Abstract
Uncontrolled activation of tumor necrosis factor receptor–associated factor (TRAF) proteins may result in profound tissue injury by linking surface signals to cytokine release. Here we show that a ubiquitin E3 ligase component, Fbxo3, potently stimulates cytokine secretion from human inflammatory cells by destabilizing a sentinel TRAF inhibitor, Fbxl2. Fbxo3 and TRAF protein in circulation positively correlated with cytokine responses in subjects with sepsis, and we identified a polymorphism in human Fbxo3, with one variant being hypofunctional. A small-molecule inhibitor targeting Fbxo3 was sufficient to lessen severity of cytokine-driven inflammation in several mouse disease models. These studies identified a pathway of innate immunity that may be useful to detect subjects with altered immune responses during critical illness or provide a basis for therapeutic intervention targeting TRAF protein abundance.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Inoue, J. et al. Tumor necrosis factor receptor-associated factor (TRAF) family: adaptor proteins that mediate cytokine signaling. Exp. Cell Res. 254, 14–24 (2000).
Xu, L.G., Li, L.Y. & Shu, H.B. TRAF7 potentiates MEKK3-induced AP1 and CHOP activation and induces apoptosis. J. Biol. Chem. 279, 17278–17282 (2004).
Alvarez, S.E. et al. Sphingosine-1-phosphate is a missing cofactor for the E3 ubiquitin ligase TRAF2. Nature 465, 1084–1088 (2010).
London, N.R. et al. Targeting Robo4-dependent Slit signaling to survive the cytokine storm in sepsis and influenza. Sci. Transl. Med. 2, 23ra19 (2010).
Harrison, C. Sepsis: calming the cytokine storm. Nat. Rev. Drug Discov. 9, 360–361 (2010).
Lutgens, E. et al. Deficient CD40-TRAF6 signaling in leukocytes prevents atherosclerosis by skewing the immune response toward an antiinflammatory profile. J. Exp. Med. 207, 391–404 (2010).
Zhu, L.J. et al. Suppression of tumor necrosis factor receptor associated factor (TRAF)-2 attenuates the proinflammatory and proliferative effect of aggregated IgG on rat renal mesangial cells. Cytokine 49, 201–208 (2010).
Yamashita, M. et al. TRAF6 mediates Smad-independent activation of JNK and p38 by TGF-beta. Mol. Cell 31, 918–924 (2008).
Lamothe, B. et al. The RING domain and first zinc finger of TRAF6 coordinate signaling by interleukin-1, lipopolysaccharide, and RANKL. J. Biol. Chem. 283, 24871–24880 (2008).
Ha, H., Han, D. & Choi, Y. TRAF-mediated TNFR-family signaling. Curr. Protoc. Immunol. 11, Unit 11 9D (2009).
Tanaka, Y. et al. c-Cbl-dependent monoubiquitination and lysosomal degradation of gp130. Mol. Cell Biol. 28, 4805–4818 (2008).
Hochstrasser, M. Biochemistry. All in the ubiquitin family. Science 289, 563–564 (2000).
Tyers, M. & Willems, A.R. One ring to rule a superfamily of E3 ubiquitin ligases. Science 284, 603–604 (1999).
Zheng, N. et al. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature 416, 703–709 (2002).
Cardozo, T. & Pagano, M. The SCF ubiquitin ligase: insights into a molecular machine. Nat. Rev. Mol. Cell Biol. 5, 739–751 (2004).
Jin, J. et al. Systematic analysis and nomenclature of mammalian F-box proteins. Genes Dev. 18, 2573–2580 (2004).
Cenciarelli, C. et al. Identification of a family of human F-box proteins. Curr. Biol. 9, 1177–1179 (1999).
Chen, B.B., Coon, T.A., Glasser, J.R. & Mallampalli, R.K. Calmodulin antagonizes a calcium-activated SCF ubiquitin E3 ligase subunit, FBXL2, to regulate surfactant homeostasis. Mol. Cell Biol. 31, 1905–1920 (2011).
Chen, B.B. et al. F box protein FBXL2 targets cyclin D2 for ubiquitination and degradation to inhibit leukemic cell proliferation. Blood 119, 3132–3141 (2012).
Chen, B.B., Glasser, J.R., Coon, T.A. & Mallampalli, R.K. F-box protein FBXL2 exerts human lung tumor suppressor-like activity by ubiquitin-mediated degradation of cyclin D3 resulting in cell cycle arrest. Oncogene 31, 2566–2579 (2012).
Chen, B.B., Glasser, J.R., Coon, T.A. & Mallampalli, R.K. FBXL2 is a ubiquitin E3 ligase subunit that triggers mitotic arrest. Cell Cycle 10, 3487–3494 (2011).
Shima, Y. et al. PML activates transcription by protecting HIPK2 and p300 from SCFFbx3-mediated degradation. Mol. Cell Biol. 28, 7126–7138 (2008).
Levy, M.M. et al. 2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Crit. Care Med. 31, 1250–1256 (2003).
Hildebrand, J.M. et al. Roles of tumor necrosis factor receptor associated factor 3 (TRAF3) and TRAF5 in immune cell functions. Immunol. Rev. 244, 55–74 (2011).
Ishida, T.K. et al. TRAF5, a novel tumor necrosis factor receptor-associated factor family protein, mediates CD40 signaling. Proc. Natl. Acad. Sci. USA 93, 9437–9442 (1996).
Vallabhapurapu, S. et al. Nonredundant and complementary functions of TRAF2 and TRAF3 in a ubiquitination cascade that activates NIK-dependent alternative NF-kappaB signaling. Nat. Immunol. 9, 1364–1370 (2008).
Tseng, P.H. et al. Different modes of ubiquitination of the adaptor TRAF3 selectively activate the expression of type I interferons and proinflammatory cytokines. Nat. Immunol. 11, 70–75 (2010).
Xie, P., Stunz, L.L., Larison, K.D., Yang, B. & Bishop, G.A. Tumor necrosis factor receptor-associated factor 3 is a critical regulator of B cell homeostasis in secondary lymphoid organs. Immunity 27, 253–267 (2007).
van Eyndhoven, W.G., Gamper, C.J., Cho, E., Mackus, W.J. & Lederman, S. TRAF-3 mRNA splice-deletion variants encode isoforms that induce NF-kappaB activation. Mol. Immunol. 36, 647–658 (1999).
Habelhah, H. Emerging complexity of protein ubiquitination in the NF-kappaB pathway. Genes Cancer 1, 735–747 (2010).
Li, S. et al. Ubiquitin ligase Smurf1 targets TRAF family proteins for ubiquitination and degradation. Mol. Cell Biochem. 338, 11–17 (2010).
Nakhaei, P. et al. The E3 ubiquitin ligase Triad3A negatively regulates the RIG-I/MAVS signaling pathway by targeting TRAF3 for degradation. PLoS Pathog. 5, e1000650 (2009).
Skaar, J.R., D'Angiolella, V., Pagan, J.K. & Pagano, M. SnapShot: F Box Proteins II. Cell 137, 1358 (2009).
Bashir, T., Dorrello, N.V., Amador, V., Guardavaccaro, D. & Pagano, M. Control of the SCF(Skp2-Cks1) ubiquitin ligase by the APC/C(Cdh1) ubiquitin ligase. Nature 428, 190–193 (2004).
Galan, J.M. & Peter, M. Ubiquitin-dependent degradation of multiple F-box proteins by an autocatalytic mechanism. Proc. Natl. Acad. Sci. USA 96, 9124–9129 (1999).
Chin, K.H. et al. Preparation, crystallization and preliminary X-ray analysis of XC2382, an ApaG protein of unknown structure from Xanthomonas campestris. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 61, 700–702 (2005).
Ilyin, G.P., Rialland, M., Pigeon, C. & Guguen-Guillouzo, C. cDNA cloning and expression analysis of new members of the mammalian F-box protein family. Genomics 67, 40–47 (2000).
Liu, L., Rodriguez-Belmonte, E.M., Mazloum, N., Xie, B. & Lee, M.Y. Identification of a novel protein, PDIP38, that interacts with the p50 subunit of DNA polymerase delta and proliferating cell nuclear antigen. J. Biol. Chem. 278, 10041–10047 (2003).
Zhang, J. et al. Novel mutations in ubiquitin-specific protease 26 gene might cause spermatogenesis impairment and male infertility. Asian J. Androl. 9, 809–814 (2007).
Month, S.R. et al. Analysis of 5′ flanking regions of the gamma globin genes from major African haplotype backgrounds associated with sickle cell disease. J. Clin. Invest. 85, 364–370 (1990).
Geva, A. et al. Hemoglobin Jamaica plain–a sickling hemoglobin with reduced oxygen affinity. N. Engl. J. Med. 351, 1532–1538 (2004).
Dodge, J.A. & Burton, L. Letter: Heterozygote advantage in cystic fibrosis. Lancet 1, 572–573 (1975).
Ebong, S.J. et al. Immunopathologic responses to non-lethal sepsis. Shock 12, 118–126 (1999).
Acknowledgements
We thank A. Lagneaux and S. Barge for assistance with studies. This material is based upon work supported, in part, by the US Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development. This work was supported by a Merit Review Award from the US Department of Veterans Affairs and National Institutes of Health R01 grants HL096376, HL097376, HL081784, HL068135 and HL098174 (to R.K.M.), HL116472 (to B.B.C.), HL01916 (to Y.Z.) and P50HL084948 (F.C.S.), American Heart Association awards 12SDG9050005 (to J.Z.) and 12SDG12040330 (to C.Z.), and American Heart Association Grant-in-Aid 12GRNT11820019 (B.J.M.). The contents presented do not represent the views of the Department of Veterans Affairs or the United States Government.
Author information
Authors and Affiliations
Contributions
R.K.M. and B.B.C. jointly oversaw the studies. B.B.C. designed the study, performed experiments, analyzed the data and wrote the manuscript; T.A.C., J.R.G., J.Z, Y.Z. and C.Z. performed experiments and assisted with animal experiments; B.J.M., B.E., F.C.S. and Y.Z. assisted with human studies. R.K.M. revised the manuscript and directed the study.
Corresponding authors
Ethics declarations
Competing interests
A provisional patent application (US 61/657,423) covering Fbxo3 inhibitors and additional modifications was filed jointly through the United States Department of Veterans Affairs and the University of Pittsburgh.
Supplementary information
Supplementary Text and Figures
Supplementary Table 1, Supplementary Figures 1–7 (PDF 4085 kb)
Rights and permissions
About this article
Cite this article
Chen, B., Coon, T., Glasser, J. et al. A combinatorial F box protein directed pathway controls TRAF adaptor stability to regulate inflammation. Nat Immunol 14, 470–479 (2013). https://doi.org/10.1038/ni.2565
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ni.2565
This article is cited by
-
The deubiquitinase USP40 preserves endothelial integrity by targeting the heat shock protein HSP90β
Experimental & Molecular Medicine (2024)
-
Low-Intensity Pulsed Ultrasound: A Physical Stimulus with Immunomodulatory and Anti-inflammatory Potential
Annals of Biomedical Engineering (2024)
-
Protective role of FBXL19 in Streptococcus pneumoniae-induced lung injury in pneumonia immature mice
Journal of Cardiothoracic Surgery (2023)
-
FBXL2 promotes E47 protein instability to inhibit breast cancer stemness and paclitaxel resistance
Oncogene (2023)
-
Repositioning linifanib as a potent anti-necroptosis agent for sepsis
Cell Death Discovery (2023)