Bacterial OTU deubiquitinases regulate substrate ubiquitination upon Legionella infection

  1. Donghyuk Shin
  2. Anshu Bhattacharya
  3. Yi-Lin Cheng
  4. Marta Campos Alonso
  5. Ahmad Reza Mehdipour
  6. Gerbrand J van der Heden van Noort
  7. Huib Ovaa
  8. Gerhard Hummer
  9. Ivan Dikic  Is a corresponding author
  1. Institute of Biochemistry II, Faculty of Medicine, Goethe University Frankfurt, Germany
  2. Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Germany
  3. Max Planck Institute of Biophysics, Germany
  4. Department of Nano-Bioengineering, Incheon National University, Republic of Korea
  5. Oncode Institute and Department of Cell and Chemical Biology, Leiden University Medical Centre, Netherlands
  6. Institute of Biophysics, Goethe University Frankfurt, Germany
6 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
Identification of novel deubiquitinases (DUBs) in Legionella pneumophila.

(a) Graphical illustration of identification of novel DUBs from L. pneumophila effector proteins. (b) Predicted DUB domain of four putative Legionella DUBs. (c, e) Time-course di-ubiquitin panel cleavage assay with Lpg1621 (LotB) and Lpg2529 (LotC). (d, f) Linkage specificity diagram of Lpg1621 (LotB) and Lpg2529 (LotC). The percentage of cleaved ubiquitin species at 90 min was plotted.

Figure 1—figure supplement 1
Ubiquitin cleavage assay with putative deubiquitinases (DUBs) from Legionella.

(a) Di-ubiquitin species were incubated with putative Legionella DUBs and analyzed by immuno-blotting with ubiquitin antibody. (b) HEK293 cell lysates were treated with purified Legionella DUBs and analyzed by immuno-blotting with indicated antibodies.

Figure 2 with 1 supplement
Biochemical properties of LotB and LotC.

(a) Predicted catalytic residues on LotB and LotC. (b, c) Di-Ub cleavage activity assay with wild-type and catalytic mutants of LotB and LotC. (d, e) Activity-based probes (ABPs) test on LotB and LotC. Propargyl-Ub-ABP (Prg-ABP) and vinylmethylester-ubiquitin-ABP (VME-ABP) were incubated as indicated time-points with LotB and LotC and analyzed on SDS-PAGE with coomassie staining. (f, g) Propargyl ubiquitin or ubiquitin-like modifiers reactivity test on LotB and LotC. Prg-ABPs are incubated with LotB and LotC with indicated time points.

Figure 2—figure supplement 1
Biochemical properties of LotB and LotC.

(a, b) Propargyl (Prg) – ubiquitin or ubiquitin-like modifiers reactivity test on LotB and LotC. Prg-ABPs are incubated with LotB and LotC for 60 min. (c) Sequence alignment of ubiquitin with NEDD8. Non-conserved residues are marked with red-reversed triangle.

Figure 3 with 1 supplement
Structural comparison of Legionella OTU-deubiquitinases with other OTU-family.

(a, b) Minimal domain boundaries of catalytically active LotB and LotC. Different constructs were cloned based on the predicted OTU-domains and their activity, and were tested with di-Ub panel. (c, d) Structural comparison of LotB and LotC with the closest homologues. CCHF- (PDB: 3PHU), OTUD2 (PDB: 4BOQ), OTUD3 (PDB: 4BOU), Taggert- (PDB: 6D × 3), DGK nairo- (PDB: 6D × 2), Otubain1 (PDB: 2ZFY), Otubain2 (PDB: 4FJV). (e) Sequence alignment of LotB and LotC with their closest homologues. Catalytic cysteine and histidine are highlighted in red and conserved residues are highlighted in yellow.

Figure 3—figure supplement 1
Sequence alignment of OTU deubiquitinase family.

The helical lobe between the catalytic Cys loop and the variable loop is shown as bar. The catalytic Cys is highlighted in red and conserved residues are highlighted in yellow.

Ubiquitin-binding sites on LotB and LotC.

(a, b) Molecular docking and simulations of monoubiquitin to LotB and LotC. Shown are representative snapshots of the MD simulations. Catalytic cysteine and key residues for the interaction between ubiquitin and LotB or LotC are depicted as sticks. (c, d) Key residues mediating interactions between ubiquitin and LotB or LotC. Residues are highlighted in the structure (left). Side-chain center-of-mass distances are shown as a function of the simulation time (right). (e, f) Di-ubiquitin cleavage assay for mutants of LotB and LotC. The catalytic activity of LotB or LotC wild-type and their mutants was tested with K63- or K48-linked Ub2, respectively.

Figure 5 with 2 supplements
Host-interacting proteins and cellular localization of LotB and LotC.

(a, c) Proteomic analysis of interacting partners of LotB and LotC. Catalytically inactive FLAG-LotB (C29A) and FLAG-LotC (C24A) were transfected and immunoprecipitated. Co-precipitated interacting proteins were analyzed by mass spectrometry. (b, d) Cellular localization of LotB and LotC. FLAG-tagged LotB and LotC were ectopically expressed in U2OS cells and immune-stained with cellular organelle markers (endoplasmic reticulum: Calnexin, mitochondria: TOMM20, Golgi: GM130).

Figure 5—figure supplement 1
Cellular localization of LotB full-length and LotB-OTU.

FLAG-tagged LotB or LotC-OTU catalytic core was ectopically expressed in U2OS cells and immune-stained with cellular organelle markers (endoplasmic reticulum: Calnexin, mitochondria: TOMM20, Golgi: GM130).

Figure 5—figure supplement 2
Proteomic analysis of interacting partners of LotB and LotC together with Legionella E3s.

(a, b) Proteomic analysis of interacting partners of LotB together with SdcA and SidC, respectively. Catalytically inactive FLAG-LotB (C29A) was co-transfected with either SdcA or SidC and immunoprecipitated. Co-precipitated interacting proteins were analyzed by mass spectrometry. (c, d) Proteomic analysis of interacting partners of LotC together with SdcA and SidC, respectively. Catalytically inactive FLAG-LotC (C24A) was co-transfected with either SdcA or SidC and immunoprecipitated. Co-precipitated interacting proteins were analyzed by mass spectrometry.

Substrate identification of LotB and LotC proteomic analysis of potential substrates of LotB and LotC.

(a–c) Schematic of the experiment and subsequent validation using western blot. (d, e) Volcano plot depicting the identified proteins with corresponding fold change and p-values. Comparison was done between Mut and WT deubiquitinase (DUB). Enriched proteins with Log2 Fold change ≥ 0.5 along with −Log10 p-value ≥ 1.3 was considered for further validation. (f–h) Immunoprecipitation of myc from the infected lysates was performed to enrich the potential substrates for LotB, which are RYK, Rab13, and PCYT1A, and for LotC, which are VAT1, HMOX1, and PPP2R1A, respectively. The enriched potential substrates were further incubated with wild-type or catalytic dead mutant DUB, followed by western blotting to detect ubiquitin and myc expression.

Tables

Table 1
TOP five candidates for putative deubiquitinases (DUBs) from Legionella effector proteins.
Legionella proteinsTarget proteins
NameAligned
region
NameAligned
region
Probability (%)Identities (%)PDB ID_Chain
Lpg1621195–274Viral OTU
(CC hemorrhagic fever virus)
69–15792.59163PHU_B
195–274Human OTUD263–14092.52174BOQ_A
195–279Human OTUD359–14292.40134BOU_A
193–278Human OTUD5100–18391.18213PFY_A
192–279Viral OTU
(Farallon virus)
88–18391.08166D × 5_B
Lpg25291–310Viral OTU (Erve virus)17–15796.24185JZE_A
7–310Viral OTU (Dera Ghazi Khan orthonairovirus)25–15696.15186D × 2_B
20–310Human Otubain150–23496.02132ZFY_A
20–310Human Otubain250–23395.84134FJV_C
7–310Viral OTU (Taggert virus)23–15695.71136D × 3_D
Lpg2411110–216Yeast UCH8152–25937.94113MHS_A
183–272EntA-im
(Enterococcus faecium)
7–8937.06152BL8_B
33–94Uncharacterized protein (Corynebacterium diphtheriae)7–7236.65213KDQ_D
120–212PG0816
(Porphyromonas gingivalis)
53–13935.31162APL_A
104–114PSII reaction center protein K (Cyanidium caldarium)2–1232.96364YUU_X2
Lpg2907117–384AvrA
(Salmonella typhimurium)
59–299100116BE0_A
158–398PopP2 (Arabidopsis thaliana)99–33999.93135W3X_C
115–390HopZ1a
(Pseudomonas syringae)
54–34299.88105KLP_C
118–275XopD
(Xanthomonas campestris)
1–14895.53112OIX_A
95–276Human SENP12–17795.21162G4D_A
  1. Values are obtained from the HHpred server (MPI Bioinformatics Toolkit).

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  1. Donghyuk Shin
  2. Anshu Bhattacharya
  3. Yi-Lin Cheng
  4. Marta Campos Alonso
  5. Ahmad Reza Mehdipour
  6. Gerbrand J van der Heden van Noort
  7. Huib Ovaa
  8. Gerhard Hummer
  9. Ivan Dikic
(2020)
Bacterial OTU deubiquitinases regulate substrate ubiquitination upon Legionella infection
eLife 9:e58277.
https://doi.org/10.7554/eLife.58277