## 2 RIPK1-Mediated Pro-Survival and Inflammatory Signaling  
RIPK1 was initially reported to strongly interact with the cell surface receptor Fas/APO-1 (CD95) and weakly with TNFR1 [6]. Subsequently, various members of the TNF superfamily, including TNF\(\kappa\), FasL, and TRAIL (TNF-related apoptosis-inducing ligand), were found to induce RIPK1-mediated NF-\(\kappa\)B (nuclear factor kappa-light-chain-enhancer of activated B cells) activation [3]. Among these, the TNF\(\kappa\) cascade has been extensively studied (Figure 2) [7]. Upon TNF\(\kappa\) binding, RIPK1 and TRADD are rapidly recruited to TNFR1, initiating the assembly of complex I through mutual interactions between their death domains [8; 9; 10]. TRADD then recruits adaptor proteins TRAF2 and 5 (TNF receptor-associated factor protein 2/5), which in turn engage the E3 ubiquitin ligases cIAP1/2 (cellular inhibitor of apoptosis 1 and 2) [11; 12]. cIAP1/2 catalyzes K63 ubiquitination of RIPK1, serving as a scaffold to recruit ubiquitin-binding proteins TAB2/3 (TAK1-binding protein 2/3) and TAK1 (transforming growth factor-\(\beta\)-activated kinase 1) [13; 14]. TAK1 then activates the MAPK (mitogen-activated protein kinase) pathway, including p38 and MK2 (MAPK-activated kinase 2) [15]. Additionally, the K63 ubiquitin chain recruits another E3 complex LUBAC (the linear ubiquitin chain assembly complex), which catalyzes the M1 linear ubiquitin chains on RIPK1 and TNFR1 [16; 17]. These linear ubiquitin chains recruit adaptor protein NEMO (NF-\(\kappa\)B essential modulator), which further engages IKK\(\kappa\)/\(\beta\) (IxB kinase \(\alpha\)/\(\beta\)) and TBK1/IKK\(\kappa\)[18; 19; 20]. IKK\(\kappa\)/\(\beta\) subsequently activates the NF-\(\kappa\)B pathway [21; 22]. Both the MAPK pathway and the NF-\(\kappa\)B pathway activate gene expression that promotes inflammation and cell survival while suppressing cell death (Figure 2) [7; 15]. Figure 1: The domain structure of RIPK1 comprises a kinase domain (KD), an intermediate domain (ID), and a death domain (DD). Within the intermediate domain lies the RIP homotypic interaction motif domain (RHIM), which participates in polymerization and interacts with the RHIM domains of RIPK3, ZBP1, and TRIF. The death domain of RIPK1 facilitates homo-dimerization and interacts with the death domains of TNFR1, TRADD, and FADD. Abbreviations: RIPK1, receptor-interacting protein kinase 1; RIPK3, receptor-interacting protein kinase 3; ZBP1, Z-DNA-binding protein 1, also known as DAI (DNA-dependent activator of interferon regulatory factors) and DLM-1; TRIF, TIR-domain-containing adapter-inducing interferon \(\beta\); TNFR1, tumor necrosis factor receptor 1; TRADD, TNFR1-associated death domain protein; FADD, Fas-associated death domain. Figure 2: Pleiotropic TNF signaling pathways. (1) Inflammation and cell survival. Engagement of TNF with its receptor TNFR1 leads to the recruitment of RIPK1 and TRADD through death domain interactions to initiate complex I formation. TRADD then recruits adaptor protein TRAF/5, which binds E3 ligase cIAP1/2. cIAP1/2 catalyzes K63 ubiquitination of RIPK1, serving as a scaffold to recruit ubiquitin-binding proteins TAB2/3 and associated TAK1, activating the downstream MAPK pathway. Additionally, the K63 ubiquitin chain recruits another E3 complex, LUBAC, which catalyzes the M1 linear ubiquitin chains on RIPK1 and TNFR1. These linear ubiquitin chains recruit adaptor protein NEMO and associated IKK\(\alpha\)/\(\beta\), phosphorylating IxB\(\alpha\) to promote its degradation and subsequent NF-\(\kappa\)B activation. Both the MAPK pathway and the NF-\(\kappa\)B pathway activate gene expression, which promotes cell survival and inflammation. (2) Apoptosis and/or pyrotosis. Under TNF treatment with protein synthesis inhibition by cycloheximide (CHX), complex I is converted to complex IIa, containing TRADD, FADD, and Caspase-8, leading to oligomerization and activation of Caspase-8 and subsequent apoptosis.