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. 2015 Nov 17;6(6):e01777-15.
doi: 10.1128/mBio.01777-15.

Posttranslational Modification of HOIP Blocks Toll-Like Receptor 4-Mediated Linear-Ubiquitin-Chain Formation

James Bowman  1 , Mary A Rodgers  2 , Mude Shi  1 , Rina Amatya  1 , Bruce Hostager  3 , Kazuhiro Iwai  4 , Shou-Jiang Gao  1 , Jae U Jung  5
Affiliations

Posttranslational Modification of HOIP Blocks Toll-Like Receptor 4-Mediated Linear-Ubiquitin-Chain Formation

James Bowman et al. mBio. .

Abstract

Linear ubiquitination is an atypical posttranslational modification catalyzed by the linear-ubiquitin-chain assembly complex (LUBAC), containing HOIP, HOIL-1L, and Sharpin. LUBAC facilitates NF-κB activation and inflammation upon receptor stimulation by ligating linear ubiquitin chains to critical signaling molecules. Indeed, linear-ubiquitination-dependent signaling is essential to prevent pyogenic bacterial infections that can lead to death. While linear ubiquitination is essential for intracellular receptor signaling upon microbial infection, this response must be measured and stopped to avoid tissue damage and autoimmunity. While LUBAC is activated upon bacterial stimulation, the mechanisms regulating LUBAC activity in response to bacterial stimuli have remained elusive. We demonstrate that LUBAC activity itself is downregulated through ubiquitination, specifically, ubiquitination of the catalytic subunit HOIP at the carboxyl-terminal lysine 1056. Ubiquitination of Lys1056 dynamically altered HOIP conformation, resulting in the suppression of its catalytic activity. Consequently, HOIP Lys1056-to-Arg mutation led not only to persistent LUBAC activity but also to prolonged NF-κB activation induced by bacterial lipopolysaccharide-mediated Toll-like receptor 4 (TLR4) stimulation, whereas it showed no effect on NF-κB activation induced by CD40 stimulation. This study describes a novel posttranslational regulation of LUBAC-mediated linear ubiquitination that is critical for specifically directing TLR4-mediated NF-κB activation.

Importance: Posttranslational modification of proteins enables cells to respond quickly to infections and immune stimuli in a tightly controlled manner. Specifically, covalent modification of proteins with the small protein ubiquitin is essential for cells to initiate and terminate immune signaling in response to bacterial and viral infection. This process is controlled by ubiquitin ligase enzymes, which themselves must be regulated to prevent persistent and deleterious immune signaling. However, how this regulation is achieved is poorly understood. This paper reports a novel ubiquitination event of the atypical ubiquitin ligase HOIP that is required to terminate bacterial lipopolysaccharide (LPS)-induced TLR4 immune signaling. Ubiquitination causes the HOIP ligase to undergo a conformational change, which blocks its enzymatic activity and ultimately terminates LPS-induced TLR4 signaling. These findings provide a new mechanism for controlling HOIP ligase activity that is vital to properly regulate a proinflammatory immune response.

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Figures

FIG 1
FIG 1
Identification and characterization of HOIP ubiquitination. (A) LUBAC was purified and separated on an SDS-PAGE gel. Bands corresponding to elevated-molecular-weight HOIP species were cut, and protein was digested with trypsin for mass spectrometry analysis. A search for posttranslationally modified LUBAC peptides revealed that HOIP was modified by ubiquitin at lysine 640 and lysine 1056. While lysine 640 is not conserved between mouse and human, lysine 1056 is highly conserved in vertebrates and is located in the linear-ubiquitin chain-determining domain (LDD) of HOIP. (B) Flag-tagged HOIP constructs with various lysine-to-arginine mutations were cotransfected in human 293T cells with HA-ubiquitin constructs. Cells were lysed under denaturing conditions (1% SDS), followed by 10-fold dilution with lysis buffer for renaturation. Flag-HOIP was immunoprecipitated with anti-Flag and immunoblotted with anti-HA to detect the ubiquitinated forms of HOIP. Lysates were also used for immunoblotting with anti-Flag (bottom). (C) V5-tagged C terminus HOIP constructs were cotransfected in human 293T cells with HA-ubiquitin lysine point mutant (ubiquitin-K48R or ubiquitin-K63R) constructs. Lysates were immunoprecipitated with anti-V5, followed by immunoblotting with anti-HA. Lysates were also used for immunoblotting with anti-V5 or anti-HA. (D) V5-tagged C-terminal HOIP constructs were cotransfected in human 293T cells with HA-ubiquitin lysine mutant constructs where all lysines, except for the indicated residue, were mutated to arginine: ubiquitin-K48 or ubiquitin-K63 can form only K48- or K63-linked ubiquitin chains, respectively. Experiment was performed as described for panel B. (E) V5-tagged C-terminal HOIP constructs were cotransfected in human 293T cells with HA-ubiquitin constructs. Immunoprecipitation and immunoblotting were conducted as described for panel B. Samples were probed with ubiquitin linkage-specific antibodies to determine ubiquitin chain linkages modifying HOIP. Data are representative of three independent experiments.
FIG 2
FIG 2
Kinetics of LUBAC activity and complementation of HOIP-deficient cells. (A) Mouse A20.2J B cells were stimulated with 10 µg/ml LPS to activate TLR4 signaling and induce LUBAC enzymatic activity. Cellular linear-ubiquitin levels were determined by immunoprecipitation with a linear-ubiquitin-specific antibody. Resting cells have low levels of linear ubiquitin relative to cells stimulated with LPS for 1 h. (B) As for panel A but with earlier time points as well as immunoblotting for IRAK1. (C) Mouse A20.2J HOIP−/− cells were complemented with vector, HOIP-WT, or HOIP-KR by lentiviral infection. LUBAC expression in complemented cells was compared to that in A20.2J HOIP+/+ cells by immunoblot analysis. (D) HOIP-Flag was immunoprecipitated from lysates of cell lines generated for panel A using Flag or IgG control antibody to determine LUBAC formation. (E) To verify function of complement cell lines, cells were stimulated with 10 µg/ml LPS for 1 h and then lysed in LUIP buffer. A linear-ubiquitin-specific antibody was used to precipitate linear ubiquitin chains, followed by immunoblotting with anti-IRAK1. Data are representative of three independent experiments.
FIG 3
FIG 3
HOIP is ubiquitinated upon TLR4 stimulation. (A) A20.2J mouse B cells were stimulated with LPS and lysed under denaturing conditions. Endogenous HOIP was immunoprecipitated, separated by SDS-PAGE, and analyzed by immunoblotting. High-molecular-weight HOIP bands (>120,000) appeared after LPS stimulation. (B) Complemented mouse A20.2J HOIP−/− cells used for Fig. 2C were stimulated with LPS for the indicated times and lysed under denaturing conditions. Anti-IgG (control)- or anti-Flag (HOIP)-immunoprecipitated samples were prepared as described for panel A. A long exposure of the Flag immunoblot revealed the presence of high-molecular-weight HOIP bands. Data are representative of three independent experiments.
FIG 4
FIG 4
Mutation of HOIP lysine 1056 increases LUBAC enzymatic activity. (A) HOIP constructs were transfected into human 293T cells in the presence or absence of HOIL-1L. At 24 h after transfection, cells were lysed, and LUBAC protein expression and linear-ubiquitin-chain levels were determined by immunoblot analysis of cell lysates. (B) A20.2J HOIP−/− complemented cells from Fig. 2C were stimulated with 10 µg/ml LPS to activate TLR4 signaling and induce LUBAC enzymatic activity. Endogenous linear-ubiquitin levels were determined by immunoprecipitation with a linear-ubiquitin-specific antibody. (C) As described for panel B, but with LPS stimulation for various time points to compare linear-ubiquitin levels between WT and KR cells. (D) Densitometry analysis of the linear-ubiquitin immunoblot from panel C. All values are relative to WT cells prior to stimulation. (E) A20.2J HOIP−/− complemented cells stimulated as described for panel C were stained for linear ubiquitin using M1Ub-APC, and linear-ubiquitin-positive cells were quantified by flow cytometry. (F) A20.2J HOIP−/− complemented cells were stimulated as described for panel C. The immunoblot for IRAK1 shows the presence of linear ubiquitin chains on IRAK1 upon TLR4 activation. Data are representative of three independent experiments.
FIG 5
FIG 5
TLR4 stimulation induces HOIP conformational change. (A) Diagram of HOIP-FRET constructs with amino-terminal YFP fusions and carboxyl-terminal CFP fusions. (B) Function of HOIP-FRET constructs as determined by immunoblot for linear ubiquitins in 293T cell lysates containing either HOIP-WT or HOIP-FRET. (C) HOIP-FRET constructs were transfected into 293T cells. At 24 h after transfection, cells were lysed and FRET was measured. FRET values were calculated by subtracting background fluorescence and dividing fluorescence of YFP by fluorescence of CFP. Data are representative of three independent experiments. (D) HOIP-FRET constructs were transfected into A20.2J HOIP−/− cells by using the Neon system (Life Technologies). At 24 h after transfection, cells were resuspended in FluoroBrite medium, and fluorescence was measured using an Envision plate reader. After addition of LPS, fluorescence readings were taken at the indicated times. Relative FRET was calculated as FRET(x min)/FRET(0 min). HOIP-eCFP and HOIP-eYFP constructs were used as negative controls. Data are representative of three independent experiments.
FIG 6
FIG 6
TLR4 stimulation leads to higher NF-κB activation in HOIP-KR cells than in HOIP-WT cells. (A) A20.2J cells were stimulated with 10 µg/ml LPS and lysed at intervals up to 480 min after treatment with LPS. (B) Quantitative PCR using cDNA reverse transcribed from cells stimulated as described for panel A for the indicated times. Values were first calculated relative to 18S and then were normalized to vector cells prior to stimulation. (C) A20.2J cells were stimulated with 10 µg/ml CD40L, and lysates were analyzed up to 240 min poststimulation. (D) Quantitative PCR using cDNA reverse transcribed from A20.2J cells stimulated as described for panel C for the indicated times. Values were first calculated relative to 18S and then were normalized to vector cells prior to stimulation. Data are representative of three independent experiments.
FIG 7
FIG 7
Model of HOIP structural alteration upon TLR4 stimulation. TLR4 stimulation activates HOIP enzymatic activity. The synthesis of linear ubiquitin chains by HOIP promotes NF-κB activation. This signal, however, must be terminated to prevent sustained signaling. Ubiquitination of HOIP at lysine 1056 is triggered by TLR4 stimulation and alters HOIP conformation. The change in conformation inactivates HOIP enzymatic activity, which in turn removes a signal required for NF-κB activation, thus facilitating termination of the immune response.
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