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E3 ligase RNF99 negatively regulates TLR-mediated inflammatory immune response via K48-linked ubiquitination of TAB2

Abstract

Innate immunity is the first line to defend against pathogenic microorganisms, and Toll-like receptor (TLR)-mediated inflammatory responses are an essential component of innate immunity. However, the regulatory mechanisms of TLRs in innate immunity remain unperfected. We found that the expression of E3 ligase Ring finger protein 99 (RNF99) decreased significantly in peripheral blood monocytes from patients infected with Gram negative bacteria (G-) and macrophages stimulated by TLRs ligands, indicating the role of RNF99. We also demonstrated for the first time, the protective role of RNF99 against LPS-induced septic shock and dextran sodium sulfate (DSS)-induced colitis using RNF99 knockout mice (RNF99–/–) and bone marrow-transplanted mice. In vitro experiments revealed that RNF99 deficiency significantly promoted TLR-mediated inflammatory cytokine expression and activated the NF-κB and MAPK pathways in macrophages. Mechanistically, in both macrophages and HEK293 cell line with TLR4 stably transfection, RNF99 interacted with and degraded TAK1-binding protein (TAB) 2, a regulatory protein of the kinase TAK1, via the lysine (K)48-linked ubiquitin-proteasomal pathway on lysine 611 of TAB2, which further regulated the TLR-mediated inflammatory response. Overall, these findings indicated the physiological significance of RNF99 in macrophages in regulating TLR-mediated inflammatory reactions. It provided new insight into TLRs signal transduction, and offered a novel approach for preventing bacterial infections, endotoxin shock, and other inflammatory ills.

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Fig. 1: Gram negative (G-) infection and TLRs stimulation decreased expression of RNF99 in macrophages.
Fig. 2: RNF99 knockout significantly aggravated TLR-mediated endotoxemia and acute colitis.
Fig. 3: Regulation of TLRs-mediated inflammatory diseases by RNF99 in macrophages.
Fig. 4: RNF99 deficiency positively regulated TLRs-mediated inflammatory cytokine production and signaling pathway activation in macrophages.
Fig. 5: RNF99 overexpression negatively regulates TLRs-mediated inflammatory cytokine production and signaling pathway activation in macrophages.
Fig. 6: RNF99 affects the formation of the TAK1-TAB1-TAB2 complex via promoting TAB2 degradation.
Fig. 7: RNF99 interacts with TAB2.
Fig. 8: RNF99 targets K611 of TAB2 for K48-linked polyubiquitination.

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Data availability

The datasets are available from the corresponding author on reasonable request.

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Acknowledgements

We thank Dr. Hongwei Pan (Department of Clinical Laboratory, Qilu Hospital of Shandong University, China) for the human samples collection.

Funding

This work was supported by grants of the National Natural Science Foundation of China (No. 81970373, 31770977, 82270487, 82200502, 81920108003, 82030051 and 82200507), the Postdoctoral Science Foundation of China and Shandong Province (No. 2018M630789, 201901009 and 2021ZLGX02), the Shandong Provincial Natural Science Foundation (No. ZR2020YQ53, 2021ZDSYS05, 2021SFGC0503, ZR2022QH089 and ZR2022QH211), the Program of Introducing Talents of Discipline to Universities (BP0719033), the National Key Research and Development Program of China (2021YFF0501403), the Taishan Scholars Program of Shandong Province (Zhang M and Zhang C), the Fundamental Research Funds for the Central Universities (No. 2018JC001), the Postdoctoral Science Foundation of China (2022M710084).

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MZ, YZ, and CZ designed, supervised the study, and revision and final approval of the manuscript. JZ and LC contributed to data research, analysis, and manuscript writing. AG, LWY, QL, YPL, WQQ, YHH, WHS, and GHS contributed to the data collection and analysis. RQR acquired human samples. All the authors have read the manuscript and provided useful comments.

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Correspondence to Cheng Zhang or Meng Zhang.

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The authors declare no competing interests.

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All mice experiments were carried out following the general guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care, as approved by the Laboratory Animal Committee of Shandong University Qilu Hospital (Jinan, Shandong Province, China). And the human investigations were in accordance with the Declaration of Helsinki, and approved by the Research Ethics Committee of Shandong University Qilu Hospital after informed consent was obtained from the patients. (Permit number: KYLL-2017(KS)-121).

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Zhang, J., Cao, L., Gao, A. et al. E3 ligase RNF99 negatively regulates TLR-mediated inflammatory immune response via K48-linked ubiquitination of TAB2. Cell Death Differ 30, 966–978 (2023). https://doi.org/10.1038/s41418-023-01115-2

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