Binding of TNF to TNFR1 induces either a cell survival or cell death response. Most responses induced by TNF have been attributed to its induction of NF-?B and cell survival. Despite the demonstration that TNF can kill cells 40 years ago (Carswell et al, PNAS, 1975), the in vivo roles of TNF-induced death remain largely unknown. This is due to an incomplete mechanistic understanding of how TNFR1 dictates survival versus death, hampering our ability to genetically manipulate the pathway such that death predominates. We have established that there are two sequential cell death checkpoints in the TNFR1 pathway. The first checkpoint occurs when RIPK1 undergoes non-degradative ubiquitination, a transcription-independent event. This restricts RIPK1 from associating with death-signaling molecules. Instead, RIPK1 associates with a survival complex to propagate the survival signal. The second checkpoint occurs with the NF-?B-dependent induction of pro- survival genes, which provides a longer-lasting protection against death. Disrupting Checkpoint 1 by blocking the ubiquitination of RIPK1 unleashes its ability to induce either apoptosis or necroptosis. Our published studies demonstrated that a critical element of Checkpoint 1 is the removal of CYLD by a pro-survival CASP8. CYLD is a deubiquitinase specific for K63-linked ubiquitin and its removal leads to sustained RIPK1 ubiquitination and protection from death. However when CASP8-mediated cleavage of CYLD is inhibited, CYLD is available to deubiquitinate RIPK1 to initiate death and by virtue of the CASP8 blockade, the only option available is necroptosis. This insight led us to hypothesize that suppression of CYLD is pivotal in Checkpoint 1 and in addition to proteolysis, other mechanisms exist to suppress CYLD.
In Aim 1, we will test the hypothesis that linear ubiquitination is one such mechanism and its disruption leads to CYLD-mediated cell death. In vivo, deficiency in Sharpin (a component of the LUBAC E3 ligase that catalyzes linear ubiquitination) leads to multi-organ inflammation, and this is reversed by a compound deficiency in Cyld. We will study how linear ubiquitination suppresses CYLD.
In Aim 2, we will conduct mechanistic studies to test the hypothesis that phosphorylation of CYLD also suppresses its activity and when this is disrupted, cells die. In vivo, we will examine whether knocking out the CYLD kinase in the epidermis results in CYLD-dependent necroptosis and subsequent skin inflammation.
In Aim 3, we will examine how these post-translational mechanisms that suppress CYLD activity are controlled by external cues. We will conduct mechanistic studies to test the hypothesis that TNFR2 expression in activated macrophages acts to release these brakes on CYLD, enabling TNFR1 then to utilize CYLD to initiate necroptosis. We will test the hypothesis that necroptosis in macrophages enabled by TNFR2 induction has a beneficial role in clearing bacterial infection. Our studies will provide mechanistic insights into the pathologies described in LUBAC-deficient patients and potentially into TNF-mediated disorders such as psoriasis, IBD, RA, and the design of vaccine adjuvants.

Public Health Relevance

In the TNF signaling pathway, suppressing CYLD is required to keep cells alive but when this is disrupted, cells undergo an inflammatory cell death known as necroptosis. We will study the mechanisms that suppress CYLD and how disruption of these mechanisms can lead to inflammation. This has implications for patients with genetic defects in HOIL-1 and HOIP, and for TNF-mediated inflammatory disorders such as psoriasis, rheumatoid arthritis and IBD.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Cellular and Molecular Immunology - A Study Section (CMIA)
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Peyman, John A
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Mayo Clinic, Rochester
United States
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Ting, Adrian T; Bertrand, Mathieu J M (2016) More to Life than NF-?B in TNFR1 Signaling. Trends Immunol 37:535-545
Legarda, Diana; Justus, Scott J; Ang, Rosalind L et al. (2016) CYLD Proteolysis Protects Macrophages from TNF-Mediated Auto-necroptosis Induced by LPS and Licensed by Type I IFN. Cell Rep 15:2449-61
Justus, Scott J; Ting, Adrian T (2015) Cloaked in ubiquitin, a killer hides in plain sight: the molecular regulation of RIPK1. Immunol Rev 266:145-60
O'Donnell, Marie Anne; Ting, Adrian T (2012) NF?B and ubiquitination: partners in disarming RIPK1-mediated cell death. Immunol Res 54:214-26
O'Donnell, Marie Anne; Hase, Hidenori; Legarda, Diana et al. (2012) NEMO inhibits programmed necrosis in an NFýýB-independent manner by restraining RIP1. PLoS One 7:e41238
O'Donnell, Marie Anne; Perez-Jimenez, Eva; Oberst, Andrew et al. (2011) Caspase 8 inhibits programmed necrosis by processing CYLD. Nat Cell Biol 13:1437-42
O'Donnell, Marie Anne; Ting, Adrian T (2011) RIP1 comes back to life as a cell death regulator in TNFR1 signaling. FEBS J 278:877-87
O'Donnell, Marie Anne; Ting, Adrian T (2010) Chronicles of a death foretold: dual sequential cell death checkpoints in TNF signaling. Cell Cycle 9:1065-71
Legarda-Addison, D; Hase, H; O'Donnell, M A et al. (2009) NEMO/IKKgamma regulates an early NF-kappaB-independent cell-death checkpoint during TNF signaling. Cell Death Differ 16:1279-88
O'Donnell, Marie Anne; Legarda-Addison, Diana; Skountzos, Penelopi et al. (2007) Ubiquitination of RIP1 regulates an NF-kappaB-independent cell-death switch in TNF signaling. Curr Biol 17:418-24

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