Suppressor of IKK epsilon ( SIKE ) is associated with the type I interferon response of the innate immune system through TANK ‐binding kinase 1 ( TBK 1). Originally characterized as an endogenous inhibitor of TBK 1 when overexpressed in viral infection and pathological cardiac hypertrophic models, a mechanistic study revealed that SIKE acts as a high‐affinity substrate of TBK 1, but its function remains unknown. In this work, we report that scratch assay analysis of parental and SIKE CRISPR /Cas9 knockout HAP 1 cells showed an ~ 20% decrease in cell migration. Investigation of the SIKE interaction network through affinity purification/mass spectrometry showed that SIKE formed interactions with cytoskeletal proteins. In immunofluorescence assays, endogenous SIKE localized to cytosolic puncta in both epithelial and myeloid cells and to nuclear puncta in myeloid cells, while in epithelial cells additional staining occurred in stress fiber‐like structures and adjacent to the plasma membrane. Using cellular markers, co‐occurrence of SIKE fluorescence with actin, α‐actinin, and ezrin was detected. Reciprocal immunoprecipitation revealed a SIKE :tubulin interaction sensitive to the phosphorylation state of SIKE , but a SIKE :α‐actinin interaction was unchanged by SIKE phosphorylation. In vitro precipitation assays confirmed a direct SIKE interaction with tubulin and α‐actinin. These results indicate that SIKE may promote cell migration by directly associating with the cytoskeleton. In this role, SIKE may mediate cytoskeletal rearrangement necessary in innate immunity, but also link a key catalytic hub, TBK 1, to the cytoskeleton. Database The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD 007262.

Suppressor of IKKepsilon (SIKE) is a 207 residue protein that is implicated in the TLR3-TANKbinding kinase-1-mediated response to viral infection. SIKE's function in this pathway is unknown, but SIKE forms interactions with two distinct cytoskeletal proteins, α-actinin and tubulin, and SIKE knockout reduces cell migration. As structure informs function and in the absence of solved structural homologs, our studies were directed toward creating a structural model of SIKE through biochemical and biophysical characterization to probe and interrogate SIKE function. Circular dichroism revealed a primarily (73%) helical structure of minimal stability ( =32 C) but reversibly denatured. Limited proteolysis (LP) and chemical modification identified the N-terminal 2/3 of the protein as dynamic and accessible, whereas size exclusion chromatography (SEC) confirmed three homo-oligomeric species. SEC coupled to chemical crosslinking characterized the primary species as dimeric, a secondary hexameric species, and a higher order aggregate/polymer. Fluorescence polarization using intrinsic tryptophan fluorescence contextualized the anisotropy value for the SIKE dimer (molecular weight 51.8 kDa) among proteins of known structure, bovine serum albumin (BSA; 66 kDa), and glutamate dehydrogenase (GDH; 332 kDa). Radii of gyration for BSA and GDH provided exclusionary values for SIKE tertiary and dimeric quaternary models that otherwise conformed to secondary structure, LP, and modification data. Dimeric quaternary models were further culled using acrylamide quenching data of SIKE's single tryptophan that showed a single, protected environment. The low cooperativity of folding and regions of dynamic and potentially disordered structure advance the hypothesis that SIKE forms a conformational ensemble of native states that accommodate SIKE's interactions with multiple, distinct protein-binding partners.Additional Supporting Information may be found in the online version of this article.Significance Statement: This work describes the biochemical and biophysical characterization of suppressor of IKKepsilon (SIKE) to support an alpha helical, dimeric structural model. The low cooperativity of folding and regions of dynamic and potentially disordered structure advance the hypothesis that SIKE forms a conformational ensemble of native states that accommodate SIKE's interactions with multiple, distinct protein-binding partners.

The innate immune system rapidly responds to challenges by pathogens via activation of an inflammatory response. As a convergence point for multiple inflammatory and anti‐viral signaling pathways, TANK binding kinase 1 (TBK1) serves as a catalytic hub to initiate host defenses. Suppressor of IKK epsilon (SIKE) is a newly identified TBK1 substrate. The goal of this project was to identify the function of SIKE within the host's anti‐viral response. Co‐immunoprecipitation – tandem MS/MS analyses of the SIKE interaction network identified several interactions with cytoskeletal components. Immunofluorescence assays of endogenous SIKE with cytoskeletal markers and colocalization analyses indicated that SIKE colocalized with tubulin, actin, and a‐actinin. Reciprocal immunoprecipitation (RcIPs) studies confirmed the SIKE:tubulin and SIKE:a‐actinin interaction that appeared enhanced following dsRNA stimulation, a mimic of viral infection. ELISA assays were used to characterize the binding affinity between SIKE and the cytoskeletal proteins under unstimulated and simulated viral‐stimulated conditions. The SIKE:actin interaction was not detected by RcIP. In vitro immunoprecipitation assays mapped direct interactions between SIKE:tubulin and SIKE:a‐actinin and were used to further probe for a weak SIKE:actin interaction. Assays employed purified cytoskeletal proteins and full‐length (residues 1–207), N‐terminal (residues 1–112), C – terminal (residues 113–207) or a phosphomimetic (S6E) of a 6x‐His‐tagged SIKE construct. Together these studies establish an interaction between SIKE and cytoskeletal proteins that may provide a direct link between innate immune signaling and cytoskeletal components.Support or Funding InformationThis work was supported by NIH grant R21AI107447 and the University of San Diego SURE program.

Upon pathogen challenge multiple receptors both inside and on the surface of the cell, recognize pathogen associated molecular patterns (PAMPs) and initiate the production of proinflammatory, antiviral, and apoptotic responses. Pathways converge at key hubs that serve to amplify and regulate the signals, and are often responsible for determining the downstream response. TANK Binding Kinase 1 (TBK1) serves as a catalytic hub in the antiviral TLR3 mediated innate immune pathway. Suppressor of IKK epsilon (SIKE) is a recently identified high affinity alternative substrate of TBK1. It was initially found to inhibit TBK1 activation of type 1 interferon production. Upon subsequent study, it was found that SIKE was phosphorylated at six serine residues by TBK1. This phosphorylation of SIKE corresponds to the activation of the antiviral response, and releases SIKE from the SIKE:TBK1 interaction. The primary function of SIKE remains unknown. Examination of SIKE's interaction network has established direct interactions with cytoskeletal proteins including tubulin and α‐actinin. Migration assays have shown that chronic myelogenous leukemia (CML) cells in which SIKE has been knocked out migrate at a slower rate. Together, these studies suggest that SIKE plays a role in cytoskeletal rearrangements associated with innate immune responses such as migration and phagocytosis. The goal of this study is to define the interaction surface of SIKE as well as the binding affinities associated with these interactions. A quartz crystal microbalance with dissipation (QCM‐D) assay was developed to obtain binding affinities for the SIKE:cytoskeletal protein complexes. A gold sensor was utilized and functionalized with protein G and α‐His antibody to which 6xHis‐SIKE was immobilized. Increasing concentrations of binding partner were flowed over the chip to develop a binding curve. Prior to examining SIKE:cytoskeletal protein interactions, SIKE's oligomeric state was defined by crosslinking studies. Crosslinking with bis(sulfosuccinimidyl)suberate (BS3) shows that SIKE is a dimer. Chemical crosslinking followed by tandem mass spectrometry was employed to determine the dimeric interface of the SIKE dimer. BS3 was used in an excess of 20 and 100X. Data were analyzed with Mascot using xComb strategy. Residues 13, 66, 68, 110, and 169 were found to be accessible to BS3 modification, while in the dimer only residues 13, 110, 119, and 120 were found to be accessible. Residues 110 and 119 are found in multiple crosslinked peptides in both dimer and monomer samples. Residues 178 and 195 were found in crosslinked peptides unique to the dimer. These data have been applied to SIKE dimer models, generated by computational docking experiments to select for models consistent with the crosslinking restraints. These data define the dimer interface of SIKE as well as the surfaces available for SIKE cytoskeletal protein interactions. Together, these studies begin to reveal parameters that determine SIKE's interactions and the protein surfaces available to mediate these interactions.Support or Funding InformationStudies were funded by USD SURE, Beckman Foundation, NIH‐NIAID R21.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The focus of this project was to explore Structure Function relationships in the enzyme UDP‐GlcNAc O transferase protein and design and build a physical model that illustrates key functional features of the protein. In particular our focus is on the Cross‐Talk between O‐GlcNAcylation and Phosphorylation that plays a critical role in overall regulation. Using the pdb file 4gyy.pdb from the paper “Crystal structure of human O‐GlcNAc Transferase with UDP‐5SGlcNAc and a peptide substrate” The structure illustrates the binding sites for both UDPGlcNAc and the peptide YPGGSTPVSSANMM with the requisite PV prior to the GlcNAcylatable S. Recent work by Leney et al (Proc Natl Acad Sci U S A. 2017 Aug 29;114(35):E7255–E7261) has shown that the preceding T can be phosphorylated and that the sequence TPVS is common in proteins thought to undergo GlcNAc‐Phosphorylation cross talk. We have also used computational models to create and built models of the peptide phosphorylated at the N‐3 Threonine. These models clearly illustrate that the phosphate group clashes with the U in UDP‐GlcNAC. Since the kinetic mechanism of OGT is ordered Bi‐Bi with UDPGlcNAc as the obligate first substrate, the phosphorylated peptide can no longer bind in the peptide substrate pocket explaining why excludes GlcNAcylation. The models described here can be used to illustrate three different aspects contained in the ASBMB 8 Core Concepts of Macromolecular Structure and Function: #3. Structure and function are related, #4. Macromolecular interactions, and #6. The biological activity of macromolecules is often regulated. In addition construction and use of the models illustrates the use of core concept #8, A variety of experimental and computational approaches can be used to observe and quantitatively measure the structure, dynamics and function of biological macromolecule.Support or Funding InformationFunded in part by NSF‐DUE 1725940 for the CREST Project.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.