K63 ubiquitination in immune signaling

Abstract

Ubc13-catalyzed K63 ubiquitination is a major control point for immune signaling. Recent evidence has shown that the control of multiple immune functions, including chronic inflammation, pathogen responses, lymphocyte activation, and regulatory signaling, is altered by K63 ubiquitination. In this review, we detail the novel cellular sensors that are dependent on K63 ubiquitination for their function in the immune signaling network. Many pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can target K63 ubiquitination to inhibit pathogen immune responses; we describe novel details of the pathways involved and summarize recent clinically relevant SARS-CoV-2-specific responses. We also discuss recent evidence that regulatory T cell (Treg) versus T helper (T_H) 1 and T_H17 cell subset regulation might involve K63 ubiquitination. Knowledge gaps that merit future investigation and clinically relevant pathways are also addressed.

Keywords: E3 ligase; K63-linked ubiquitination; SARS-CoV-2; Ubc13; immune response; immune tolerance; pattern recognition receptor (PRR); ubiquitin.

Figure 1

Key figure. Ubc13-catalyzed K63-linked ubiquitination can regulate immune cell activation and immune tolerance mechanisms. Signaling sensors of K63-linked ubiquitination [including cellular inhibitor of apoptosis proteins (cIAPs), TNF receptor-associated factors (TRAFs), Nod-like receptors (NLRs), and stimulator of interferon genes (STING)] activate innate and adaptive inflammatory responses (left). FOXP3-dependent K63 ubiquitination of target substrates regulates immune tolerance mechanisms that counteract the inflammatory phenotype (right). While some NLRs negatively regulate K63-linked ubiquitination to blunt inflammatory responses, the role of NLRs in regulatory T cell (Treg)- versus T_H1/T_H17-induced T cell responses is unknown. The mechanistic role of Ubc13 on the modulators of K63 ubiquitination [including SHARPIN or Casitas B lineage lymphoma b (CBL-B)], deficiency of which blunts immune tolerance mechanisms and causes a shift from Treg-induced immunosuppression to T_H1/T_H17-induced immune activation, needs further investigation. Abbreviations: BCR, B cell receptor; TCR, T cell receptor; TLR, Toll-like receptor.

Figure 2

K63 ubiquitination in immune cell signaling cascades. K63-linked polyubiquitin chains (red ovals) and linear polyubiquitin chains (green ovals) catalyzed by Ubc13-Uev1a and UbcH5/linear ubiquitin chain assembly complex (LUBAC) are necessary for efficient activation of immune and antiviral cell signaling pathways. (1) The tumor necrosis factor receptor 1 (TNFR1)-induced non-canonical mechanism involves K48-ubiquitin chain-mediated degradation of TNF receptor-associated factor 3 (TRAF3) by cellular inhibitor of apoptosis proteins 1/2 (cIAP1/2), release of TRAF2/TRAF6 for K63 ubiquitination, and activation of non-canonical nuclear factor KappaB (NF-κB)/MAPK. (2,3) TNFR1-induced K63-ubiquitination of RIPK1 drives two opposing TNFR1 canonical signaling pathways: canonical NF-κB activation modulated by TRAF2-cIAP1-UbcH5-LUBAC-Ubc13-RIPK1 (shown as Complex-I) and classical apoptosis mediated by RIPK1, Caspase-8 and FADD (shown as Complex-II), which is regulated by RIPK1 kinase activity or necroptosis mediated by RIPK1-RIPK3 activity. (4) Stimulation of Toll-like receptors (e.g., TLR4) triggers MyD88-dependent TRAF6- cIAP1/2-Ubc13-catalyzed K63 ubiquitination of downstream substrates followed by NF-κB/MAPK activation. (5) T/B cell receptor (TCR/BCR) activation recruits the caspase recruitment domain-containing membrane-associated guanylate kinase protein1–B cell lymphoma 10–mucosa-associated lymphoid tissue protein 1 (paracaspase) [CARMA1-BCL10-MALT1 (CBM)] complex and activates E2-E3 ligase-mediated K63 ubiquitination-dependent downstream pathways. (6) Stimulation of Nod-like receptors (NLRs) recruits the adaptor proteins to trigger K63 ubiquitination of E3 ligases and their target substrates (including RIPK2) for activation of downstream pathways. The deubiquitinase A20 and some NLR family proteins [e.g., Nod-like receptor Card domain 3 (NLRC3) and NOD-, LRR-, and pyrin domain-containing protein 12 (NLRP12)] negatively regulate K63 ubiquitination-mediated immune cell activation.

Figure 3

Ubiquitination signaling pathways in innate and adaptive immunity. The black arrows indicate stimulation of a pathway and the red arrows denote either inhibition or functional implications due to inhibition. The small red ovals attached to proteins are K63-linked ubiquitin chains. Membrane Toll-like receptors (TLRs) and cytokine receptors [including canonical tumor necrosis factor (TNF) receptors] activate ubiquitinated TNF receptor-associated factor 2/6 (TRAF2/6) pathways to stimulate cytokine and interferon (IFN) production. Single-strand (ss)RNA, double-strand (ds)RNA and mitochondrial (mt)DNA activate cytosolic receptors that are also controlled by ubiquitination. Stimulator of interferon genes (STING) is an intermediate for activation of downstream IFN-activating genes, IFN regulatory factor 3/7 (IRF3/7) by retinoic acid inducible gene-I (RIG-I), mitochondrial antiviral-signaling protein (MAVS), melanoma differentiation-associated protein 5 (MDA-5) through TANK-binding kinase 1 (TBK1), cyclic GMP-AMP (cGAMP) sensing dsRNA. K63 ubiquitination of SHANK-associated RH domain interacting protein in postsynaptic density (SHARPIN) and Casitas B-lineage Lymphoma-b (CBL-B) promotes regulatory T cell (Treg) lineage commitment and deficiency in K63-ubiquitinated SHARPIN or CBL-B stimulates helper T cell (T_H) 1 and T_H17 inflammatory responses. K63 ubiquitination of NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) has a role in the activation of the inflammasome. Nod-like receptor card domain 3 (NLRC3) blocks both STING activation and TNF receptor-associated factor 6 (TRAF6) activation. NLRC5 and NLRX1 inhibit IKK activation, enhancement of nuclear factor KappaB (NF-κB) and inflammation signaling by TLRs. NLRP12 negatively regulates non-canonical NF-κB pathway. NLRC5, NLRX1, and NLRP12 negatively regulate RIG-induced antiviral immune responses. Inhibition of NF-κB, RIG-I, and MDA-5 signaling (by ssRNA) is blocked when NLRC5 is K63-ubiquitinated.

Figure 4

Stimulator of interferon genes (STING) in interferon (IFN) responses. STING has a central role in stimulating innate immunity, Type 1 and 3 IFN production, and also inflammation via nuclear factor KappaB (NF-κB). IFN production by STING is dependent on K63 ubiquitination (red ovals). STING acts as a linker for activation of downstream IFN activating genes (IFN regulatory factor 3; IRF3,) by retinoic acid inducible gene-1 (RIG-I), mitochondrial antiviral-signaling protein (MAVS), melanoma differentiation-associated protein 5 (MDA-5) through TANK-binding kinase 1 (TBK1) and cyclic GMP-AMP (cGAMP), which senses RNA viruses. Additionally, TLR7/8 and TLR3 signal IFN production by single-strand (ss)RNA and double-strand (ds)RNA and these signal through K63-ubiquitinated TNF receptor-associated factor 3/6 (TRAF3/6; not shown here). The four identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame proteins that inhibit K63 ubiquitination and innate responses are 3CL, ORF9, NSP8, and Papain-like protease (PLPro) (in red). Unknown mechanisms are in purple. The purple arrows show a feedback loop that turns off STING and is inhibited by the NSP9,10 proteins, which can block the K63 ubiquitination of STING that is key for IFN 1 and 3 production but not for NF-κB activation.