Abstract
The mammalian tumor necrosis factor (TNF) cytokine is a central mediator of inflammatory events. Recent studies revealed a number of complex and sophisticated interactions between the TNF pathway and the enzymatic activities encoded by ubiquitin ligases and deubiquitylation enzymes. However, very little is known about the identity of the ubiquitin pathway members that control the extent of ubiquitylation in TNF responses. To address this deficit, we conducted an unbiased, high-content screen of the human ubiquitin pathway for gene products that control defining features of the cellular response to TNF. In particular, we sought to identify ubiquitin modifying enzymes that alter the ability of TNF to regulate the nuclear accumulation of nuclear factor kappa B. In this screen, we identified and validated several novel regulators of the TNF pathway. We believe these regulators constitute potential targets for pharmacological interventions that manipulate TNF-dependent inflammation.
Highlights
The mammalian tumor necrosis factor (TNF) cytokine directs immunological responses to pathogenic microbes and is a central feature of numerous inflammatory illnesses [1]
In an unbiased siRNA screen of the entire complement of deubiquitylation enzymes, and E1, E2, and E3 ubiquitin ligases in the human genome, we identified a number of enzymes that modify the extent to which TNF directs the nuclear accumulation of NFκB
A HIGH-CONTENT ASSAY FOR THE NUCLEAR LOCALIZATION OF NF-κB We developed a rapid and sensitive high-content assay to identify ubiquitin modifiers that regulate intracellular signals in response to TNF (Figure 1A)
Summary
The mammalian tumor necrosis factor (TNF) cytokine directs immunological responses to pathogenic microbes and is a central feature of numerous inflammatory illnesses [1]. Given the critical links between cytokine signaling, immunity, and disease, there is considerable interest in the molecular pathways that translate detection of a cytokine into induction of a host response. Myeloid cells such as dendritic cells, macrophages, and mast cells release soluble TNF homotrimers in response to the molecular signatures of pathogenic invasion [6]. The release of SODD exposes TNFR death domains that nucleate a complex arrangement of adaptor proteins, kinases, and ubiquitin ligases. Complex I contains TNFR, TRADD, the adaptor molecule receptor-interacting serine/threonine–protein kinase-1 [RIPK1 [10]], the ubiquitin ligase TNF receptor associated factor 2 [TRAF2 [11]], and the ubiquitin ligase cellular inhibitor of apoptosis proteins 1 and 2 [c-IAP1/2 [12]]
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