TRK-fused gene (TFG) regulates ULK1 stability via TRAF3-mediated ubiquitination and protects macrophages from LPS-induced pyroptosis

Jian-Hong Shi,Ying-Hui Tan,Ting-Ting Wang,Wen-Wen Liu,Yan Qin,Nai-Peng Cui,Chen Ling,Li-Nuo Zhang,Zhi-Yu Ni

doi:10.1038/s41419-022-04539-9

Abstract

TRK-fused gene (TFG) is known to be involved in protein secretion and plays essential roles in an antiviral innate immune response. However, its function in LPS-induced inflammation and pyroptotic cell death is still unknown. Here, we reported that TFG promotes the stabilization of Unc-51 like autophagy activating kinase (ULK1) and participates in LPS plus nigericin (Ng) induced pyroptotic cell death. Our results showed that TFG-deficient THP-1 macrophages exhibit higher mitochondrial ROS production. LPS/Ng stimulation triggers a much higher level of ROS and induces pyroptotic cell death. ULK1 undergoes a rapid turnover in TFG-deficient THP-1 cells. TFG forms complex with an E3 ligase, tumor necrosis factor receptor-associated factor 3 (TRAF3), and stabilizes ULK1 via disturbing ULK1-TRAF3 interaction. Knockdown of TFG facilitates the interaction of ULK1 with TRAF3 and subsequent K48-linked ULK1 ubiquitination and proteasome degradation. Rescue of ULK1 expression blocks LPS/Ng-induced cell death in TFG-deficient THP-1 macrophages. Taken together, TFG plays an essential role in LPS/Ng-induced pyroptotic cell death via regulating K48-linked ULK1 ubiquitination in macrophages.

Highlights

Inflammatory responses and cell death are major hosts respond to infection
TRK-fused gene (TFG)-deficiency promotes mitochondrial ROS production and LPS/Ng-induced pyroptosis in THP-1 cells TFG is known to play a vital role in secretory cargo traffic from the endoplasmic reticulum (ER) to the Golgi apparatus [17, 18] and participate nuclear factor κB (NF-κB) signaling [37], which are essential processes in macrophage-mediated inflammation
LPS/Ng treatment triggered a higher level of mitochondrial ROS (Fig. 1A, B, shTFG vs. shTFG LPS/Ng) and promoted pyroptotic cell death (Fig. 1C, D) in TFG-knockdown THP-1 cells

Summary

Introduction

Inflammatory responses and cell death are major hosts respond to infection. TLRs triggers downstream immune signaling, including nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling cascades and inducing expression of precursor forms of cytokines IL-1β and IL-18 [2]. NLRs stimulate caspase-1 activation and trigger a form of cell death called pyroptosis, which is accompanied by IL-1β processing and releasing [3, 4]. Disordered inflammatory response and fulminant pyroptotic cell death of macrophages are involved in many diseases, such as acute lung injury [5], arthritis [6, 7], SARS-CoV-2-associated cytokine storm [8, 9]. Blocking pyroptotic cell death signaling cascades may benefit patients with infectious and autoinflammatory diseases by limiting tissue damage. Our and other research groups have previously identified tumor necrosis factor receptor-associated factor 3 (TRAF3), a TRAF family member with E3 ligase activity, plays an essential role in NLRP3 inflammasome and TLR induced pyroptosis in macrophages [10,11,12,13,14]

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Results

Conclusion