RNF144A inhibits autophagy by targeting BECN1 for degradation during L. monocytogenes infection.

Abstract

Listeria monocytogenes (L. monocytogenes, Lm) is widely used in the laboratory as an infection model for the research on pathogenesis and host defense against gram-positive intracellular bacteria. Macroautophagy (called simply "autophagy" hereafter), is important in the host defense against pathogens, such as bacteria, viruses, and parasites. BECN1 plays a pivotal role in the initiation of autophagy and accumulating evidence indicates that post-translational modifications of BECN1 provide multiple strategies for autophagy regulation. In this study, we demonstrated that the RING1-IBR-RING2 (RBR) family member RNF144A (ring finger protein 144A), which was induced by Lm infection, promoted Lm infection in an autophagy-dependent but STING1-independent pattern. rnf144a deficiency in mice protected mice from Lm infection with inhibited innate immune responses. Interestingly, RNF144A decreased Lm-induced autophagosome accumulation. Mechanistic investigation indicated that RNF144A interacted with BECN1 and promoted its K48-linked ubiquitination, leading to the subsequent proteasome-dependent degradation of BECN1 and reduced autophagosome accumulation. Further study demonstrated that RNF144A promoted the ubiquitination of BECN1 at K117 and K427, and these two ubiquitination sites were essential to the role of BECN1 in autophagy and Lm infection. Thus, our findings suggested a new regulator in intracellular bacterial infection and autophagy, which may contribute to our understanding of host defense against intracellular bacterial infection via autophagy.Abbreviations: ATG3: autophagy related 3; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG10: autophagy related 10; ATG12: autophagy related 12; ATG16L1: autophagy related 16 like 1; Baf A₁: bafilomycin A₁; BECN1: beclin 1; BMDC: bone marrow-derived dendritic cell; BMDM: bone marrow-derived macrophage; CFUs: colony-forming units; CHX: cycloheximide; CQ: chloroquine; CXCL10/IP-10: C-X-C motif chemokine ligand 10; EBSS: Earle's balanced salt solution; ELISA: enzyme-linked immunosorbent assay; IFIT1/ISG56: interferon induced protein with tetratricopeptide repeats 1; IFNB/IFN-β: interferon beta; IL6: interleukin 6; IRF3, interferon regulatory factor 3; Lm: L. monocytogenes; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MOI: multiplicity of infection; PLA: proximity ligation assay; PMA: phorbol myristate acetate; PMA-THP1, PMA-differentiated THP1; PMs: peritoneal macrophages; PTMs: posttranslational modifications; RBR: RING1-IBR-RING2; RNF144A: ring finger protein 144A; STING1, stimulator of interferon response cGAMP interactor 1; TBK1, TANK binding kinase 1; TNF/TNF-α: tumor necrosis factor.