Abstract
Incontinentia Pigmenti (IP) is a rare X-linked disease characterized by early male lethality and multiple abnormalities in heterozygous females. IP is caused by NF-κB essential modulator (NEMO) mutations. The current mechanistic model suggests that NEMO functions as a crucial component mediating the recruitment of the IκB-kinase (IKK) complex to tumor necrosis factor receptor 1 (TNF-R1), thus allowing activation of the pro-survival NF-κB response. However, recent studies have suggested that gene activation and cell death inhibition are two independent activities of NEMO. Here we describe that cells expressing the IP-associated NEMO-A323P mutant had completely abrogated TNF-induced NF-κB activation, but retained partial antiapoptotic activity and exhibited high sensitivity to death by necroptosis. We found that robust caspase activation in NEMO-deficient cells is concomitant with RIPK3 recruitment to the apoptosis-mediating complex. In contrast, cells expressing the ubiquitin-binding mutant NEMO-A323P did not recruit RIPK3 to complex II, an event that prevented caspase activation. Hence NEMO, independently from NF-κB activation, represents per se a key component in the structural and functional dynamics of the different TNF-R1-induced complexes. Alteration of this process may result in differing cellular outcomes and, consequently, also pathological effects in IP patients with different NEMO mutations.
Highlights
Tumor necrosis factor α (TNF) is a major cytokine promoting inflammation and innate immune responses
To test whether expression of NEMO-A323P has functional consequences for TNF-induced cytotoxicity, we reconstituted NEMO-deficient murine embryonic fibroblast (MEF) with empty vector, wild-type NEMO (NEMO-WT) or NEMO harboring the A323P mutation (NEMO-A323P)
To gain mechanistic insight on how the phosphorylation of MLKL occurs, we investigated whether RIPK3 is recruited to the FADD complex in NEMO-A323P cells after TNF/zVAD stimulation
Summary
Tumor necrosis factor α (TNF) is a major cytokine promoting inflammation and innate immune responses. LUBAC in turn mediates the formation of linear ubiquitin chains which enhances recruitment of the IκB-kinase (IKK) complex, composed of the adaptor protein NEMO (NF-κB essential modulator) and the kinases IKK1 and IKK2.2 NEMO binds to non-degradative ubiquitin chains within the TNF-R1 signaling complex through the cooperation of two domains: the NEMO ubiquitin-binding domain (NUB, called UBAN) and the zinc-finger domain Together, they enable efficient recruitment and retention of IKK1/2 and their activation by the TAK1.3–5 The IKK complex subsequently activates NF-κB signaling, leading to transcription of prosurvival genes, such as cIAP1/2, and the cellular FLICE-like inhibitor protein (cFLIP), an enzyme-inactive homolog of Caspase-8.6–8. After the discovery of the NUB domain, it was clearly shown that this mutation abolished NEMO’s ability to bind to ubiquitin.[25,26,27]
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