*3.1. TNFR1 Signaling Pathways*

The pathways triggered upon TNFR1 activation are better known. TNFR1 contains an intracellular death domain (DD) that, in the absence of ligand, interacts with a cytosolic silencer of death domains (SODD) [50]. Upon binding to TNF-α, the inhibitory protein SODD is released and the DD of TNFR1 is recognized by the TNF receptor-associated death domain, TRADD, which recruits two additional adaptor proteins, TNF receptorassociated factor 1 or 2 (TRAF1/2) and receptor interacting serine/threonine-protein kinase 1 (RIPK1) [51–53]. The assembling of different signaling pathways that activate distinct downstream responses will depend on the ubiquitination state of RIPK1 [54]. Thus, RIPK1 is the major regulator of the cellular decision between TNF-mediated pro-survival signaling or death.

Ubiquitinated RIPK1 allows the activation of complex I, comprising TRADD, RIPK1, TRAF2, cellular inhibitor of apoptosis protein 1 or 2 (cIAP1/2), and linear ubiquitin chain assembly complex (LUBAC) [55,56]. Both cIAPs and LUBAC promote poly-ubiquitination of RIPK1 [55,56], which leads to the recruitment of two complexes: transforming growth factor-beta-activated kinase 1 (TAK1) complex, comprising TAK-binding proteins (TAB) 2 and 3; and inhibitor of kappa B kinase (IKK) complex, involving two kinases IKKα and IKKβ, and the regulatory subunit NF-κB essential modulator (NEMO, also known as

IKKγ) [57]. The recruitment of these two complexes (TAK1 and IKK) leads to the activation of mitogen activated protein kinases (MAPKs) and the canonical NF-κB pathway.

The activation of the IKK complex requires NEMO ubiquitination, by LUBAC [56,58], and IKKβ phosphorylation, by TAK1 [55,59]. In turn, the phosphorylated IKKβ initiates phosphorylation and proteosomal degradation of NF-kB inhibitor (IκB), unmasking the p65 subunit of NF-κB and enabling the translocation of the NF-κB heterodimer, composed of a p65 and a p50 subunit, into the nucleus, where it activates the transcription of various proinflammatory, anti-apoptotic, and pro-survival genes [60]. Another TAK1-dependent mechanism that upregulates proinflammatory gene expression involves the phosphorylation of mitogen activated protein kinases (MAPKs), such as c-jun kinase (JNK) and p38 [59,61], which further induces activator protein-1 (AP-1) transcription factor [62].

Alternatively, TNF-*A* binding to TNFR1 can induce two types of programmed cell death, apoptosis or necroptosis, when death-inducing signaling complexes (IIa, IIb, or IIc) are assembled in the cytosol [43]. TNF-TNFR1 mediated NF-κB signaling induces cell survival and requires polyubiquitination of RIPK1 bound to TRADD [63,64]. Therefore, when RIPK1 is not ubiquitinated, it dissociates from complex I, which favors the formation of death complexes. In these NF-κB inhibited conditions, TRADD recruits Fas-associated death domain (FADD) [53], a pro-caspase 8 dimer, and a heterodimer of pro-caspase 8 and the long form of cellular FLICE-inhibitory protein (c-FLIP) [57], forming complex IIa.

The depletion of the cIAP1 and 2 also reduces, or prevents, RIPK1 ubiquitination [65], resulting in apoptosis, through complex IIb. This cytoplasmic complex, formed by nonubiquitinated RIPK1 and FADD, recruits RIPK3, pro-caspase 8 dimer, and c-FLIP-pro-caspase 8 heterodimer, and induces apoptotic cell death, similarly to complex IIa [57].

Furthermore, the aggregation of RIPK1 and RIPK3 leads to the activation of mixed lineage kinase domain-like protein (MLKL) [66,67], through complex IIc. Several mechanisms by which phosphorylated MLKL induces necrotic cell death have been proposed [68], such as mitochondrial fragmentation and/or plasma membrane rupture with a subsequent influx of positively charged ions.

Ubiquitination of the proteins involved in TNFR1-signaling cascades has a major role in determining TNF-induced downstream outcomes. Ubiquitination status of RIPK1 determines whether TNF-TNFR1 signaling mediates cell survival or apoptosis, since RIPK1 ubiquitination prevents complex IIa and IIb from assembling. Several ubiquitin-modifying proteins that act on RIPK1 have been identified. The ubiquitin-modifying enzyme A20 is able to bind and remove polyubiquitin chains from RIPK1 and LUBAC, blocking NF-kB activation. Cylindromatosis (CYLD) is another deubiquitylating enzyme that acts on several proteins, such as TRAF2, RIPK1, and IKKγ, to regulate the NF-kB and JNK pathways [43,69]. In addition, cellular degradation or depletion of cIAPs prevents RIPK1 ubiquitination.
