Immunity by ubiquitylation: a reversible process of modification

Abstract

The conjugation of ubiquitin, a 76-amino-acid peptide, to a protein substrate provides a tag that either marks the labelled protein for degradation or modulates its function. The process of protein ubiquitylation--which is catalysed by coordinated enzymatic reactions that are mediated by enzymes known as E1, E2 and E3--has an important role in the modulation of immune responses. Importantly, protein ubiquitylation is a reversible process, and removal of ubiquitin molecules is mediated by de-ubiquitylating enzymes: for example, A20, which has been implicated in the regulation of immune responses. In addition, the conjugation of ubiquitin-like molecules, such as ISG15 (interferon-stimulated protein of 15 kDa), to proteins is also involved in immune regulation. This Review covers recent progress in our understanding of protein ubiquitylation in the immune system.

Figure 1. The ubiquitin-conjugation cycle and the E3 ligases.

a | Ubiquitin is activated by a ubiquitin-activating enzyme (known as enzyme 1, E1) in the presence of ATP and becomes bound to this E1. The activated ubiquitin molecule is then transferred to a ubiquitin-conjugating enzyme (known as E2). Ubiquitin–protein ligases (known as E3 ligases) recruit both the ubiquitin–E2 complex and the substrate protein, and they help to transfer ubiquitin from E2 to the substrate. Substrate-conjugated ubiquitin is removed by de-ubiquitylating enzymes (DUBs). b | A schematic representation of several RING (really interesting new gene)-type and HECT (homologous to the E6-associated protein carboxy terminus)-type E3 ligases is shown. RING-type E3 ligases can be divided into those that contain both the RING-finger domain and the substrate-recruiting domains (which are known as single-protein RING-type E3 ligases) and those that are multisubunit complexes composed of scaffold proteins, substrate-recruiting proteins and a RING-finger-domain-only protein. For example, suppressor of cytokine signalling (SOCS) proteins function as the substrate-recruiting component of the E3 ligase known as the ECS complex (elongin-B–elongin-C–cullin-5–SOCS-protein complex). A20 functions both as an E3 ligase — through its zinc finger (Zn) domain, which both recruits ubiquitin–E2 complexes and transfers the ubiquitin molecule to the substrate — and as a DUB, through its OTU (ovarian tumour) domain. For further details, see the main text. C2 domain, protein-kinase-C-related domain 2; CBL-B, Casitas B-lineage lymphoma B; GRAIL, gene related to anergy in lymphocytes; ITCH, itchy; PP_i, pyrophosphate; ROQ domain, roquin domain; SH2 domain, SRC-homology-2 domain; TKB domain, protein-tyrosine-kinase-binding domain; TRAF, tumour-necrosis-factor-receptor-associated factor; WW domain, domain that contains two conserved tryptophan residues.

Figure 2. Protein ubiquitylation in T-cell tolerance.

a | Autoimmune regulator (AIRE), a RING (really interesting new gene)-type E3 ligase, is implicated in expression of tissue-specific antigens (TSAs) by medullary thymic epithelial cells (mTECs), which present antigens (including TSAs) to developing thymocytes to induce central tolerance to those antigens. In developing thymocytes, signalling through the T-cell receptor (TCR) causes negative selection by inducing thymocyte apoptosis through expression of NUR77 and BIM (B-cell-lymphoma-2-interacting mediator of cell death). The E3 ligase CBL (Casitas B-lineage lymphoma) can function as an E3 ligase for BIM. b | In mature peripheral T cells, ligation of the TCR in the absence of the appropriate co-stimulatory signals results in augmented expression of three E3 ligases — CBL-B, ITCH (itchy) and GRAIL (gene related to anergy in lymphocytes) — and this promotes the conjugation of ubiquitin to phospholipase C-γ1 (PLC-γ1) and protein kinase C-θ (PKC-θ), leading to the induction of T-cell anergy. This pathway seems to define at least one crucial state of T-cell anergy. Another RING-type E3 ligase, roquin, has been implicated in regulating the stability and/or translation of mRNA encoding inducible T-cell co-stimulator (ICOS) and interleukin-21 (IL-21). APC, antigen-presenting cell; Ca²⁺, calcium ions.

Figure 3. The JNK-signalling pathway in differentiation of CD4⁺ T cells into T_H2 cells.

Co-ligation of the T-cell receptor (TCR) and CD28 activates the signalling cascade that involves MEKK1 (mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase kinase 1), MAPK kinase 7 (MAPKK7) and JNK (JUN amino-terminal kinase), and this results in the phosphorylation and activation of the E3 ligase itchy (ITCH). Activated ITCH then targets JUNB, a transcription factor that is required for induction of the gene encoding interleukin-4 (IL-4), for ubiquitylation and degradation. So, this pathway inhibits the expression of IL-4 and the differentiation of CD4⁺ T cells into T helper 2 (T_H2) cells.

Figure 4. Ubiquitin conjugation regulates NF-κB activation.

Triggering of various cell-surface receptors leads to assembly of a multimolecular complex that includes the RING (really interesting new gene)-type E3 ligase TRAF6 (tumour-necrosis-factor receptor (TNFR)-associated factor 6). TRAF6 recruits the ubiquitin-loaded E2 ubiquitin-conjugating enzyme 13 (UBC13) and promotes K63 (Lys63)-linked polyubiquitylation of IKK-γ (inhibitor of NF-κB (IκB) kinase-γ) and activation of the IKK complex. Phosphorylation of IκB by the IKK complex recruits an SCF complex (S-phase kinase-associated protein 1 (SKP1)–cullin-1 (CUL1)–F-box-protein complex), which is an E3 ligase that induces K48-linked polyubiquitylation of IκB. This results in the proteasome-dependent degradation of IκB and the release of NF-κB (nuclear factor-κB), which is required for the transactivation of genes by NF-κB. BCL-10, B-cell lymphoma 10; CARMA1, caspase-recruitment domain (CARD)–membrane-associated guanylate kinase (MAGUK) protein 1; IL-1R, interleukin-1 receptor; IRAK, IL-1R-associated kinase; MALT1, mucosa-associated-lymphoid-tissue lymphoma-translocation gene 1; MyD88, myeloid differentiation primary-response protein 88; PDK1, 3-phosphoinositide-dependent protein kinase 1; PKC-θ, protein kinase C-θ; RIP1, receptor-interacting protein 1; TCR, T-cell receptor; TLR, Toll-like receptor; TRADD, TNFR-associated via death domain; TRIF, Toll/IL-1R (TIR)-domain-containing adaptor protein inducing interferon-β.

Figure 5. A20 as a dual enzyme in NF-κB signalling.

A | Stimulation through tumour-necrosis factor (TNF) receptors (TNFRs) causes TNFR-associated factor 2 (TRAF2)-mediated K63 (Lys63)-linked polyubiquitylation of receptor-interacting protein 1 (RIP1) (a), which results in the downstream activation of nuclear factor-κB (NF-κB). A20 then functions first as a de-ubiquitylating enzyme to remove K63-linked polyubiquitin chains (b), which terminates TNF-mediated signalling (c). At this stage, A20 then functions as an E3 ligase through recruitment of the ubiquitin-bound E2 ubiquitin-conjugating enzyme H5 (UBCH5), facilitating the transfer of ubiquitin from UBCH5 to RIP1 and promoting K48-linked polyubiquitylation of RIP1 (d) and subsequent degradation of RIP1. B | A20 also functions as a de-ubiquitylating enzyme to remove K63-linked polyubiquitin chains from TRAF6, a mediator of Toll-like receptor (TLR) signalling. TRADD, TNFR-associated via death domain; TRIF, Toll/interleukin-1 receptor (TIR)-domain-containing adaptor protein inducing interferon-β.

Figure 6. SOCS1 as a component of the E3 ligase the ECS complex.

Triggering of interferon (IFN) or interleukin-6 (IL-6) receptors results in the recruitment and activation of Janus kinases (JAKs) and, subsequently, in the phosphorylation of downstream signal transducer and activator of transcription (STAT) proteins. Activated JAKs also associate with suppressor of cytokine signalling 1 (SOCS1) through the SRC homology 2 (SH2) domain of SOCS1. The SOCS-box domain of SOCS1 forms a complex with the elongin-B–elongin-C complex and cullin-5 (CUL5), to form an ECS complex (elongin-B–elongin-C–CUL5–SOCS-protein complex), which is an E3 ligase that promotes polyubiquitylation and degradation of JAKs and inhibits STAT-mediated gene transcription. RBX2, RING (really interesting new gene)-box-2.