Journal: The Journal of Biological Chemistry

Article Title: Outer membrane protein 25 of Brucella suppresses TLR-mediated expression of proinflammatory cytokines through degradation of TLRs and adaptor proteins

doi: 10.1016/j.jbc.2023.105309

Figure Lengend Snippet: Outer membrane protein 25 (Omp25) interacts with TLRs and their adaptor proteins except for MYD88. A , yeast two-hybrid assays showing the interaction of Omp25 with TLRs and adaptor proteins. AH109 yeast strain was transformed with bait plasmid harboring Omp25d fused with DNA-binding domain (BD) and the prey plasmid harboring TIRAP/TLR2/TLR4/TLR9/TRIF/MYD88 fused with the DNA activation domain (AD). Both the bait and prey plasmids were selected on SD/-Leu/-Trp amino acid dropout medium, followed by analyzing their interaction by streaking the yeast on the same media with X-α-Gal or on the triple amino acid dropout media, SD/-His/-Leu/-Trp containing X-α-Gal. The growth of yeast with blue color on the amino acid dropout media indicates the positive interaction. AH109 yeast harboring the plasmid expressing lamin fused with BD and T-antigen fused with AD served as the negative control, whereas the yeast carrying p53 fused with BD and T-antigen fused with AD served as the positive control. B – H , confirmation of interaction between Omp25 and TLRs or adaptor proteins by coimmunoprecipitation (co-IP). The lysate of HEK293T cells overexpressing FLAG-tagged TLR2/TLR4/TLR9/TIRAP/TRIF/TRAM/MYD88 was mixed with purified MBP-Omp25d or MBP alone, followed by IP of FLAG-tagged proteins using the anti-FLAG antibody and immunoblotting. The co-IP of MBP-Omp25d with FLAG-tagged TLRs or adaptor proteins suggests the positive interaction. No interaction was observed between MBP-Omp25 and FLAG-MYD88. The immunoblots are representative of two independent experiments. AD, activation domain; BD, binding domain; HEK293T, human embryonic kidney 293T cell line; MBP, maltose-binding protein; TLR, Toll-like receptor; WCL, whole-cell lysate.

Article Snippet: FLAG-tagged TIRAP (gifted by Dr Douglas Golenbock), TLR2, TLR4 (a gift from Ruslan Medzhitov; Addgene plasmid #13087), TLR9, TRIF (a gift from Kate Fitzgerald & Tom Maniatis; Addgene plasmid #41550), TRAM (a gift from Kate Fitzgerald & Doug Golenbock; Addgene plasmid #41551), and MYD88 (gifted by Dr Douglas Golenbock) plasmids were used to amplify respective TLR or adaptor protein genes and cloned into pGADT7 vector in fusion with DNA activation domain to generate prey plasmids.

Techniques: Membrane, Transformation Assay, Plasmid Preparation, Binding Assay, Activation Assay, Expressing, Negative Control, Positive Control, Co-Immunoprecipitation Assay, Purification, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Outer membrane protein 25 of Brucella suppresses TLR-mediated expression of proinflammatory cytokines through degradation of TLRs and adaptor proteins

doi: 10.1016/j.jbc.2023.105309

Figure Lengend Snippet: Outer membrane protein 25 (Omp25) induces degradation of TLRs and their adaptor proteins except for M Y D88. A – H , HEK293T cells were cotransfected with indicated concentrations of FLAG-tagged TLR2/TLR3/TLR4/TLR9/TIRAP/MYD88/TRIF/TRAM and MYC/HA-tagged Omp25 or its variants. Cells were lysed 24 h post-transfection, followed by immunoblotting. Omp25 and its variants induced degradation of TLRs and their adaptor proteins except for MYD88. I , Omp25 promotes the degradation of endogenous TIRAP in macrophages. RAW264.7 cells were transfected with indicated concentrations of MYC-Omp25 expressing plasmid. Twenty-four hours post-transfections, cells were harvested, followed by immunoblotting and detection of endogenous TIRAP and MYD88. J , the levels of endogenous TIRAP and MYD88 in macrophages transduced with lentivirus particles harboring Omp25 expression construct. RAW264.7 cells were transducted with lentiviral particles harboring Omp25d or empty vector. Fourty-eight hours post-transduction, cells were collected, followed by immunoblotting and detection of endogenous TIRAP and MYD88. Omp25d delivered through the lentivirus promoted the degradation of TIRAP. K – N , Omp25 does not affect the expression of its target proteins. RAW264.7 cells were transfected with indicated concentrations of Omp25 or its variants. Twenty-four hours post-transfections, total RNA was extracted from the cells, followed by cDNA synthesis and qPCR analysis. The transcript levels of endogenous TLR2, TLR4, TIRAP, or MYD88 were not altered in the presence of Omp25 or its variants. O and P , pulse-chase analysis of TIRAP degradation by Omp25d. HEK293T cells were cotransfected with FLAG-TIRAP and HA-Omp25d or empty vector for 24 h, followed by treatment with cycloheximide. Subsequently, the cells were harvested at the indicated time points, lysed, and subjected to immunoblotting. The gradual degradation of FLAG-TIRAP was detected in the presence of HA-Omp25d with increasing time points in cycloheximide-treated cells. Q , the recombinant MBP-Omp25d protein induces degradation of endogenous TIRAP. RAW264.7 cells were treated with purified MBP-Omp25d or MBP alone for 5 h. Subsequently, the cells were lysed and subjected to immunoblotting to detect MBP-Omp25d, endogenous TIRAP or MYD88, and actin. The recombinant MBP-Omp25d protein induced degradation of endogenous TIRAP but did not affect the level of MYD88. Actin served as the loading control for all the immunoblots. Right panel of each blot indicates the densitometry of TLRs or their adaptor protein bands, which were normalized with actin. The immunoblots are representative of three independent experiments. cDNA, complementary DNA; EV, empty vector; HA, hemagglutinin; HEK293T, human embryonic kidney 293T cell line; MBP, maltose-binding protein; qPCR, quantitative PCR; TLR, Toll-like receptor.

Article Snippet: FLAG-tagged TIRAP (gifted by Dr Douglas Golenbock), TLR2, TLR4 (a gift from Ruslan Medzhitov; Addgene plasmid #13087), TLR9, TRIF (a gift from Kate Fitzgerald & Tom Maniatis; Addgene plasmid #41550), TRAM (a gift from Kate Fitzgerald & Doug Golenbock; Addgene plasmid #41551), and MYD88 (gifted by Dr Douglas Golenbock) plasmids were used to amplify respective TLR or adaptor protein genes and cloned into pGADT7 vector in fusion with DNA activation domain to generate prey plasmids.

Techniques: Membrane, Transfection, Western Blot, Expressing, Plasmid Preparation, Transduction, Construct, cDNA Synthesis, Pulse Chase, Recombinant, Purification, Control, Binding Assay, Real-time Polymerase Chain Reaction