TAB1: A Target of Triptolide in Macrophages

Slides:



Advertisements
Similar presentations
Figure S1. Production of recombinant NS1 protein
Advertisements

Figure S1 A B Figure S1. SPATA2 is required for TNFα or zVAD.fmk induced necroptosis in L929 cells. (A) L929 cells were transfected with a pool of four.
IL-18 Downregulates Collagen Production in Human Dermal Fibroblasts via the ERK Pathway  Hee Jung Kim, Seok Bean Song, Jung Min Choi, Kyung Moon Kim,
Volume 114, Issue 5, Pages (May 1998)
LPS-Induced Upregulation of SHIP Is Essential for Endotoxin Tolerance
Volume 133, Issue 1, Pages (July 2007)
Takashi Tanaka, Michelle A. Soriano, Michael J. Grusby  Immunity 
Volume 25, Issue 6, Pages (December 2006)
Volume 4, Issue 3, Pages (September 2006)
Volume 69, Issue 4, Pages (February 2006)
Volume 24, Issue 5, Pages (May 2006)
Volume 22, Issue 3, Pages (May 2006)
Volume 28, Issue 3, Pages (September 2015)
Volume 30, Issue 3, Pages (March 2009)
Volume 26, Issue 1, Pages (January 2007)
Volume 23, Issue 1, Pages (July 2006)
Volume 19, Issue 4, Pages (April 2017)
A Mechanism for Inhibiting the SUMO Pathway
Volume 36, Issue 1, Pages (January 2012)
Volume 35, Issue 6, Pages (December 2011)
ASK1 Is Essential for JNK/SAPK Activation by TRAF2
Volume 19, Issue 7, Pages (July 2012)
Volume 15, Issue 1, Pages (January 2012)
IGF-II-Mediated COX-2 Gene Expression in Human Keratinocytes Through Extracellular Signal-Regulated Kinase Pathway  Hye Jung Kim, Tae-Yoon Kim  Journal.
Volume 19, Issue 6, Pages (September 2005)
Volume 6, Issue 6, Pages (March 2014)
Yongli Bai, Chun Yang, Kathrin Hu, Chris Elly, Yun-Cai Liu 
Volume 19, Issue 11, Pages (June 2017)
Volume 19, Issue 9, Pages (September 2012)
Volume 14, Issue 7, Pages (July 2007)
MUC1 Oncoprotein Stabilizes and Activates Estrogen Receptor α
Volume 19, Issue 4, Pages (April 2012)
Mechanisms of cross hyporesponsiveness to toll-like receptor bacterial ligands in intestinal epithelial cells  Jan-Michel Otte, Elke Cario, Daniel K.
PARP1 Represses PAP and Inhibits Polyadenylation during Heat Shock
Volume 47, Issue 4, Pages e3 (October 2017)
Yuming Wang, Jennifer A. Fairley, Stefan G.E. Roberts  Current Biology 
Volume 17, Issue 1, Pages (January 2010)
Lisheng Ge, Ziqiu Wang, Meifang Wang, Siddhartha Kar, Brian I. Carr 
Volume 23, Issue 2, Pages (February 2016)
MUC1 Oncoprotein Stabilizes and Activates Estrogen Receptor α
Regulation of YAP by Mechanical Strain through Jnk and Hippo Signaling
C-Jun Downregulation by HDAC3-Dependent Transcriptional Repression Promotes Osmotic Stress-Induced Cell Apoptosis  Yan Xia, Ji Wang, Ta-Jen Liu, W.K.
Volume 69, Issue 3, Pages e5 (February 2018)
Granulin Is a Soluble Cofactor for Toll-like Receptor 9 Signaling
Interleukin-6-Resistant Melanoma Cells Exhibit Reduced Activation of STAT3 and Lack of Inhibition of Cyclin E-Associated Kinase Activity  Markus Böhm,
Volume 19, Issue 5, Pages (November 2003)
Volume 24, Issue 11, Pages e8 (November 2017)
c-Src Activates Endonuclease-Mediated mRNA Decay
Volume 30, Issue 3, Pages (March 2009)
Volume 19, Issue 9, Pages (September 2012)
A Critical Role for Noncoding 5S rRNA in Regulating Mdmx Stability
Volume 32, Issue 4, Pages (April 2010)
Yi Tang, Jianyuan Luo, Wenzhu Zhang, Wei Gu  Molecular Cell 
IL-18 Downregulates Collagen Production in Human Dermal Fibroblasts via the ERK Pathway  Hee Jung Kim, Seok Bean Song, Jung Min Choi, Kyung Moon Kim,
TopBP1 Activates the ATR-ATRIP Complex
Volume 35, Issue 1, Pages (July 2009)
Volume 57, Issue 3, Pages (February 2015)
Rsk1 mediates a MEK–MAP kinase cell survival signal
Volume 21, Issue 6, Pages (November 2017)
Volume 10, Issue 4, Pages (April 1999)
Volume 13, Issue 12, Pages (December 2006)
Hua Gao, Yue Sun, Yalan Wu, Bing Luan, Yaya Wang, Bin Qu, Gang Pei 
Volume 34, Issue 5, Pages (June 2009)
PU.1 Expression Delineates Heterogeneity in Primary Th2 Cells
Phosphorylation of CBP by IKKα Promotes Cell Growth by Switching the Binding Preference of CBP from p53 to NF-κB  Wei-Chien Huang, Tsai-Kai Ju, Mien-Chie.
A Direct HDAC4-MAP Kinase Crosstalk Activates Muscle Atrophy Program
Volume 129, Issue 5, Pages (June 2007)
Volume 22, Issue 3, Pages (May 2006)
Volume 41, Issue 4, Pages (February 2011)
Volume 10, Issue 2, Pages (February 1999)
Presentation transcript:

TAB1: A Target of Triptolide in Macrophages Yan Lu, Ya Zhang, Long Li, Xiujing Feng, Sen Ding, Wei Zheng, Jianxin Li, Pingping Shen  Chemistry & Biology  Volume 21, Issue 2, Pages 246-256 (February 2014) DOI: 10.1016/j.chembiol.2013.12.009 Copyright © 2014 Elsevier Ltd Terms and Conditions

Chemistry & Biology 2014 21, 246-256DOI: (10. 1016/j. chembiol. 2013 Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 1 Identification of TAB1 as a TP-Binding Protein Ex Vivo (A) TP-binding proteins were pulled down from the total cell lysate, which had been incubated with TP (30 μM), resolved by SDS gel electrophoresis, and visualized by silver staining. (B) Anti-TAB1 and protein A/G agarose was used to pull down the TAB1-binding molecules in the lysate, which had been incubated with TP (15 μM) or without TP. Then the complex of TAB1 and TP was destroyed by methanol and sonication. The existence of TP in this coimmunoprecipitation was detected by ESI-MS. (C) Western blot analysis using the anti-TAB1 antibody and the samples from (A) validates TAB1 as the TP target protein. (D) TP and TAB1 were observed by confocal microscopy. TP-mmc (15 μM) was added to Ana-1 cells (upper panel: bar, 5 μm; lower panel: bar, 8 μm), and TP-mmc (15 μM) with/without TP (15 μM) was added to RAW 264.7 cells (middle panel: bar, 50 μm) for 2 hr, followed by the addition of anti-TAB1 antibody (upper and middle panels: 10 μl) or anti-caspase-1 (lower panel: 10 μl) and the fluorescently labeled secondary antibody. (E) TP dose-dependently and specifically protects the target TAB1 from degradation by proteases in vitro. Different doses of TP (30 μM, 0.030 μM) or DMSO (0.1%) were added to the protein lysate without protease inhibitors for 4 hr at 37°C. The levels of TAB1 and GAPDH in the sample were determined by western blot. The numbers under each western blotting band mean the grayscale value of the band. (F) The recombinant TAB1 or BSA was incubated with DMSO, TP (1.2, 12 μM) for 18 hr at 4°C and then digested with subtilisin at room temperature for 20 min. The reactions were stopped by adding SDS loading buffer and boiling for 5 min. Samples were separated by SDS-PAGE and stained with Coomassie brilliant blue. Chemistry & Biology 2014 21, 246-256DOI: (10.1016/j.chembiol.2013.12.009) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 2 Identification of Recombinant TAB1 as a TP-Binding Protein In Vitro (A) Expression and purification of TAB1-his in bacteria. Lane A, load sample; lane B, flowthrough; lane C, wash with 20 mM imidazole; lane D, elution with 50 mM imidazole; lane E, elution with 100 mM imidazole; lane F, elution with 250 mM imidazole. (B) ITC data for binding of TP to TAB1. The upper panels show the raw data, and the lower panels show the corresponding binding isotherm fitted according to the “three binding site” model. Reference titration of ligand into buffer was used to correct for heat of dilution. The thermodynamic parameters (K, ΔH, and ΔS) are indicated on the right. (C) Synchronous fluorescence spectrum of TAB1-his (T = 290 K, pH 7.40), CTAB1-his = 0.36 μM, CTP = 0–7 μM, Δλ = 15 nm (left panel), Δλ = 60 nm (right panel). (D) Binding of TP to the deletion mutants of TAB1. TP (30 μM) was incubated with cell lysates from 293T cells that were transfected with the mutants of TAB1-FLAG-pCDNA3.1 plasmids. Immunoprecipitation using anti-TP and immunoblotting with anti-FLAG were performed as in Figure 1C. Lanes 1, 3, 5, and 7, input; lanes 2, 4, 6, and 8, coIP sample. Chemistry & Biology 2014 21, 246-256DOI: (10.1016/j.chembiol.2013.12.009) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 3 TP Inhibits TAK1 Kinase Activity through Repression of the TAB1-TAK1 Interaction in LPS-Induced Cell Lines Ana-1 cells were pretreated with TP (30 nM or the indicated concentrations) or DMSO for 12 hr or indicated time, followed by exposure to 0.5 μg/ml LPS for 4 hr. Negative control cell group is only treated with DMSO. Then the whole-cell lysates were prepared. (A) TP reduces the LPS-induced endogenous TAK1-TAB1 interaction in Ana-1 cells. Ana-1 cells were pretreated with TP (30 nM) or DMSO for 1 hr, followed by exposure to 0.5 μg/ml LPS for 2 hr. Whole-cell lysates were subjected to immunoprecipitation with an anti-TAB1 or anti-TAK1 antibody. Immunoprecipitates were analyzed by western blot using an anti-TAK1 or anti-TAB1 antibody. (B) The phosphorylation of TAK1 was inhibited by different concentration of TP (7.5, 15, and 30 nM) and at the different pretreatment time (0.5, 1, 2, 4, 8, 12, and 18 hr). Whole-cell lysates were subjected to western blot analysis using an anti-TAK1 or anti-pTAK1 antibody. (C) TP reduces phosphorylation of the TAK1 kinase substrate and signaling pathways downstream of TAK1/MAPK. Whole-cell lysates were subjected to western blot analysis using anti-MKK3/6, anti-pMKK3/6, anti-MKK4, anti-pMKK4, anti-pp38, anti-p38, anti-p-JNK, anti-JNK, anti-p-ERK1/2, anti-ERK1/2, anti-p-c-Jun, or anti-c-Jun antibodies. (D) TP suppresses the DNA-binding activity of AP-1. Nuclear proteins were extracted and subjected to an EMSA to evaluate AP-1 activation as described in the Supplemental Experimental Procedures. (E) TP inhibits the kinase activity of TAK1. TAK1 kinase activity in each group was determined using the cellular TAK1/TAB1 kinase activity assay kit in accordance with the manufacturer’s instructions. Each bar represents averaged results, n = 3 biological replicates, assayed three times each; error bars, SE. ∗∗p < 0.01; ∗∗∗p < 0.001. The red numbers under each western blotting band mean the grayscale value of the band. Chemistry & Biology 2014 21, 246-256DOI: (10.1016/j.chembiol.2013.12.009) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 4 The Antagonism of TAB1 to the Biological Activity of TP (A and B) The 293T cells that were transfected with TAB1-FLAG-pCDNA3.1 plasmids were exposed to TP (30 nM) and TNF-α (50 ng/ml), and the whole-cell lysates were prepared. (A) Overexpression of TAB1 promotes the expression of pro-inflammatory cytokines, which were inhibited by TP. The expression of TNF-α, IL-1β, IL-6, and IL-10 mRNA was measured by real-time PCR. (B) Overexpression of TAB1 promotes phosphorylation of TAK1 and signaling molecules downstream of TAK1/MAPK, which had been inhibited by the TP. The whole-cell lysates were subjected to western blot analysis using anti-P-TAK1, anti-TAK1, anti-pp38, anti-p38, anti-p-JNK, anti-JNK, anti-MKK3/6, anti-pMKK3/6, anti-MKK4, and anti-pMKK4 antibodies. (C) Schematic diagram represents the mechanism by which TP affects the cascade of signaling events through targeting TAB1 in macrophages. Each bar represents averaged results, n = 3 biological replicates, assayed three times each; error bars, SE. ∗∗p < 0.01; ∗∗∗p < 0.001. The numbers under each western blotting band mean the grayscale value of the band. Chemistry & Biology 2014 21, 246-256DOI: (10.1016/j.chembiol.2013.12.009) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 5 TP Inhibits TAK1 Kinase Activity through Repression of the TAB1-TAK1 Interaction In Vivo Mice were injected with or without TP (100 μg/kg, intraperitoneal [i.p.]) and then stimulated with LPS (4 mg/kg, i.p.) after 18 hr. The animals were euthanized at 4 hr after LPS injection. Mouse peritoneal macrophages were isolated, and whole-cell lysates were prepared. The “con” means negative control group. (A) TP reduces the LPS-induced endogenous TAK1-TAB1 interaction in vivo. Whole-cell lysates were subjected to immunoprecipitation with an anti-TAB1 antibody. Immunoprecipitates and whole-cell lysates were analyzed by western blot using an anti-TAK1 or anti-TAB1 antibody. (B) TP inhibits phosphorylation of TAK1. Whole-cell lysates were subjected to western blot analysis using an anti-TAK1 or anti-pTAK1 antibody. (C) TP reduces phosphorylation of the TAK1 kinase substrate and affects signaling pathways downstream of TAK1/MAPK. Whole-cell lysates were subjected to western blot analysis using anti-MKK3/6, anti-pMKK3/6, anti-MKK4, anti-pMKK4, anti-pp38, anti-p38, anti-p-JNK, anti-JNK, anti-p-c-Jun, and anti-c-Jun antibodies. Each bar represents averaged results, n = 3 biological replicates, assayed three times each; error bars, SE. ∗∗p < 0.01; ∗∗∗p < 0.001. Chemistry & Biology 2014 21, 246-256DOI: (10.1016/j.chembiol.2013.12.009) Copyright © 2014 Elsevier Ltd Terms and Conditions

Figure 6 TP Inhibits LPS-Induced Macrophage Activation and Inflammatory Responses (A) Effects of TP on cell proliferation. Ana-1 cells were treated with indicated concentrations of TP, and cell proliferation was determined by MTT proliferation assay. (B) TP decreases NO, IL-6, and phagocytic activity in LPS-induced Ana-1 cells. Ana-1 cells were pretreated with the indicated concentrations of TP for 12 hr and then exposed to 0.5 μg/ml LPS for 4 hr, except for NO assay. (Left panel) The concentration of IL-6 from the culture supernatant was measured by ELISA. (Middle panel) NO production was determined using an assay of the cell-free culture supernatant (0.1 μg/ml LPS for 24 hr) for nitrite and the Griess reagent. (Right panel) The phagocytic activity of macrophages was measured by the neutral red uptake (NRU) assay. Each value represents the mean ± SE of three separate experiments. (C) The effects of TP (100 μg/kg, i.p.) on TNF-a, IL-6, IL-1β, IL-13, IL-12, and IL-4 production in the serum in LPS-induced mice (4 mg/kg, i.p.). The profile of cytokine release was determined using commercially available ELISA kits. (D) The effects of TP on the phagocytic ability of peritoneal macrophages in LPS-induced mice prepared as in (C). The phagocytic ability of peritoneal macrophages in each group was tested using latex beads. Each bar represents averaged results, n = 3 biological replicates, assayed three times each; error bars, SE. ∗∗p < 0.01; ∗∗∗p < 0.001. Chemistry & Biology 2014 21, 246-256DOI: (10.1016/j.chembiol.2013.12.009) Copyright © 2014 Elsevier Ltd Terms and Conditions