Ubiquitin-Mediated Regulation of Cell Death, Inflammation, and Defense of Homeostasis

Abstract

Cell death and inflammation are ancient processes of fundamental biological importance in both normal physiology and human disease pathologies. The recent observation that apoptosis regulatory components have dual roles in cell death and inflammation suggests that these proteins function, not primarily to kill, but to coordinate tissue repair and remodeling. This perspective unifies cell death components as positive regulators of tissue repair that replaces malfunctioning or damaged tissues and enhances the resilience of epithelia to insult. It is now recognized that cells that die by apoptosis do not do so silently, but release a variety of paracrine signals to communicate with their cellular environment to ensure tissue regeneration, and wound healing. Moreover, inflammatory signaling pathways, such as those emanating from the TNF receptor or Toll-related receptors, take part in cell competition to eliminate developmentally aberrant clones. Ubiquitylation has emerged as crucial mediator of signal transduction in cell death and inflammation. Here, we focus on recent advances on ubiquitin-mediated regulation of cell death and inflammation, and how this is used to regulate the defense of homeostasis.

Keywords: Caspases; Homeostasis; IAPs; RIPK1; Ubiquitin.

Figure 1. Ubiquitylation as mediator and regulator of signal transduction in cell death, inflammation, and defense of homeostasis.

(A) Tissue malfunction results in a secretery programme and inflammatory response whose purpose it is to restore homeostasis. Tissue homeostasis is regulated by a collective decision mechanism that influences cell death and proliferation across tissues. These include cell competition and apoptosis-induced compensatory proliferation. (B) Tissue stress response and inflammation underlie a common principle in which the conjugation of typical Ub-chains produces robust networks that are decoded by Ub-receptors whose actions serve to coordinate adaptation to tissue stress. (C) Ribbon Structure of Ub. Lysines at position 48 and 63 of Ub are highlighted. (D, E)Topology of K48- (C) and K63-linked (D) tetra Ub chains.

Figure 2. Evolutionary conservation of TNF-induced cell death and the defense of homeostasis.

(A) TNF signaling and the transition of complex-I to complex-II. Upon TNF-binding, cIAPs are recruited to the TNF-R1 signaling complex (Complex-I) via TRADD/TRAF2. cIAPs ubiquitylate several molecules within the complex. RIPK1 ubiquitylation is the most readily observed. Ubiquitylation of components of complex-I, such as RIPK1, drives the recruitment of HOIL-1/HOIP/Sharpin that together form the Linear Ubiquitin Assembly Complex (LUBAC). LUBAC generates linear Ub chains on NEMO and RIPK1 that in turn recruits more NEMO molecules via its linear Ub binding UBAN domain. NEMO is probably constitutively associated with IKKa/IKKb and IKKb is phosphorylated and activated by TAK1 that is independently recruited to ubiquitylated complex-I via its Ub receptors TAB2 and TAB3 that bind only to K63-linked Ub chains. Phosphorylated and activated IKKb in turn phosphorylates IκBα, which leads to recruitment of a HECT E3 ligase. This E3 ligase promotes K48-linked ubiquitylation and proteasomal degradation of IκBα, allowing translocation of NFkB subunits p50/p65 to drive production of cytokines. p50/p65 also promote expression of IκBα, to cause feedback inhibition, as well as genes such as cFLIP that are required to protect cells from complex-II-induced cell death. The numbered arrows provide a tentative indication of temporal sequence. Complex-II is most likely generated from complex-I, in an as yet undefined manner, and comprises RIPK1, FADD and caspase-8. Deubiquitylation by CYLD is thereby a decisive step in the transition of complex-I to complex-II. Caspase-8 limits Complex-II formation by cleaving and inactivating RIPK1. Consequently, loss of IAPs, LUBAC or caspase-8 activity results in formation of Complex-II that is able to drive necroptosis. Formation of complex-II, Necrosome or ripoptosome can also occur following stimulation of Pattern Recognition receptors or genotoxic stress. (B) Eiger-mediated signalling that regulates a variety of cellular and tissue processes, including the elimination of polarity mutant cells. Eiger mediates its effect through binding to its cognate receptor Grindelwald. This results in activation of JNK in a DTRAF2/Bendless/dUev1A dependent manner. The Drosophila homologue of TAB2/3 (dTAB2) links TAK1 to the presumptive Ub chains conjugated by DTRAF2. dCYLD influences the decision as to whether JNK drives cell death or non-cell death processes (see text for further details).

Figure 3. IAP-mediated regulation of caspases in Drosophila.

(A) Binding profile of DIAP1 with caspases and IAP antagonists. Direct physical interaction with the effector caspases drICE or DCP-1 and the initiator caspase DRONC is mediated through DIAP1’s BIR1 and BIR2 domains, respectively. Following their activation, drICE and DCP-1 expose an NH₂-terminal IBM (depicted as A), which allows their binding to BIR1. (B) Sequence alignment of IBM-bearing proteins. Identical residues are highlighted in black. Residues conserved in four or more IBM-proteins are indicated in grey. (C) DIAP1’s BIR2-DRONC association is essential for DIAP1 to neutralise DRONC. Following binding, DIAP1’s RING-finger promotes Ub conjugation of DRONC, leading to its inactivation through non-degradative ubiquitylation of monomeric DRONC (left panel), and by targeting apoptosome-associated active DRONC for degradation (right panel). (D-E) Mechanism of effector caspase (drICE) inactivation by DIAP1 (D) and DIAP2 (E). (D) Full-length wild-type DIAP1 is held in an inactive conformation, and requires caspase-mediated proteolytic cleavage at residue 20 for its activation. After cleavage, BIR-mediated caspase-binding occurs more efficiently. Cleavage also facilitates recruitment of N-end rule UBR E3 ligases, which together with DIAP1’s RING domain, promote ubiquitylation and inactivation of drICE and DCP-1. (E) drICE is also subject to regulation by DIAP2. drICE binds to the BIR3 of DIAP2 in an IBM-dependent manner, and following binding cleaves DIAP2 at D100. DIAP2 cleavage results in a covalent adduct between D100 and the catalytic machinery of drICE, trapping the caspase. Full inactivation of drICE is achieved through RING-mediated ubiquitylation.

Figure 4. Model depicting Ub-dependent activation of NF-κB during Imd signalling.

Ligand binding induces recruitment of Imd and dFADD to PGRP-LC. This allows binding of DREDD to dFADD, which leads to dimerisation-induced activation of DREDD. Active DREDD subsequently cleaves IMD, which leads to the exposure of an IBM at the neo-amino terminus of cleaved IMD. This in turn allows recruitment of DIAP2 into the signalling complex, followed by DIAP2-mediated ubiquitylation of various components of this complex, such as IMD and DREDD. Ubiquitylation of DREDD is key for DREDD-mediated cleavage of RELISH. The Ub chains of DREDD may serve as a scaffold for the recruitment of IKK. Since IKK binds to RELISH, as evidenced by its ability to phosphorylate RELISH, IKK might bring RELISH into close proximity of DREDD for proteolysis. Ubiquitylation of components of the IMD pathway also leads to activation of TAK1 and IKK, which in turn phosphorylates RELISH. (6.) Phosphorylated and cleaved RELISH subsequently translocates to the nucleus where it drives expression of RELISH target genes.

Figure 5. XIAP-mediated regulation of caspases and NF-κB.

(A) XIAP directly inhibits the effector caspase-3 and caspase-7, and the initiator caspase-9. The sequence preceding the BIR2 domain of XIAP occupies the catalytic pocket of caspase-3 or caspase-7, thereby blocking substrate entry. In addition, the BIR2 domain interacts with the IBM of caspase-3 or caspase-7 that is exposed following their proteolytic activation (shown as an arrow). XIAP-mediated inhibition of caspase-9 requires proteolytic cleavage of caspase-9, which exposes an IBM that binds to the BIR3 of XIAP. Caspase-9 activity is blocked because XIAP prevents caspase 9 dimerization, a prerequisite for initiator caspasenactivity. The RING domain of XIAP also contributes to caspase inhibition. (B) NOD-mediated activation of NF-kB and MAPK signalling. Detection of bacterial peptidoglycans by NOD1 and NOD2 results in the formation of an oligomeric signalling complex that recruits RIPK2, and XIAP. XIAP mediated ubiquitylation of RIPK2 allows the recruitment of TAB2/TAB3/TAK1 and IKKs, thereby triggering NF-kB and MAPK signalling.