Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation.

Activation of nuclear factor-kappaB (NF-kappaB), a key mediator of inducible transcription in immunity, requires binding of NF-kappaB essential modulator (NEMO) to ubiquitinated substrates. Here, we report that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO selectively binds linear (head-to-tail) ubiquitin chains. Crystal structures of the UBAN motif revealed a parallel coiled-coil dimer that formed a heterotetrameric complex with two linear diubiquitin molecules. The UBAN dimer contacted all four ubiquitin moieties, and the integrity of each binding site was required for efficient NF-kappaB activation. Binding occurred via a surface on the proximal ubiquitin moiety and the canonical Ile44 surface on the distal one, thereby providing specificity for linear chain recognition. Residues of NEMO involved in binding linear ubiquitin chains are required for NF-kappaB activation by TNF-alpha and other agonists, providing an explanation for the detrimental effect of NEMO mutations in patients suffering from X-linked ectodermal dysplasia and immunodeficiency.

Fluorescence-based sensors to monitor localization and functions of linear and K63-linked ubiquitin chains in cells.

Ubiquitin chains modify a major subset of the proteome, but detection of ubiquitin signaling dynamics and localization is limited due to a lack of appropriate tools. Here, we employ ubiquitin-binding domain (UBD)-based fluorescent sensors to monitor linear and K63-linked chains in vitro and in vivo. We utilize the UBD in NEMO and ABIN (UBAN) for detection of linear chains, and RAP80 ubiquitin-interacting motif (UIM) and TAB2 Npl4 zinc finger (NZF) domains to detect K63 chains. Linear and K63 sensors decorated the ubiquitin coat surrounding cytosolic Salmonella during bacterial autophagy, whereas K63 sensors selectively monitored Parkin-induced mitophagy and DNA damage responses in fixed and living cells. In addition, linear and K63 sensors could be used to monitor endogenous signaling pathways, as demonstrated by their ability to differentially interfere with TNF- and IL-1-induced NF-κB pathway. We propose that UBD-based biosensors could serve as prototypes to track and trace other chain types and ubiquitin-like signals in vivo.

A20 inhibits LUBAC-mediated NF-κB activation by binding linear polyubiquitin chains via its zinc finger 7.

Linear polyubiquitination of proteins has recently been implicated in NF-κB signalling and is mediated by the linear ubiquitin chain assembly complex (LUBAC), consisting of HOIL-1, HOIP and Sharpin. However, the mechanisms that regulate linear ubiquitination are still unknown. Here, we show that A20 is rapidly recruited to NEMO and LUBAC upon TNF stimulation and that A20 inhibits LUBAC-induced NF-κB activation via its C-terminal zinc-finger 7 (ZF7) domain. Expression of a polypeptide corresponding to only ZF7 was sufficient to inhibit TNF-induced NF-κB activation. Both A20 and ZF7 can form a complex with NEMO and LUBAC, and are able to prevent the TNF-induced binding of NEMO to LUBAC. Finally, we show that ZF7 preferentially binds linear polyubiquitin chains in vitro, indicating A20-ZF7 as a novel linear ubiquitin-binding domain (LUBID). We thus propose a model in which A20 inhibits TNF- and LUBAC-induced NF-κB signalling by binding to linear polyubiquitin chains via its seventh zinc finger, which prevents the TNF-induced interaction between LUBAC and NEMO.

Analysis of nuclear factor-κB (NF-κB) essential modulator (NEMO) binding to linear and lysine-linked ubiquitin chains and its role in the activation of NF-κB.

Nuclear factor-κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of κB kinase (IKK) complex, controls NF-κB signaling by binding to ubiquitin chains. Structural studies of NEMO provided a rationale for the specific binding between the UBAN (ubiquitin binding in ABIN and NEMO) domain of NEMO and linear (Met-1-linked) di-ubiquitin chains. Full-length NEMO can also interact with Lys-11-, Lys-48-, and Lys-63-linked ubiquitin chains of varying length in cells. Here, we show that purified full-length NEMO binds preferentially to linear ubiquitin chains in competition with lysine-linked ubiquitin chains of defined length, including long Lys-63-linked deca-ubiquitins. Linear di-ubiquitins were sufficient to activate both the IKK complex in vitro and to trigger maximal NF-κB activation in cells. In TNFα-stimulated cells, NEMO chimeras engineered to bind exclusively to Lys-63-linked ubiquitin chains mediated partial NF-κB activation compared with cells expressing NEMO that binds to linear ubiquitin chains. We propose that NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. This phenomenon could explain quantitatively distinct NF-κB activation patterns in response to numerous cell stimuli.

Human Wrnip1 is localized in replication factories in a ubiquitin-binding zinc finger-dependent manner.

Wrnip1 (Werner helicase-interacting protein 1) has been implicated in the bypass of stalled replication forks in bakers' yeast. However, the function(s) of human Wrnip1 has remained elusive so far. Here we report that Wrnip1 is distributed inside heterogeneous structures detectable in nondamaged cells throughout the cell cycle. In an attempt to characterize these structures, we found that Wrnip1 resides in DNA replication factories. Upon treatments that stall replication forks, such as UVC light, the amount of chromatin-bound Wrnip1 and the number of foci significantly increase, further implicating Wrnip1 in DNA replication. Interestingly, the nuclear pattern of Wrnip1 appears to extend to a broader landscape, as it can be detected in promyelocytic leukemia nuclear bodies. The presence of Wrnip1 into these heterogeneous subnuclear structures requires its ubiquitin-binding zinc finger (UBZ) domain, which is able to interact with different ubiquitin (Ub) signals, including mono-Ub and chains linked via lysine 48 and 63. Moreover, the oligomerization of Wrnip1 mediated by its C terminus is also important for proper subnuclear localization. Our study is the first to reveal the composite and regulated topography of Wrnip1 in the human nucleus, highlighting its potential role in replication and other nuclear transactions.