Structure of fish TRAF4 and its implication in TRAF4-mediated immune cell and platelet signaling.

Due to an increasing interest in immunity and signal transduction in teleost fish, important key signaling molecules associated with the immune response, including TRAF molecules, have been recently cloned and characterized. To better understand the role of TRAF4 in fish immune signaling and compare it with the human system, our study cloned the TRAF4 gene from the Antarctic yellowbelly rockcod Notothenia coriiceps (ncTRAF4) and purified the protein. Here, we report the first crystal structure of teleost fish TRAF4. Based on biochemical characterization, our findings elucidated the mechanisms through which signaling molecules gain cold adaptivity. Additionally, we identified a platelet receptor GPIbß homolog in N. coriiceps (ncGPIbß) and found that the "RRFERLFKEARRTS" region of this homolog directly binds to ncTRAF4, indicating that ncTRAF4 also recognizes the "RLXA" motif for receptor interactions and further TARF4-mediated cellular signaling. Collectively, our findings provide novel insights into the mechanisms of TRAF4-mediated immune cell and platelet signaling in fish and the structural flexibility-mediated cold adaptiveness of signaling molecules.

TRAF4 binds to the juxtamembrane region of EGFR directly and promotes kinase activation.

The activation of the epidermal growth factor receptor (EGFR) is crucial for triggering diverse cellular functions, including cell proliferation, migration, and differentiation, and up-regulation of EGFR expression or activity is a key factor in triggering the development of cancer. Here we show that overexpression of a scaffold protein, tumor necrosis factor receptor (TNF-R)-associated factor 4 (TRAF4), promotes EGF-induced autophosphorylation of EGFR (activation) and downstream signaling, whereas TRAF4 deficiency attenuates EGFR activation and EGF-driven cell proliferation. Using structure-based sequence alignment and NMR spectroscopy, we identified a TRAF4 binding site in the C-terminal half of the juxtamembrane (JM) segment of EGFR, a region known to promote asymmetric dimerization and subsequent activation. Deletion of the TRAF4 binding site led to dramatic defects in EGFR activation and EGF-driven cell proliferation. Specific point mutations in the TRAF4 binding site also resulted in significant attenuation of EGFR activation. Detailed structural examination of the inactive versus active forms of EGFR suggests that TRAF4 binding probably induces a conformational rearrangement of the JM region to promote EGFR dimerization. These results identify a novel mechanism of TRAF4-mediated EGFR activation and signaling.

Molecular basis for unique specificity of human TRAF4 for platelets GPIbß and GPVI.

Tumor necrosis factor (TNF)-receptor associated factor 4 (TRAF4), an adaptor protein with E3-ligase activity, is involved in embryogenesis, cancer initiation and progression, and platelet receptor (GPIb-IX-V complex and GPVI)-mediated signaling for reactive oxygen species (ROS) production that initiates thrombosis at arterial shears. Disruption of platelet receptors and the TRAF4 interaction is a potential target for therapeutic intervention by antithrombotic drugs. Here, we report a crystal structure of TRAF4 (amino acid residues 290∼470) in complex with a peptide from the GPIbß receptor (amino acid residues 177∼181). The GPIbß peptide binds to a unique shallow surface composed of two hydrophobic pockets on TRAF4. Further studies revealed the TRAF4-binding motif Arg-Leu-X-Ala. The TRAF4-binding motif was present not only in platelet receptors but also in the TGF-ß receptor. The current structure will provide a template for furthering our understanding of the receptor-binding specificity of TRAF4, TRAF4-mediated signaling, and related diseases.

Structure of the TRAF4 TRAF domain with a coiled-coil domain and its implications for the TRAF4 signalling pathway.

The TNF receptor-associated factor (TRAF) proteins are structurally similar scaffold proteins that mediate between members of the TNF receptor (TNFR) family and downstream effector molecules such as kinases in the immune signalling pathway. Seven TRAFs have been identified, including TRAF4, which is a unique member that participates in many ontogenic processes, including nerve-system development. TRAFs commonly contain the TRAF domain, which mediates interaction with target receptors and effectors. As a first step towards elucidating the molecular mechanisms of the TRAF4-mediated signalling pathway, the first crystal structure of the human TRAF4 TRAF domain with a coiled-coil domain is reported at 2.3 Šresolution.

Preliminary X-ray crystallographic studies of the short form of the human TRAF4 TRAF domain.

TNF (tumour necrosis factor) receptor-associated factor 4 (TRAF4) is a unique TRAF protein that participates in morphogenetic and developmental function and cell migration. TRAF-family proteins contain a TRAF domain for target interaction. In this study, the short form of the human TRAF4 TRAF domain, corresponding to amino acids 290-462, was overexpressed in Escherichia coli using engineered C-terminal His tags. The short form of the TRAF4 TRAF domain was purified to homogeneity and crystallized at 293 K. Finally, X-ray diffraction data were collected to a resolution of 4.2 Å from a crystal belonging to the hexagonal space group P32, with unit-cell parameters a = b = 147.17, c = 202.69 Å.

Structural biology study of human TNF receptor associated factor 4 TRAF domain.

TRAF4 is a unique member of TRAF family, which is essential for innate immune response, nervous system and other systems. In addition to be an adaptor protein, TRAF4 was identified as a regulator protein in recent studies. We have determined the crystal structure of TRAF domain of TRAF4 (residues 292-466) at 2.60 Å resolution by X-ray crystallography method. The trimericly assembled TRAF4 resembles a mushroom shape, containing a super helical "stalk" which is made of three right-handed intertwined α helixes and a C-terminal "cap", which is divided at residue L302 as a boundary. Similar to other TRAFs, both intermolecular hydrophobic interaction in super helical "stalk" and hydrogen bonds in "cap" regions contribute directly to the formation of TRAF4 trimer. However, differing from other TRAFs, there is an additional flexible loop (residues 421-426), which contains a previously identified phosphorylated site S426 exposing on the surface. This S426 was reported to be phosphorylated by IKKα which is the pre-requisite for TRAF4-NOD2 complex formation and thus to inhibit NOD2-induced NF-κB activation. Therefore, the crystal structure of TRAF4-TRAF is valuable for understanding its molecular basis for its special function and provides structural information for further studies.

Preliminary X-ray crystallographic studies of the TRAF domain of human TRAF4.

TNF receptor-associated factor (TRAF) proteins were initially identified as tumour necrosis factor receptor (TNFR)-interacting proteins that perform critical functions in the regulation of inflammation, antiviral responses and apoptosis. Although TRAF4 is a canonical TRAF protein, it contains a unique domain boundary and functions differently in the cell. In this study, the human TRAF4 TRAF domain, corresponding to amino acids 290-470, was overexpressed in Escherichia coli using engineered C-terminal His tags. The TRAF4 TRAF domain was then purified to homogeneity and crystallized at 293 K. Finally, X-ray diffraction data were collected to a resolution of 2.3 Šfrom a crystal belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 58.9, b = 87.9, c = 117.3 Å, α = ß = γ = 90°.

IκB kinase α phosphorylation of TRAF4 downregulates innate immune signaling.

Despite their homology, IκB kinase α (IKKα) and IKKß have divergent roles in NF-κB signaling. IKKß strongly activates NF-κB while IKKα can downregulate NF-κB under certain circumstances. Given this, identifying independent substrates for these kinases could help delineate their divergent roles. Peptide substrate array technology followed by bioinformatic screening identified TRAF4 as a substrate for IKKα. Like IKKα, TRAF4 is atypical within its family because it is the only TRAF family member to negatively regulate innate immune signaling. IKKα's phosphorylation of serine-426 on TRAF4 was required for this negative regulation. Binding to the Crohn's disease susceptibility protein, NOD2, is required for TRAF4 phosphorylation and subsequent inhibition of NOD2 signaling. Structurally, serine-426 resides within an exaggerated ß-bulge in TRAF4 that is not present in the other TRAF proteins, and phosphorylation of this site provides a structural basis for the atypical function of TRAF4 and its atypical role in NOD2 signaling.

Genomic and functional uniqueness of the TNF receptor-associated factor gene family in amphioxus, the basal chordate.

The TNF-associated factor (TRAF) family, the crucial adaptor group in innate immune signaling, increased to 24 in amphioxus, the oldest lineage of the Chordata. To address how these expanded molecules evolved to adapt to the changing TRAF mediated signaling pathways, here we conducted genomic and functional comparisons of four distinct amphioxus TRAF groups with their human counterparts. We showed that lineage-specific duplication and rearrangement were responsible for the expansion of amphioxus TRAF1/2 and 3 lineages, whereas TRAF4 and 6 maintained a relatively stable genome and protein structure. Amphioxus TRAF1/2 and 3 molecules displayed various expression patterns in response to microbial infection, and some of them can attenuate the NF-kappaB activation mediated by human TRAF2 and 6. Amphioxus TRAF4 presented two unique functions: activation of the NF-kappaB pathway and involvement in somite formation. Although amphioxus TRAF6 was conserved in activating NF-kappaB pathway for antibacterial defense, the mechanism was not the same as that observed in humans. In summary, our findings reveal the evolutionary uniqueness of the TRAF family in this basal chordate, and suggest that genomic duplication and functional divergence of the TRAF family are important for the current form of the TRAF-mediated signaling pathways in humans.

Tumor necrosis factor-receptor-associated factor-4 is a positive regulator of transforming growth factor-beta signaling that affects neural crest formation.

The transforming growth factor (TGF)-beta superfamily regulates cell proliferation, apoptosis, differentiation, migration, and development. Canonical TGFbeta signals are transduced to the nucleus via Smads in both major signaling branches, bone morphogenetic protein (BMP) or Activin/Nodal/TGFbeta. Smurf ubiquitin (Ub) ligases attenuate these pathways by targeting Smads and other signaling components for degradation by the 26S proteasome. Here, we identify tumor necrosis factor (TNF)-receptor-associated factor-4 (TRAF4) as a new target of Smurf1, which polyubiquitylates TRAF4 to trigger its proteasomal destruction. Unlike other TRAF family members, which mediate signal transduction by TNF, interleukin, or Toll-like receptors, we find that TRAF4 potentiates BMP and Nodal signaling. In the frog Xenopus laevis, TRAF4 mRNA is stored maternally in the egg animal pole, and in the embryo it is expressed in the gastrula marginal zone, neural plate, and cranial and trunk neural crest. Knockdown of embryonic TRAF4 impairs signaling, neural crest development and neural folding, whereas TRAF4 overexpression boosts signaling and expands the neural crest. In human embryonic kidney 293 cells, small interfering RNA knockdown of Smurf1 elevates TRAF4 levels, indicating endogenous regulation of TRAF4 by Smurf1. Our results uncover new functions for TRAF4 as a Smurf1-regulated mediator of BMP and Nodal signaling that are essential for neural crest development and neural plate morphogenesis.

TRAF4, the unique family member.

The fourth member of the TRAF protein family (TRAF4) presents several characteristics that distinguish it from the other members of the family. These characteristics concern the primary sequence of the protein, a strong evolutionary conservation, and a tightly regulated physiological expression during development. The subcellular localization of TRAF4 is controversial as it has been detected at the cell membrane, in the cytoplasm and in the nucleus. Using mouse and fly models, it has been established that TRAF4 is a key molecule in diverse ontogenic processes, particularly in the nervous system. However, the molecular mechanisms of action of TRAF4 remain evasive as it was found to interact with diverse types of proteins, leading either to pro-apoptotic or anti-apoptotic functions. Finally, few studies implicated TRAF4 in human diseases.