Regulation of autophagy by E3 ubiquitin ligase RNF216 through BECN1 ubiquitination

Abstract

These authors contribute equally to this work.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 a; 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.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.

TRAF Family Proteins Regulate Autophagy Dynamics by Modulating AUTOPHAGY PROTEIN6 Stability in Arabidopsis

“…In addition to TRAF6, several other regulatory cofactors, such as BCL-2, DAPK, Cul4, Nedd5, and RNF216, also associate with Beclin-1 to regulate its activity (He and Levine, 2010;Wirawan et al, 2012;Mei et al, 2016). Of these, TRAF6/Cul4 and Nedd4/ RNF216 are two types of E3 ligases that promote the stabilization and degradation, respectively, of Beclin-1 and subsequently regulate autophagy dynamics in mammals (Shi and Kehrl, 2010;Platta et al, 2012;Xia et al, 2013;Xu et al, 2014). Upon induction of autophagy, TRAF6 and Cul4 ubiquitinate Beclin-1 at the K63 residue, which allows it to interact with VPS34 and then activate autophagy (Shi and Kehrl, 2010;Xia et al, 2013).…”

“…TRAF6 also mediates K63-linked ubiquitination of ULK1, which subsequently promotes the stabilization and self-association of ULK1 and therefore activates autophagy (Nazio et al, 2013). However, recent studies have revealed that K11-and K48-linked ubiquitination of Beclin-1 by the E3 ligases Nedd4 and RNF216, respectively, have an opposite effect from that of K63-linked ubiquitination, instead leading to degradation of Beclin-1 and the suppression of autophagy (Platta et al, 2012;Xu et al, 2014). Although the regulation of autophagosome formation by ubiquitination of ATG proteins has been extensively studied in mammalian cells, it is still unclear whether plant autophagy involves a similar regulatory mechanism.…”

The ubiquitin system: a critical regulator of innate immunity and pathogen–host interactions

“…For example, RNF216 binds to and destabilizes Beclin-1 through K48-linked ubiquitination, whereas USP19 maintains Beclin-1 protein levels through K11-specific deubiquitination. 61,62 Upon induction, xenophagy, reminiscent of other selective autophagic routes, such as mitophagy, utilizes autophagic receptors that bind both a molecular tag attached to the bacterial cargo and the autophagic modifier LC3 to selectively bridge bacterial cargo to the autophagosomal membrane. 63 Ubiquitin appears to represent a common 'eat-me' signal in various selective autophagic pathways including xenophagy.…”

Hepatitis B virus dampens autophagy maturation via negative regulation of Rab7 expression

“…Fluorescence confocal microscopy was performed as previously described (16). Cells grown on coverslips were fixed in BD Cytofix/Cytoperm solution (BD Biosciences) at room temperature for 15 min.…”

“…Cells were directly lysed with RIPA containing protease and phosphatase inhibitors (Roche) and proteins were separated by SDS-PAGE after denaturation (16). Immunoblot analysis was performed by initial transfer of proteins onto polyvinylidenefluoride filters using a Mini Trans-Blot (Bio-Rad) followed by a blocking step with 5% nonfat dried milk plus 0.1% Tween 20 for 2 hours at room temperature and exposed to primary antibodies diluted 2,000-fold that recognized microtubule-associated protein light chain 3 (LC3, Cell Signal), autophagyrelated protein 5 (Atg5, Cell Signal), Rab7 (Cell Signal), or 5000-fold for GAPDH (Santa Cruz) overnight at 4°C and subsequently washed.…”