USP19 Modulates Autophagy And Antiviral Immune Responses By Deubiquitinating Beclin-1

Autophagy is a highly conserved degradation pathway in mammalian cells, which eliminates toxic protein aggregates, dysfunctional mitochondria and invading pathogens. Autophagy has three different forms: macroautophgy, microautophagy and molecular chaperon-mediated autophagy. Macroautophagy(hereafter autophagy) is the most common form of autophagy and lead to the formation of double-membrane organelle called ‘autophagosome’, which can engulf the substrates in the cytoplasm. The autophagosome then fuses with lysosome, causing the the degradation of inner contents by lysosomal hydrolases. In mammalian cells, a number of highly conserved autophagy-related proteins(ATGs) to yeast have been found to orchestrate autophagy process by assembly of autopahgic complex. It has also been reported that proteins involved in autophagy have regulatory functions in immune responses, such as inflammatory responses, antiviral response, antigen processing and presentation.The agonists can induce autophagy process through inhibition of the mammalian target of rapamycin(m TOR), which resides in a macromolecular complex, m TORC1. m TORC1 negatively regulates ULK1 complex, which consists of ULK1, ATG13, ATG101, and FIP200. Conversely, nutrient depletion or energy exhaustion inhibits m TORC1, which then activates ULK1, leading to the initiation of autophagy and phosphorylation of Beclin-1. The Beclin-1 complex consists of Beclin-1, phosphatidylinositol 3-kinase(VPS34), VPS15 and ATG14 L. Stimulation of this complex products phosphatidylinositol-3-phosphate, which is essential for the nucleation of autophagosomal membrane. Furthermore, AMPK directly activates the VPS34 lipid kinase in autophagy through phosphorylation of Beclin-1, thus promoting the generation of phosphatidylinositol-3-phosphate. Beclin-1 serves as a signaling bub that responds to various signals to activate VPS34 in autophagy. Considering the central role of Beclin-1 in autophagy, the study of post-translation modification of Beclin-1 is important. Previous study of the post-translation modification about Beclin-1 mainly focused on phosphorylation, while the ubiquitination modification of Beclin-1 remains largely unknown.Ubiquitin is a highly conserved polypeptide containing 76 amino acid, can be linked to the lysine of intracellular protein or other ubiquitin to form a ubiquitin chain via several enzymes(E1, E2 and E3). Ubiquitination and deubiqutination control a number of cellular processes of target proteins, such as protein degradation, DNA repair, chromatin remodeling, cell cycle regulation, endocytosis and kinase cascade signaling pathway. Recently, it has been revealed that ubiquitination modification is involved in the regulation of autophagy. TRAF6 promotes the K63-linked ubiquitination of Beclin-1, hence attenuating the interaction between Beclin-1 and Bcl-2. TRAF6 can also associate with AMBRA1 and mediate the stabilization, self-association and function of ULK1 through K63-linked ubiquitination. Additionally, a research showed that half of TRIMs modulate autophagy. TRIMs interacts with autophagy factors and act as platforms assembling ULK1 and Beclin-1 in their activated states. Furthermore, TRIM5α functions as a selective cargo receptor and delivers its cognate cytosolic target for autophagic degradation. With the further study, the importance of ubiquitination in the regulation of autophagy net web is becoming clear, but the detail roles of ubiquitination in autophagy and ATGs’ regulation need to be further discovered.Considering the essential role of ubiquitination in autophagy, we speculate deubiquitinating enzymes associated with autophagy process. In this study, we identified USP19 as a positive regulator in autophagy, further revealed USP19-Beclin-1-mediated the cross talk between autophagy and antiviral responses:1. USP19 functions as positive regulator in autophagy, we performed a screen and identified USP19 as a positive regulator of autophagy. We further validated that USP19 positively regulates autophagy in a deubiquitinase dependent manner.2. USP19 targets Beclin-1. Exogenous and endogenous data suggested that USP19 specifically interacts with Beclin-1 during autophagy.3. USP19 stabilizes Beclin-1. USP19 specifically mediates the stabilization of Beclin-1, but not affects the concentration of other members of Beclin-1 complex. USP19 stabilized Beclin-1 in a USP13-independent manner and protects Beclin-1 from proteasomal degradation through its protease activity.4. USP19 removes the K11-linked ubiquitin chains of Beclin-1. USP19 cleaves the K11-linked ubiquitination of Beclin-1 and the catalytic activity of USP19 is essential for Beclin-1 deubiquitination through a K11 linkage.5. Lys437 in Beclin-1 is essential for its ubiquitination and degradation. K437 in Beclin-1 is an essential residue for USP19-mediated K11-linked deubiquitination and stabilization of Beclin-1, thereby regulating autophagic flux.6. USP19 inhibits RNA virus-induced type I IFN activation. USP19 is a negative regulator of RIG-I-mediated type I IFN signaling as well as antiviral immunity, relying on the non-autophagic role of Beclin-1.7. USP19-Beclin-1 inhibit the interaction between RIG-I-MAVS. USP19-mediated stabilization of Beclin-1 suppresses RIG-I-MAVS, further negatively regulating type I IFN signaling.K11-linked ubiquitination controls the degradation of Beclin-1 through proteasome pathway, while USP19 reverses this modification and rescue Beclin-1 degradation, thus promoting the autophagic flux. In RLR-mediated type I IFN signaling, Beclin-1 associates with MAVS, blocking its interaction with RIG-I, thereby inhibiting the activation of type I interferon signaling.