Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination

Abstract

Autophagy is an evolutionarily conserved biological process involved in an array of physiological and pathological events. Without proper control, autophagy contributes to various disorders, including cancer and autoimmune and inflammatory diseases. It is therefore of vital importance that autophagy is under careful balance. Thus, additional regulators undoubtedly deepen our understanding of the working network, and provide potential therapeutic targets for disorders. In this study, we found that RNF216 (ring finger protein 216), an E3 ubiquitin ligase, strongly inhibits autophagy in macrophages. Further exploration demonstrates that RNF216 interacts with BECN1, a key regulator in autophagy, and leads to ubiquitination of BECN1, thereby contributing to BECN1 degradation. RNF216 was involved in the ubiquitination of lysine 48 of BECN1 through direct interaction with the triad (2 RING fingers and a DRIL [double RING finger linked]) domain. We further showed that inhibition of autophagy through overexpression of RNF216 in alveolar macrophages promotes Listeria monocytogenes growth and distribution, while knockdown of RNF216 significantly inhibited these outcomes. These effects were confirmed in a mouse model of L. monocytogenes infection, suggesting that manipulating RNF216 expression could be a therapeutic approach. Thus, our study identifies a novel negative regulator of autophagy and suggests that RNF216 may be a target for treatment of inflammatory diseases.

Keywords:

• autophagy
• BECN1
• protein degradation
• RNF216
• ubiquitination

Abbreviations:

• Atg, autophagy-related
• BALF, bronchoalveolar lavage fluid
• BMDM, bone marrow-derived macrophage
• CFU, colony-forming unit
• GFP, green fluorescent protein
• HRP, horseradish peroxidase
• i.t., intratracheally
• LPS, lipopolysaccharide
• MAP1LC3A, microtubule-associated protein 1 light chain 3 α
• MOI, multiplicity of infection
• NFKB, nuclear factor of kappa light polypeptide gene enhancer in B-cells
• PBS, phosphate-buffered saline
• RIPK1, receptor (TNFRSF)-interacting serine-threonine kinase 1
• RNF216, ring finger protein 216;TIRAP, toll-interleukin 1 receptor (TIR) domain containing adaptor protein
• TLR, toll-like receptor
• TNF, tumor necrosis factor
• TRAF, TNF receptor-associated factor
• TICAM1/TRIF, toll-like receptor adaptor molecule 1
• TICAM2, toll-like receptor adaptor molecule 2
• shRNA, short hairpin RNA
• Triad, 2 RING fingers and a DRIL (double RING finger linked)
• Ub, ubiquitin

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We would like to thank Dr. Rennolds S. Ostrom (University of Tennessee Health Science Center in Memphis, TN) for the constructive suggestions and critical review of this manuscript.

Funding

This work was supported by the Ministry of Science and Technology of China 2011CB966200 (to Y.Z.), Strategic Priority Research Program of the Chinese Academy of Sciences XDA01040000 (to Y.Z.), National Natural Science Foundation of China 81130057, 81071748 (to Y.Z.), 81190133 (to C.X.), 31300708 (to H.S.), Natural Science Foundation of Shanghai Municipality 12ZR1415900 (to C.X.), 11JC1411400, 11431920900 (to Y.Z.), and Shanghai Municipal Education Commission J50207 (to Y.Z. and C.X.), 14YZ036 (to C.X.), Shanghai Bureau of Public Health 2012187 (to C.X.).