biomolecules Review Targeting RIP Kinases in Chronic Inflammatory Disease Mary Speir 1,2, Tirta M. Djajawi 1,2 , Stephanie A. Conos 1,2, Hazel Tye 1 and Kate E. Lawlor 1,2,* 1 Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, VIC 3168, Australia; [email protected] (M.S.); [email protected] (T.M.D.); [email protected] (S.A.C.); [email protected] (H.T.) 2 Department of Molecular and Translational Science, Monash University, Clayton, VIC 3168, Australia * Correspondence: [email protected]; Tel.: +61-85722700 Abstract: Chronic inflammatory disorders are characterised by aberrant and exaggerated inflam- matory immune cell responses. Modes of extrinsic cell death, apoptosis and necroptosis, have now been shown to be potent drivers of deleterious inflammation, and mutations in core repressors of these pathways underlie many autoinflammatory disorders. The receptor-interacting protein (RIP) kinases, RIPK1 and RIPK3, are integral players in extrinsic cell death signalling by regulating the production of pro-inflammatory cytokines, such as tumour necrosis factor (TNF), and coordinating the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, which underpin patholog- ical inflammation in numerous chronic inflammatory disorders. In this review, we firstly give an overview of the inflammatory cell death pathways regulated by RIPK1 and RIPK3. We then discuss how dysregulated signalling along these pathways can contribute to chronic inflammatory disorders of the joints, skin, and gastrointestinal tract, and discuss the emerging evidence for targeting these RIP kinases in the clinic. Keywords: apoptosis; necroptosis; RIP kinases; chronic inflammatory disease; tumour necrosis factor; Citation: Speir, M.; Djajawi, T.M.; interleukin-1 Conos, S.A.; Tye, H.; Lawlor, K.E. Targeting RIP Kinases in Chronic Inflammatory Disease. Biomolecules 2021, 11, 646. https://doi.org/ 1. Introduction 10.3390/biom11050646 Common chronic inflammatory and autoinflammatory diseases are caused by immune Academic Editor: Vladimir dysregulation, the aetiology of which involves a complex interplay between genetic and N. Uversky environmental factors that is incompletely understood [1]. Unmistakably, however, the underlying cause of distinct tissue pathologies is a failure to resolve inflammation arising Received: 9 April 2021 from the excessive production of inflammatory cytokines and chemokines, as well as Accepted: 22 April 2021 danger-associated molecular patterns (DAMPs) that are released from dying cells. Tumour Published: 28 April 2021 necrosis factor (TNF) is the archetypal death ligand and a pivotal pathogenic cytokine in many common inflammatory diseases. Accordingly, a number of TNF antagonists, as Publisher’s Note: MDPI stays neutral well as other anti-inflammatory biological agents that dampen its activity, have met with with regard to jurisdictional claims in considerable success in the clinic. Yet, many patients still fail to respond, or else develop published maps and institutional affil- severe adverse reactions to these therapies, highlighting the need for a both a greater iations. understanding of TNF signalling on a cell-by-cell level and also the need for alternative strategies to target TNF signalling pathways in disease. Over the last decade, the serine-threonine receptor-interacting protein (RIP) kinases -1 and -3 have emerged as key regulators of innate immunity via their integral roles in Copyright: © 2021 by the authors. cell death signalling during cellular stress and following exposure to inflammatory and Licensee MDPI, Basel, Switzerland. infectious stimuli [2–4]. RIPK1 has a critical scaffolding role in tumour necrosis factor This article is an open access article receptor-1 (TNFR1) and Toll-like receptor 3/4 (TLR3/4) pro-inflammatory signalling [5–7], distributed under the terms and as well as a kinase-dependent role in both apoptotic and necroptotic cell death [8]. The conditions of the Creative Commons carboxy-terminal death domain (DD) of RIPK1 can facilitate its interaction with other DD- Attribution (CC BY) license (https:// containing proteins to promote formation of death receptor signalling complexes [9–11], creativecommons.org/licenses/by/ while its RIP homotypic interaction motif (RHIM) domain (also shared with RIPK3) enables 4.0/). Biomolecules 2021, 11, 646. https://doi.org/10.3390/biom11050646 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 646x FOR PEER REVIEW 2 2of of 23 22 RIPK1/3RIPK1/3 interactions interactions with with other other cell cell death death and/or and/or immune immune a adaptorsdaptors [12], [12], e.g. e.g.,, TIR TIR domain domain-- containingcontaining adaptor adaptor-inducing-inducing IFN-IFN-β (TRIF) [5,13] [5,13] and the the dsDNA dsDNA receptor receptor Z Z-DNA-DNA binding proteinprotein-1-1 (ZBP1; also known as DAI) [[14]14].. TNFTNF ligation ligation of of TNFR1 TNFR1 typically typically recruits recruits RIPK1 RIPK1 into into a membrane a membrane bound bound receptor receptor sig- nallingsignalling complex, complex, referred referred to as to complex as complex I, that I, that activates activates NF NF--B-κmediatedB-mediated transcription transcription of proof pro-survival-survival genes genes (Figure (Figure 1). Along1). Along with withRIPK1, RIPK1, TNFR1 TNFR1-associated-associated death domain death domain protein (TRADD)protein (TRADD) is recruited is recruited to TNFR1 to TNFR1via their via common their common DD [15,16]. DD [The15,16 adaptor]. The adaptor TNFR-associ- TNFR- atedassociated factor factor2 (TRAF2) 2 (TRAF2) subsequently subsequently binds binds to TRADD to TRADD and andrecruits recruits the thecellular cellular inhibitor inhibitor of apoptosisof apoptosis proteins proteins (cIAP) (cIAP)-1-1 and and cIAP2 cIAP2 [17,18]. [17,18]. The The E3 E3 ubiquitin ubiquitin ligase ligase activity ofof cIAP1/2cIAP1/2 attachesattaches K63 K63-- and K11 K11-linked-linked polyubiquitin chains to RIPK1, allowing for the recruitment ooff the linear ubiquitinubiquitinchain chain assembly assembly complex complex (LUBAC), (LUBAC), which which is ais heterotrimeric a heterotrimeric complex com- plexmade made up of up haeme-oxidized of haeme-oxidized IRP2 ubiquitinIRP2 ubiquitin ligase 1ligase (HOIL-1), 1 (HOIL HOIL-1-interacting-1), HOIL-1-interacting protein protein(HOIP), (HOIP), and SHANK-associated and SHANK-associated RH domain-interacting RH domain-interacting protein protein (SHARPIN). (SHARPIN). Subsequent Sub- sequentM1-linked M1- ubiquitylationlinked ubiquitylation of complex of complex I components I components by LUBAC by LUBAC stabilises stabilises the the complex com- plexand promotesand promotes the TAK1-the TAK1 and- IKK-dependentand IKK-dependent activation activation of canonical of canonical NF- κNFB transcrip--B tran- scriptiontion [19– 21[19].–21]. Post-translational Post-translational modification modification of of RIPK1 RIPK1 within within complex complex I, I, as as well well asas the selectselectiveive cleavage cleavage of of RIPK1 RIPK1 and and RIPK3 RIPK3 (and (and cylindromatosis cylindromatosis [CYLD]) [CYLD]) by by caspase caspase-8,-8, main- main- taintain TNF TNF-mediated-mediated pro pro-survival-survival signalling signalling and and block the transition to pro-apoptoticpro-apoptotic cell deathdeath complex complex II [22 [22––29]29].. This This critical critical pro pro-survival-survival scaffolding scaffolding role for RIPK1 is evide evidencednced byby the fact that RIPK1 RIPK1-deficient-deficient mice die shortly after birth due to systemic multi-organmulti-organ inflammationinflammation mediated mediated by unrestrained apoptosis and necroptosis [[3030–3636].]. Figure 1. Regulation of RIPK1 RIPK1-- and RIPK3 RIPK3-mediated-mediated cell death and inflammation. inflammation. TNF TNF ligation ligation of of its its receptor, receptor, TNFR1, typically induces complex 1 formation, where TNFR1 associates with RIPK1 and TRADD. TRADD subsequently interacts typically induces complex 1 formation, where TNFR1 associates with RIPK1 and TRADD. TRADD subsequently interacts with TRAF2 to facilitate cIAP1 and LUBAC recruitment to the complex. The cIAPs and LUBAC ubiquitylate RIPK1 to with TRAF2 to facilitate cIAP1 and LUBAC recruitment to the complex. The cIAPs and LUBAC ubiquitylate RIPK1 to facilitate a signalling cascade that induces pro-survival and pro-inflammatory gene expression. If either RIPK1 phosphor- ylationfacilitate or a ubiquitylation signalling cascade are impaired, that induces FADD pro-survival-RIPK1(RIPK3) and pro-inflammatory-caspase-8 associate gene to expression.form the pro If- eitherapoptotic RIPK1 complex phosphory- II (or thelation ripoptos or ubiquitylationome). If caspase are impaired,-8 activity FADD-RIPK1(RIPK3)-caspase-8 is compromised, a further complex, associate termed to form the thenecrosome, pro-apoptotic forms complex in a RIPK1 II (or- and the RIPK3ripoptosome). kinase-dependent If caspase-8 manner. activity Within is compromised, this complex, a further RIPK3 complex,-mediated termed phosphorylation the necrosome, and forms activation in a RIPK1- of MLKL and induces RIPK3 necroptosis, a lytic inflammatory form of cell death. Of note, TLR4 ligation can also induce similar inflammatory (e.g., Biomolecules 2021, 11, 646 3 of 22 kinase-dependent manner. Within this complex, RIPK3-mediated phosphorylation and activation of MLKL induces necroptosis, a lytic inflammatory form of cell death. Of note, TLR4 ligation can also induce similar inflammatory