Jessica K. Bell

The TLR3 signaling complex forms by cooperative receptor dimerization

Toll-like receptors (TLRs) initiate immune responses by recognizing pathogen-associated molecules, but the molecular basis for recognition is poorly understood. In particular, it is unclear how receptor-ligand interactions lead to the initiation of downstream signaling. Here, we describe the mechanism by which TLR3 recognizes its ligand, double-stranded RNA (dsRNA), and forms an active signaling complex. We show that dsRNA binds saturably, specifically, and reversibly to a defined ligand-binding site (or sites) on the TLR3 ectodomain (TLR3ecd). Binding affinities increase with both buffer acidity and ligand size. Purified TLR3ecd protein is exclusively monomeric in solution, but through a highly cooperative process, it forms dimers when bound to dsRNA, and multiple TLR3ecd dimers bind to long dsRNA strands. The smallest dsRNA oligonucleotides that form stable complexes with TLR3ecd (40–50 bp) each bind one TLR3ecd dimer, and these are also the smallest oligonucleotides that efficiently activate TLR3 in cells. We conclude that TLR3 assembles on dsRNA as stable dimers and that the minimal signaling unit is one TLR3 dimer.

DEOXYHYPUSINE HYDROXYLASE IS A Fe(II)-DEPENDENT, HEAT-REPEAT ENZYME: IDENTIFICATION OF AMINO ACID RESIDUES CRITICAL FOR Fe(II) BINDING AND CATALYSIS

Deoxyhypusine hydroxylase (DOHH) catalyzes the final step in the post-translational synthesis of hypusine (Nϵ-(4-amino-2-hydroxybutyl)lysine) in eIF5A. DOHH is a HEAT-repeat protein with eight tandem helical hairpins in a symmetrical dyad. It contains two potential iron coordination sites (one on each dyad) composed of two strictly conserved His-Glu motifs. The purified human recombinant DOHH was a mixture of active holoenzyme containing 2 mol of iron/mol of DOHH and inactive metal-free apoenzyme. The two species could be distinguished by their different mobilities upon native gel electrophoresis. The DOHH apoenzyme exhibited markedly reduced levels of iron and activity. DOHH activity could be restored only by the addition of Fe2+ to the apoenzyme but not by other metals including Cd2+,Co2+,Cr2+,Cu2+,Mg2+,Mn2+,Ni2+, and Zn2+. The role of the strictly conserved His-Glu residues was evaluated by site-directed mutagenesis. Substitution of any single amino acid in the four His-Glu motifs with alanine abolished the enzyme activity. Of these eight alanine substitutions, six, including H56A, H89A, E90A, H207A, H240A, and E241A, caused a severe reduction in the iron content. Our results provide strong evidence that Fe(II) is the active-site-bound metal critical for DOHH catalysis and that the strictly conserved His-Glu motifs are essential for iron binding and catalysis. Furthermore, the iron to DOHH stoichiometry and dependence of iron binding on each of the four conserved His-Glu motifs suggest a binuclear iron mediated reaction mechanism, distinct from that of other Fe(II)-dependent protein hydroxylases, such as prolyl 4-hydroxylase or lysyl hydroxylases.

Cisplatin Enhances Protein Kinase R-Like Endoplasmic Reticulum Kinase- and CD95-Dependent Melanoma Differentiation-Associated Gene-7/Interleukin-24–Induced Killing in Ovarian Carcinoma Cells

Melanoma differentiation associated gene-7/interleukin 24 (mda-7/IL-24) is a unique interleukin (IL)-10 family cytokine displaying selective apoptosis-inducing activity in transformed cells without harming normal cells. The present studies focused on defining the mechanism(s) by which recombinant adenoviral delivery of MDA-7/IL-24 inhibits cell survival of human ovarian carcinoma cells. Expression of MDA-7/IL-24 induced phosphorylation of protein kinase R-like endoplasmic reticulum kinase (PERK) and eukaryotic initiation factor2α (eIF2α). In a PERK-dependent fashion, MDA-7/IL-24 reduced ERK1/2 and AKT phosphorylation and activated c-Jun NH2-terminal kinase (JNK) 1/2 and p38 mitogen-activated protein kinase (MAPK). MDA-7/IL-24 reduced MCL-1 and BCL-XL and increased BAX levels via PERK signaling; cell-killing was mediated via the intrinsic pathway, and cell killing was primarily necrotic as judged using Annexin V/propidium iodide staining. Inhibition of p38 MAPK and JNK1/2 abolished MDA-7/IL-24 toxicity and blocked BAX and BAK activation, whereas activation of mitogen-activated extracellular-regulated kinase (MEK) 1/2 or AKT suppressed enhanced killing and JNK1/2 activation. MEK1/2 signaling increased expression of the MDA-7/IL-24 and PERK chaperone BiP/78-kDa glucose regulated protein (GRP78), and overexpression of BiP/GRP78 suppressed MDA-7/IL-24 toxicity. MDA-7/IL-24-induced LC3-green fluorescent protein vesicularization and processing of LC3; and knockdown of ATG5 suppressed MDA-7/IL-24-mediated toxicity. MDA-7/IL-24 and cisplatin interacted in a greater than additive fashion to kill tumor cells that was dependent on a further elevation of JNK1/2 activity and recruitment of the extrinsic CD95 pathway. MDA-7/IL-24 toxicity was enhanced in a weak additive fashion by paclitaxel; paclitaxel enhanced MDA-7/IL-24 + cisplatin lethality in a greater than additive fashion via BAX. Collectively, our data demonstrate that MDA-7/IL-24 induces an endoplasmic reticulum stress response that activates multiple proapoptotic pathways, culminating in decreased ovarian tumor cell survival.

Structure of Factor H-binding Protein B (FhbB) of the Periopathogen, Treponema denticola INSIGHTS INTO PROGRESSION OF PERIODONTAL DISEASE

Background: The Treponema denticola FhbB protein binds FH, a complement regulator. Results: The structure of FhbB was solved, and its interaction with FH was further defined. Conclusion: The structurally unique FhbB protein interacts with CCP7 of FH through electrostatic interactions. Significance: The T. denticola/FH interaction may perturb complement regulation resulting in conditions that favor the development of periodontal disease. Periodontitis is the most common disease of microbial etiology in humans. Periopathogen survival is dependent upon evasion of complement-mediated destruction. Treponema denticola, an important contributor to periodontitis, evades killing by the alternative complement cascade by binding factor H (FH) to its surface. Bound FH is rapidly cleaved by the T. denticola protease, dentilisin. In this report, the structure of the T. denticola FH-binding protein, FhbB, was solved to 1.7 Å resolution. FhbB possesses a unique fold that imparts high thermostability. The kinetics of the FH/FhbB interaction were assessed using surface plasmon resonance. A KD value in the micromolar range (low affinity) was demonstrated, and rapid off kinetics were observed. Site-directed mutagenesis and sucrose octasulfate competition assays collectively indicate that the negatively charged face of FhbB binds within FH complement control protein module 7. This study provides significant new insight into the molecular basis of FH/FhbB interaction and advances our understanding of the role that T. denticola plays in the development and progression of periodontal disease.

Lateral Clustering of TLR3:dsRNA Signaling Units Revealed by TLR3ecd:3Fabs Quaternary Structure.

Toll-like receptor 3 (TLR3) recognizes dsRNA and initiates an innate immune response through the formation of a signaling unit (SU) composed of one double-stranded RNA (dsRNA) and two TLR3 molecules. We report the crystal structure of human TLR3 ectodomain (TLR3ecd) in a quaternary complex with three neutralizing Fab fragments. Fab15 binds an epitope that overlaps the C-terminal dsRNA binding site and, in biochemical assays, blocks the interaction of TLR3ecd with dsRNA, thus directly antagonizing TLR3 signaling through inhibition of SU formation. In contrast, Fab12 and Fab1068 bind TLR3ecd at sites distinct from the N- and C-terminal regions that interact with dsRNA and do not inhibit minimal SU formation with short dsRNA. Molecular modeling based on the co-structure rationalizes these observations by showing that both Fab12 and Fab1068 prevent lateral clustering of SUs along the length of the dsRNA ligand. This model is further supported by cell-based assay results using dsRNA ligands of lengths that support single and multiple SUs. Thus, their antagonism of TLR3 signaling indicates that lateral clustering of SUs is required for TLR3 signal transduction.

Innate immune agonist, dsRNA, induces apoptosis in ovarian cancer cells and enhances the potency of cytotoxic chemotherapeutics

Ovarian cancer is the most lethal gynecological cancer. Here we show that innate immune agonist, dsRNA, directly induces ovarian cancer cell death and identify biomarkers associated with responsiveness to this targeted treatment. Nuclear staining and MTT assays following dsRNA stimulation revealed two subpopulations, sensitive (OVCAR‐3, CAOV‐3; patient samples malignant 1 and 2) and resistant (DOV‐13, SKOV‐3). Microarray analysis identified 75 genes with differential expression that further delineated these two subpopulations. qPCR and immunoblot analyses showed increased dsRNA receptor expression after stimulation as compared to resistant and immortalized ovarian surface epithelial cells (e.g., 70‐fold with malignant 2, 43‐fold with OVCAR‐3). Using agonists, antagonists, and shRNA‐mediated knockdown of dsRNA receptors, we show that TLR3, RIG‐I, and mda5 coordinated a caspase 8/9‐ and interferon‐dependent cell death. In resistant cells, dsRNA receptor overexpression restored dsRNA sensitivity. When dsRNA was combined with carboplatin or paclitaxel, cell viability significantly decreased over individual treatments (1.5‐to 7.5‐fold). Isobologram analyses showed synergism in dsRNA combinations (CI=0.4–0.82) vs. an additive effect in carboplatin/paclitaxel treatment (CI= 1.5–2). Our data identify a predictive marker, dsRNA receptor expression, to target dsRNA responsive populations and show that, in dsRNA‐sensitive cells, dsRNA induces apoptosis and enhances the potency of cytotoxic chemotherapeutics.—Van, D. N., Roberts, C. F., Marion, J. D., Lépine, S., Harikumar, K. B., Schreiter, J., Dumur, C. I., Fang, X., Spiegel, S., Bell, J. K. Innate immune agonist, dsRNA, induces apoptosis in ovarian cancer cells and enhances the potency of cytotoxic chemotherapeutics. FASEB J. 26, 3188–3198 (2012). www.fasebj.org

Mechanism of endogenous regulation of the type I interferon response by suppressor of IκB kinase epsilon (SIKE), a novel substrate of TANK-binding kinase 1 (TBK1).

Background: Suppressor of IκB kinase ϵ (SIKE) inhibits a key innate immune effector molecule, TANK-binding kinase 1 (TBK1), through an undefined mechanism. Results: SIKE is a TBK1 substrate. Conclusion: SIKE controls TBK1 activity by acting as a high affinity substrate. Significance: SIKE attenuates phosphorylation of interferon regulatory factor 3 (IRF3) by serving as an alternative, high affinity substrate for TBK1. TANK-binding kinase 1 (TBK1) serves as a key convergence point in multiple innate immune signaling pathways. In response to receptor-mediated pathogen detection, TBK1 phosphorylation promotes production of pro-inflammatory cytokines and type I interferons. Increasingly, TBK1 dysregulation has been linked to autoimmune disorders and cancers, heightening the need to understand the regulatory controls of TBK1 activity. Here, we describe the mechanism by which suppressor of IKKϵ (SIKE) inhibits TBK1-mediated phosphorylation of interferon regulatory factor 3 (IRF3), which is essential to type I interferon production. Kinetic analyses showed that SIKE not only inhibits IRF3 phosphorylation but is also a high affinity TBK1 substrate. With respect to IRF3 phosphorylation, SIKE functioned as a mixed-type inhibitor (Ki, app = 350 nm) rather than, given its status as a TBK1 substrate, as a competitive inhibitor. TBK1 phosphorylation of IRF3 and SIKE displayed negative cooperativity. Both substrates shared a similar Km value at low substrate concentrations (∼50 nm) but deviated >8-fold at higher substrate concentrations (IRF3 = 3.5 μm; SIKE = 0.4 μm). TBK1-SIKE interactions were modulated by SIKE phosphorylation, clustered in the C-terminal portion of SIKE (Ser-133, -185, -187, -188, -190, and -198). These sites exhibited striking homology to the phosphorylation motif of IRF3. Mutagenic probing revealed that phosphorylation of Ser-185 controlled TBK1-SIKE interactions. Taken together, our studies demonstrate for the first time that SIKE functions as a TBK1 substrate and inhibits TBK1-mediated IRF3 phosphorylation by forming a high affinity TBK1-SIKE complex. These findings provide key insights into the endogenous control of a critical catalytic hub that is achieved not by direct repression of activity but by redirection of catalysis through substrate affinity.

Homology model and targeted mutagenesis identify critical residues for arachidonic acid inhibition of Kv4 channels.

Polyunsaturated fatty acids such as arachidonic acid (AA) exhibit inhibitory modulation of Kv4 potassium channels. Molecular docking approaches using a Kv4.2 homology model predicted a membrane-embedded binding pocket for AA comprised of the S4-S5 linker on one subunit and several hydrophobic residues within S3, S5 and S6 from an adjacent subunit. The pocket is conserved among Kv4 channels. We tested the hypothesis that modulatory effects of AA on Kv4.2/KChIP channels require access to this site. Targeted mutation of a polar residue (K318) and a nonpolar residue (G314) within the S4-S5 linker as well as a nonpolar residue in S3 (V261) significantly impaired the effects of AA on K+ currents in Xenopus oocytes. These residues may be important in stabilizing (K318) or regulating access to (V261, G314) the negatively charged carboxylate moiety on the fatty acid. Structural specificity was supported by the lack of disruption of AA effects observed with mutations at residues located near, but not within the predicted binding pocket. Furthermore, we found that the crystal structure of the related Kv1.2/2.1 chimera lacks the structural features present in the proposed AA docking site of Kv4.2 and the Kv1.2/2.1 K+ currents were unaffected by AA. We simulated the mutagenic substitutions in our Kv4.2 model to demonstrate how specific mutations may disrupt the putative AA binding pocket. We conclude that AA inhibits Kv4 channel currents and facilitates current decay by binding within a hydrophobic pocket in the channel in which K318 within the S4-S5 linker is a critical residue for AA interaction.

Predicting toll-like receptor structures and characterizing ligand binding

Toll-like receptor (TLR) ligand-binding domains comprise 18-25 tandem copies of a 24-residue motif known as the leucine-rich repeat (LRR). Unlike other LRR proteins, TLRs contain significant numbers of non-consensus LRR sequences, which makes their identification by computer domain search programs problematic. Here, we provide methods for identifying non-consensus LRRs. Using the location of these LRRs, hypothetical models are constructed based on the known molecular structures of homologous LRR proteins. However, when a hypothetical model for TLR3 is compared with the molecular structure solved by x-ray crystallography, the solenoid curvature, planarity, and conformations of the LRR insertions are incorrectly predicted. These differences illustrate how non-consensus LRR motifs influence TLR structure. Since the determination of molecular structures by crystallography requires substantial amounts of protein, we describe methods for producing milligram amounts of TLR3 extracellular domain (ECD) protein. The recombinant TLR3-ECD previously used to solve the molecular structure of TLR3-ECD has also been used to study the binding of TLR3-ECD to its ligand, double-stranded RNA (dsRNA). In the last section, we describe the preparation of defined TLR3 ligands and present methods for characterizing their interaction with TLR3-ECD.

Measuring the effect of ligand binding on the interface stability of multimeric proteins using dynamic light scattering

We have demonstrated that an approach using guanidine hydrochloride at low concentrations to progressively disrupt protein-protein interactions can be quantitated using dynamic light scattering. This approach is sensitive enough to detect ligand-induced changes of subunit-subunit interactions for homo-hexameric glutamate dehydrogenase, allowing ΔΔG of reversible subunit dissociation to be calculated. The use of dynamic light scattering makes this approach generally applicable to soluble proteins to monitor the relative strength of protein-protein interactions with a particular emphasis on assessing the impact of ligand binding on such interfaces.