Exclusivity and Compensation in NFκB Dimer Distributions and IκB Inhibition.

The NFκB transcription factor family members RelA, p50, and cRel form homo- and heterodimers that are inhibited by IκBα, IκBβ, and IκBε. These NFκB family members have diverse biological functions, and their expression profiles differ, leading to different concentrations in different tissue types. Here we present definitive biophysical measurements of the NFκB dimer affinities and inhibitor affinities to better understand dimer exchange and how the presence of inhibitors may alter the equilibrium concentrations of NFκB dimers in the cellular context.

Suppressor of IKKepsilon forms direct interactions with cytoskeletal proteins, tubulin and α-actinin, linking innate immunity to the cytoskeleton.

Unlabelled: Suppressor of IKKepsilon (SIKE) is associated with the type I interferon response of the innate immune system through TANK-binding kinase 1 (TBK1). Originally characterized as an endogenous inhibitor of TBK1 when overexpressed in viral infection and pathological cardiac hypertrophic models, a mechanistic study revealed that SIKE acts as a high-affinity substrate of TBK1, but its function remains unknown. In this work, we report that scratch assay analysis of parental and SIKE CRISPR/Cas9 knockout HAP1 cells showed an ~ 20% decrease in cell migration.

Cyclic-di-GMP binding induces structural rearrangements in the PlzA and PlzC proteins of the Lyme disease and relapsing fever spirochetes: a possible switch mechanism for c-di-GMP-mediated effector functions.

The c-di-GMP network of Borrelia burgdorferi, a causative agent of Lyme disease, consists of Rrp1, a diguanylate cyclase/response regulator; Hpk1, a histidine kinase; PdeA and PdeB, c-di-GMP phosphodiesterases; and PlzA, a PilZ domain c-di-GMP receptor. Borrelia hermsii, a causative agent of tick-borne relapsing fever, possesses a putative c-di-GMP regulatory network that is uncharacterized. While B.

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).

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.

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.

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).

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.