Ubiquitin-specific Peptidase 21 Inhibits Tumor Necrosis Factor α-induced Nuclear Factor κB Activation via Binding to and Deubiquitinating Receptor-interacting Protein 1

Abstract

Ubiquitination and deubiquitination of receptor-interacting protein 1 (RIP1) play an important role in the positive and negative regulation of the tumor necrosis factor ␣ (TNF␣)-induced nuclear factor B (NF-B) activation. Using a combination of functional genomic and proteomic approaches, we have identified ubiquitin-specific peptidase 21 (USP21) as a deubiquitinase for RIP1. USP21 is constitutively associated with RIP1 and deubiquitinates RIP1 in vitro and in vivo. Notably, knockdown of USP21 in HeLa cells enhances TNF␣-induced RIP1 ubiquitination, IB kinase ␤ (IKK␤), and NF-B phosphorylation, inhibitor of NF-B ␣ (IB␣) phosphorylation and ubiquitination, as well as NF-B-dependent gene expression. Therefore, our results demonstrate that USP21 plays an important role in the down-regulation of TNF␣-induced NF-B activation through deubiquitinating RIP1. Transcription factor nuclear factor B (NF-B)3 plays an important role in controlling the expression of survival factors, cytokines, and proinflammatory molecules in a broad range of cellular responses (1-3). NF-B is sequestered in the cytoplasm by a family of inhibitory proteins called inhibitor of NF-B (IB) proteins in inactivated cells. Many intercellular stimuli are capable of triggering the activation of a signal transduction pathway that leads to the degradation of IB proteins through the 26 S proteasome (4 -6). Degradation of the IB proteins allows NF-B translocation from cytoplasm to the nucleus and activates the expression of the target genes (7).Upon binding of tumor necrosis factor ␣ (TNF␣), TNF receptor 1 (TNFR1) recruits several adaptor proteins, including receptorinteracting protein 1 (RIP1/RIPK1) and TNF receptor-associated factor 2 (TRAF2), to form a complex (8, 9). This TNFR1-associated complex initiates the activation of IB kinase (IKK), which phosphorylates IB protein and activates NF-B (10 -17).Protein ubiquitination is a crucial regulatory mechanism in various cellular processes, including cell cycle progression, the DNA damage response, and immune responses (18 -20). In the TNF␣-induced NF-B signal transduction pathway, the Lys 63 -linked polyubiquitination of RIP1 protein mediated by TRAF2 E3 ligase is essential for TNF␣-induced IKK/NF-B activation, whereas phosphorylation of the IB proteins by activated IKK leads to their Lys 48 -linked polyubiquitination, which labels it for its degradation by the 26 S proteasome (21).Several deubiquitinating enzymes, including CYLD, A20, Cezanne, ubiquitin-specific peptidase 15 (USP15), and USP31, have been suggested to be involved in the down-regulation of TNF␣-induced NF-B activation (22-26). However, it remains unclear how deubiquitination plays a role in the down-regulation of TNF␣-induced NF-B activation.The USPs belong to a subclass of the protein-deubiquitinating enzyme (DUB) superfamily that are categorized into five subclasses based on their ubiquitin-protease domains in the human genome and have been shown to be involved in a broad range of biological activities (27). Even though the USP subclass o...

The deubiquitinating enzyme CYLD controls apical docking of basal bodies in ciliated epithelial cells

“…USP 21 is a good candidate for carrying this function, as it has recently been shown to localize at the centrosome and affect primary cilia formation 50 . Moreover, it shares a similar target with CYLD in the regulation NF-kB signalling pathway 51 .…”

The roles of FADD in extrinsic apoptosis and necroptosis

“…Similar to apoptosis induction, necroptosis also requires the removal of the K63-linked polyubiquitin chain of RIP1, which provides a docking site for TAK/TAB and IKK complexes and induces the NF-kB pathway (42,43,85). CYLD is required for necroptosis induction by deubiquitinating RIP1, whereas the involvements of other deubiquitinating enzymes that trigger RIP1 deubiquitination such as A20, USP21 and cezanne in necroptosis induction are currently unknown (46)(47)(48)86). Inhibition of cIAP function, which is critical for RIP1 polyubiquitination, by genetic deletion or pharmacological methods, has been reported to sensitize cells to necroptotic cell death by preventing RIP1 K63-linked ubiquitination (49,78,(87)(88)(89)(90).…”

“…(OTUB7B) are also able to induce complex II formation (45)(46)(47)(48). The conformation changes of complex I after receptor internalization result in two kinds of cytosolic complex II (i.e., TRADD-dependent complex IIA and RIP1-dependent complex IIB), both of which can initiate apoptosis (38,44).…”

A20 and CYLD Do Not Share Significant Overlapping Functions during B Cell Development and Activation

“…Several deubiquitinases (DUBs) have been reported to negatively regulate NF-kB. Among them are A20, CYLD, Cezanne, and USP21 (3)(4)(5)(6).…”