Formation of SUMO3-conjugated chains of MAVS induced by poly(dA:dT), a ligand of RIG-I, enhances the aggregation of MAVS that drives the secretion of interferon-β in human keratinocytes

https://doi.org/10.1016/j.bbrc.2019.11.189Get rights and content

Highlights

  • Poly(dA:dT)-induced secretion of IFN-β is dependent on RIG-I and MAVS in human keratinocytes.

  • Inhibition of SUMOylation by Ginkgolic acid and Ubc9 siRNA inhibits poly(dA:dT)-induced secretion of IFN-β in human keratinocytes.

  • Poly(dA:dT) induces the formation of SUMO3-conjugated chains of MAVS in human keratinocytes.

  • The conjugation of SUMO3 to MAVS induced by poly(dA:dT) enhances the aggregation of MAVS that drives the secretion of IFN-β in human keratinocytes.

Abstract

The retinoic-acid inducible gene (RIG)-I is a cytoplasmic pattern recognition receptor that senses single-stranded (ss) or double-stranded (ds) RNA. RIG-I also senses AT-rich dsDNA, poly(dA:dT), through the action of an RNA polymerase III-transcribed RNA intermediate. Upon the binding of an RNA ligand, RIG-I binds to the mitochondrial antiviral-signaling protein (MAVS) and induces the formation of filamentous aggregates of MAVS, leading to the formation of a signaling complex that drives Type I interferon (IFN) responses. In the current study, we investigated the issue of whether the SUMOylation of MAVS induced by poly(dA:dT) affects the aggregation of MAVS in the RIG-I/MAVS pathway in human keratinocytes. Our results show that the poly(dA:dT)-induced secretion of IFN-β was dependent on RIG-I and MAVS. The inhibition of SUMOylation by Ginkgolic acid or Ubc9 siRNA was found to inhibit the poly(dA:dT)-induced secretion of IFN-β, suggesting that the SUMOylation is required for the poly(dA:dT)-activated RIG-I/MAVS pathway, which drives the secretion of IFN-β. In addition, treatment with poly(dA:dT) enhanced the formation of polymeric chains of small-ubiquitin like modifiers (SUMO)3, but not SUMO1 and SUMO2, on MAVS. Our results also show that the conjugation of SUMO3 to MAVS induced by poly (dA:dT) enhanced the aggregation of MAVS. These collective results show that the formation of SUMO3-conjugated chains of MAVS induced by poly (dA:dT), a ligand of RIG-I, enhances the aggregation of MAVS which, in turn, drives the secretion of IFN-β in human keratinocytes.

Introduction

In the cytoplasm, two essential pattern recognition receptors, the retinoic-acid inducible gene (RIG)-I and melanoma differentiation-associated (MDA) 5 recognize viral or endogenous RNA. RIG-I recognizes relatively short (<2000 bp) single-stranded (ss) RNA with 5′-triphosphate and small double-stranded (ds) RNA (21–27 bp), whereas MDA5 preferentially binds long dsRNA (>2000 bp) [1]. In addition, RIG-I also senses AT-rich dsDNA, poly(dA:dT), through the action of an RNA polymerase III-transcribed RNA intermediate [2]. RIG-I is a RNA helicase, comprised of two N-terminal caspase recruitment domains (CARDs), a central DExD/H-box ATPase/helicase domain and a C-terminal regulatory domain [3]. Upon the binding of RNA ligand, RIG-I undergoes a conformational change and binds to the mitochondrial antiviral-signaling protein (MAVS), a mitochondrial adaptor protein, through the binding of its CARD domain to the CARD domain of MAVS [4]. MAVS is primarily localized on the mitochondrial outer membrane and contains an N-terminal CARD domain, a proline-rich region (PRR) and a C-terminal transmembrane (TM) domain [5]. The interaction of CARD domains between RIG-I and MAVS induces the filamentous aggregation of MAVS on the mitochondria. The MAVS aggregate forms a signaling complex with TANK-binding kinase 1 (TBK1) and Iκ-B kinase ε (IKK-ε), leading to the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3) and production of type I interferons (IFNs) [5].

MAVS, an essential adaptor protein in RIG-I signaling pathway, links the upstream recognition of RNA ligand to downstream signal transduction, IRF3/type I IFNs. It has been reported that MAVS function is regulated through its phosphorylation or ubiquitination at the post-translational level [5]. The SUMOylation is also a post-translational modification that modulates protein functions via cross-talk with other post-translational modifications, such as phosphorylation, ubiquitination and acetylation [6]. Although, to our knowledge, there is no direct evidence to show that the function of MAVS is regulated by SUMOylation, it has been reported that incubation of a recombinant fusion protein, a small ubiquitin-like modifier (SUMO)-CARD domain of MAVS lacking the PRR and TM domain, with mitochondria induced the activation or aggregation of MAVS and the dimerization of IRF3 [7]. Given this finding, we hypothesized that SUMOylation at the CARD domain of MAVS may affect the RIG-I/MAVS pathway that drives the secretion of type I IFNs.

The SUMO proteins, ∼12 kDa in size, are covalently attached to and detached from various proteins [8]. Mammals produce, three SUMO proteins, SUMO1, SUMO2 and SUMO3. The conjugation of SUMO to a substrate protein is promoted by a set of activating (E1), conjugating (E2) and ligating (E3) enzymes, and the deconjugation of SUMO from proteins is regulated by specific isopeptidase, sentrin/SUMO-specific proteases (SENPs) [8,9]. SUMO1 is covalently conjugated to proteins as a single moiety, whereas SUMO2 and SUMO3 are covalently attached to proteins in the form of polymeric SUMO chains since SUMO2 and SUMO3 have also a consensus SUMO modification site ΨKxE (Ψ; a hydrophobic amino acid, K; lysine, x; any amino acid and E; an acidic residue) and could be used as substrates for the conjugation of SUMO [6,10].

In addition to dendritic cells (DCs), keratinocytes are another major source of type I IFNs, especially IFN-β, in skin inflammatory/immune lesions [11]. The role of keratinocytes in the defense against viral infection and the development of autoimmune diseases has attracted increasing attention in both basic and clinical studies. In the current study, we report on an investigation of the issue of whether poly(dA:dT) induces the SUMOylation of MAVS in human keratinocytes. We then assessed the effects of the SUMOylation of MAVS on the RIG-I/MAVS pathway in human keratinocytes.

Section snippets

Reagents and antibodies

The reagents and antibodies used in this study are described in Supplementary Material.

Cell cultures

Human primary epidermal keratinocytes from neonatal foreskin, HEKn (Invitrogen, Carlsbad, CA, USA), and the Human keratinocyte cell line HaCaT (CLS, Eppelheim, Germany) were grown in serum-free EpiLife medium. The method for cell culture is described in more detail in Supplementary Material.

Treatment with poly(dA:dT) or Ginkgolic acid

Cells was treated with a poly(dA:dT) in a transfection reagent (Xtreme GENE9 DNA transfection reagent, Roche, Basel,

Poly(dA:dT)-induced secretion of IFN-β is dependent on RIG-I and MAVS

We initially examined the effect of poly(dA:dT), a synthetic analog of microbial dsDNA, on the secretion of IFN-β in human primary keratinocytes, HEKn cells and a human immortalized keratinocyte cell line, HaCaT cells. Treatment with poly(dA:dT) induced the secretion of IFN-β in HEKn and HaCaT cells, in a time- and dose-dependent manner (Supplementary Fig. S1).

We then examined the issue of whether the treatment with poly(dA:dT)-induced secretion of IFN-β is dependent on the RIG-I/MAVS pathway

Discussion

The findings reported herein indicate that the formation of SUMO3-conjugated chains of MAVS induced by poly(dA:dT), a repetitive synthetic dsDNA, enhances the aggregation of MAVS in turn, drives the secretion of IFN-β in human keratinocytes (Supplementary Fig. S1 and Fig. 4).

Hu et al. [14] reported that the SUMOylation of RIG-I and MDA5 by the Tripartite motif-containing protein (TRIM38), the E3 ubiquitin-protein ligase, suppresses K-48 ubiquitination and the degradation of RIG-I in uninfected

Declaration of competing interest

The authors state no conflicts of interest.

Acknowledgment

This research was supported by grants from the Korea Centers for Disease Control & Prevention, Korea Ministry of Health & Welfare, Republic of Korea (5-2019-A0082-00002).

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