S, a catalytically inactive mutant.

(E) A model of NLRP3 inflammasome regulation by PLK4 and Spata2/CYLD. Polyubiquitination of PLK4 suppresses PLK4 binding to and phosphorylation of NEK7. This polyubiquitination-mediated suppression can be reversed by Spata2 and CYLD-mediated deubiquitination of PLK4, thereby allowing for PLK4 phosphorylation of NEK7, which attenuates NEK7 binding to NLRP3 and NLRP3 inflammasome activation. Spata2 or CYLD deficiency would cause accumulation of PLK4 polyubiquitination, reduction of NEK7 phosphorylation, increase of NEK7 binding to NLRP3, and eventually hyperactivation of NLRP3 inflammasome.

Data information: For all Immunoblotting data, molecular weights in kDa are indicated to the left.
Mycobacterium tuberculosis protein kinase G acts as an unusual ubiquitinating enzyme to impair host immunity (2021)
Figure 1

Figure 1. Mtb PknG interacts with the E2 protein UbcH7 through the Ubl domain.

A Yeast two-hybrid assay for the interaction of PknG with UbcH7. Yeast strains were transformed with the indicated plasmids, wherethe TAK1-TAB2 interaction served as a positive control. Left, low stringency. Right, high stringency.

B Immunoprecipitation (IP) of UbcH7 by Mtb PknG in U937 cells. Cells were infected with wild-type (WT) Mtb or Mtb ΔpknG at an MOI of 1. Non-infected cells were used as controls. After 4 hours, the cells were lysed and immunoprecipitated with the antibody against PknG. IP products were immunoblotted with the antibody against UbcH7.

C Pull-down of His-tagged E2s (5 μg each) by GST-PknG (10 μg each). Pull-down products were immunoblotted using the antibody against the His-tag.

D Schematic diagram of the Mtb PknG domains.

E, F Topology diagrams of the β-grasp folds of ubiquitin (E, left), the ubiquitin-fold domain (UFD) of the E1 enzyme (E, right), and the putative Ubl domain in PknG (F, left) are shown, and a cartoon depiction of the β-grasp fold of PknG is included (F, right). Strands in the topology diagrams are illustrated as cyan arrows, and the helices are highlighted in magenta.

G IP of UbcH7 by PknG or PknGΔUbl in U937 cells. Cells were infected with WT, ΔpknG, or ΔpknG:pknGΔUbl Mtb strains as in B.

H IP of UbcH7 by PknG or PknG E190A in U937 cells. Cells were infected with WT, ΔpknG, or ΔpknG:pknG E190A Mtb strains as in B.
Mycobacterium tuberculosis protein kinase G acts as an unusual ubiquitinating enzyme to impair host immunity (2021)
Figure 2

Figure 2. Mtb PknG catalyzes the conjugation of ubiquitin onto UbcH7 in vitro.

A In vitro ubiquitin conjugation assay of UbcH7. UbcH7 was incubated with PknG, Ub, and ATP/Mg2+ at 37°C for 30 min. Reaction products were analyzed on a 12% SDS-PAGE gel and stained with Coomassie Brilliant Blue.

B In vitro ubiquitin conjugation assay of UbcH7 catalyzed by PknG over time. The reaction products were analyzed as in A.

C The ADP/ATP ratios in the ubiquitination reaction mixtures. The reaction mixtures containing 2 μM PknG K181M and 5 μM ATP were incubated at 37°C for 30 min in the absence (reaction 1) or presence (reaction 2) of 5 μM Ub and 5 μM UbcH7. LC-MS MultiQuant software was used to calculate the normalized abundance of each compound (including AMP, ADP, and ATP) by measuring the peak area intensity.

D In vitro ubiquitin conjugation assay of UbcH7 catalyzed by GST-tagged PknG or its mutants at 37°C for 30 min. Reaction products were immunoblotted with antibodies against UbcH7 (top panel), Ub (middle panel), and the GST tag on PknG (bottom panel).

E In vitro ubiquitin conjugation assay of UbcH7 catalyzed by PknG or His-UBA1 at 37°C for 30 min. The reaction products were analyzed by immunoblotting with antibodies against Ub (top panel), the His-tag on UBA1 (middle panel), and PknG (bottom panel), or by staining with Coomassie Brilliant Blue (bottom). For reducing conditions, samples were treated with 500 mM β-mercaptoethanol (βME) prior to SDS-PAGE gel analysis.

F In vitro ubiquitin conjugation assay of UbcH7 catalyzed by PknG or its E1 activity-dead mutant (PknG P57A). Top, the schematic of Mtb PknG. Red arrow, Pro57 site; Bottom, reaction products were analyzed as in A.
Mycobacterium tuberculosis protein kinase G acts as an unusual ubiquitinating enzyme to impair host immunity (2021)
Figure 3

Figure 3. Mtb PknG is an unconventional E1 with isopeptidase activity in vitro.

A MS/MS analysis of UbcH7 in the absence (top) or presence (bottom) of PknG to reveal ubiquitination sites. Tryptic peptides spanning residues ranging from 74 to 100 were identified as the high-confidence peptides possessing di-Gly modifications. The ubiquitinated residue (Lys82) in the peptide sequence is highlighted in red. The peak heights are the relative abundances of the corresponding fragmentation ions. Matched amino terminus-containing ions (b ions) are indicated in red, and matched carboxyl terminus-containing ions (y ions) are indicated in blue. Only the major peaks that were identified are labeled.

B Ubiquitin conjugation assay of UbcH7 or its mutants catalyzed by GST-tagged PknG at 37°C for 30 min. Reaction products were immunoblotted using antibodies against UbcH7 (top panel), Ub (middle panel), and the GST tag on PknG (bottom panel).

C In vitro ubiquitin discharge assay of UbcH7~Ub with or without PknG. The reaction products were immunoblotted as in B.

D In vitro isopeptidase assays of PknG with or without ubiquitin-aldehyde (Ub-Ad). The reactions were performed at 37°C for 30 min and analyzed as in B.

E In vitro ubiquitin conjugation assay of UbcH7 by PknG, PknG P57A, or PknG I87A. The reaction products were immunoblotted as in B.

F In vitro ubiquitin discharge assay of UbcH7~Ub by PknG or its isopeptidase activity-dead mutant (PknG I87A). The reaction products were immunoblotted as in B.
Mycobacterium tuberculosis protein kinase G acts as an unusual ubiquitinating enzyme to impair host immunity (2021)
Figure 4

Figure 4. Mtb PknG inhibits NF-κB activation in a E190 residue-dependent manner.

A Luciferase assay of NF-κB activation in HEK293T cells transfected with WT or mutant Mtb PknG. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05, **P < 0.01. Data were analyzed according to one-way analysis of variance (ANOVA) using GraphPad Prism 7.

B, C qPCR analysis of Tnf mRNA (B) and Il6 mRNA (C) in primary human monocyte-derived macrophages. Cells were infected with WT, ΔpknG, ΔpknG:pknG, ΔpknG:pknG K181M, ΔpknG:pknG E190A, or ΔpknG:pknG K181M, E190A Mtb strains at an MOI of 1 for 0-24 hours. The fold changes obtained from each sample were normalized to those of GAPDH. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05, **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7.

D Survival of Mtb strains in primary human monocyte-derived macrophages treated as in B and C. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05, **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7.
Mycobacterium tuberculosis protein kinase G acts as an unusual ubiquitinating enzyme to impair host immunity (2021)
Figure 5

Figure 5. Mtb PknG targets TRAF2 and TAK1 for degradation by binding to UbcH7.

A Dual-luciferase assay of TRAF2-, TAB2-, TAB3-, TAK1-, or TAB1-induced NF-κB activation in the absence or presence of Mtb PknG in HEK293T cells. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05. Data were analyzed according to a two-tailed unpaired t-test using GraphPad Prism 7.

B IP of TRAF2 and TAK1 by Mtb PknG in U937 cells. Cells were infected with the indicated Mtb strains at an MOI of 1. Non-infected cells were used as controls. After 4 hours, the cells were lysed and immunoprecipitated with the antibody against PknG. The immunoprecipitated proteins were immunoblotted with TRAF2, TAK1, TAB2 (negative control), or TAB3 (negative control) antibodies.

C IP of TRAF2 and TAK1 by GFP-tagged Mtb PknG or its truncated forms in transfected HEK293T cells.

D Immunoblotting analysis of TRAF2, TAK1, and PknG from lysates of U937 cells infected with the indicated Mtb strains at an MOI of 1 for 0-24 hours. GAPDH was used as the loading control. Densitometric quantification of immunoblots is indicated below the immunoblots.

E Immunoblotting of TRAF2, TAK1, and PknG from lysates of U937 cells infected with the indicated Mtb strains at an MOI of 1 for 8 hours. Cells were treated with CHX (50 μg/mL) with or without MG132 (5 μM). Cell lysates were analyzed by immunoblotting for TRAF2 (anti-TRAF2), TAK1 (anti-TAK1), and PknG (anti-PknG). GAPDH was used as the loading control. Densitometric quantification of immunoblots is indicated below the immunoblots.
Mycobacterium tuberculosis protein kinase G acts as an unusual ubiquitinating enzyme to impair host immunity (2021)
Figure 6

Figure 6. Mtb PknG promotes Lys48-linked poly-ubiquitination of TRAF2 and TAK1 depending on UbcH7-binding.

A, B In vitro ubiquitination of Flag-TRAF2 (A) and Flag-TAK1 (B) in the presence of Ub-conjugated UbcH7 (catalyzed by PknG) and PknG or its mutants, with or without ATP as an energy source. The reaction products were analyzed by immunoblotting with antibodies against Flag-tagged TRAF2 and TAK1.

C, D In vivo ubiquitination assay of TRAF2 (C) and TAK1 (D) in U937 cells infected with WT, ΔpknG, ΔpknG:pknG, ΔpknG:pknG K181M, or ΔpknG:pknG E190A Mtb strains at an MOI of 1 for 6 hours. Endogenous ubiquitinated proteins were enriched using TUBE reagents and immunoblotted with antibodies against TRAF2 (C, top panel) or TAK1 (D, top panel).

E, F In vitro ubiquitination of TRAF2 (E) and TAK1 (F) in the presence of UbcH7, Ub, and

PknG or its mutants. The reaction products were immunoblotted using antibodies against TRAF2 or TAK1.

G, H In vivo ubiquitination assay of TRAF2 (G) and TAK1 (H) in the absence or presence of PknG in HEK293T cells transfected with the indicated plasmids for 24 hours. Cells were lysed and immunoprecipitated using the antibody against Flag. Immunoprecipitated proteins were immunoblotted with the antibody against HA.
Mycobacterium tuberculosis protein kinase G acts as an unusual ubiquitinating enzyme to impair host immunity (2021)
Figure 7

Figure 7. The Ubl domain of PknG contributes to host innate immune suppression in vivo.

A, B qPCR analysis of Tnf mRNA (A) and Il6 mRNA (B) in splenic cells from C57BL/6 mice infected with WT or mutant Mtb stains for 0-20 days. Data are representative of one experiment with two independent biological replicates (mean ± SEM of n = 6 mice per group). *P < 0.05 and **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7.

C Bacterial load in lungs from C57BL/6 mice treated as in A. Data are representative of one experiment with two independent biological replicates (mean ± SEM of n = 6 mice per group). *P < 0.05 and **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7.

D H&E staining of lungs and livers of C57BL/6 mice treated as in A. Arrows indicate puncta of cellular infiltration. Scale bars, 200 μm.

E Proposed model depicting Mtb PknG-mediated host innate immune suppression. During mycobacterial infection, PknG binds to ATP to catalyze a unique two-step ubiquitination cascade through unconventional E1 and E3 enzyme activities with the release of ADP instead of AMP, ultimately leading to degradation of TRAF2 and TAK1 and suppression of host innate immune responses.

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