Antibodies targeting the shared cytokine receptor IL-1 receptor accessory protein invoke distinct mechanisms to block all cytokine signaling.

Interleukin-1 (IL-1)-family cytokines are potent modulators of inflammation, coordinating a vast array of immunological responses across innate and adaptive immune systems. Dysregulated IL-1-family cytokine signaling, however, is involved in a multitude of adverse health effects, such as chronic inflammatory conditions, autoimmune diseases, and cancer. Within the IL-1 family of cytokines, six-IL-1α, IL-1ß, IL-33, IL-36α, IL-36ß, and IL-36γ-require the IL-1 receptor accessory protein (IL-1RAcP) as their shared co-receptor. Common features of cytokine signaling include redundancy of signaling pathways, sharing of cytokines and receptors, pleiotropy of the cytokines themselves, and multifaceted immune responses. Accordingly, targeting multiple cytokines simultaneously is an emerging therapeutic strategy and can provide advantages over targeting a single cytokine pathway. Here, we show that two monoclonal antibodies, CAN10 and 3G5, which target IL-1RAcP for broad blockade of all associated cytokines, do so through distinct mechanisms and provide therapeutic opportunities for the treatment of inflammatory diseases.

Combinatorially restricted computational design of protein-protein interfaces to produce IgG heterodimers.

Redesigning protein-protein interfaces is an important tool for developing therapeutic strategies. Interfaces can be redesigned by in silico screening, which allows for efficient sampling of a large protein space before experimental validation. However, computational costs limit the number of combinations that can be reasonably sampled. Here, we present combinatorial tyrosine (Y)/serine (S) selection (combYSelect), a computational approach combining in silico determination of the change in binding free energy (ΔΔG) of an interface with a highly restricted library composed of just two amino acids, tyrosine and serine. We used combYSelect to design two immunoglobulin G (IgG) heterodimers-combYSelect1 (L368S/D399Y-K409S/T411Y) and combYSelect2 (D399Y/K447S-K409S/T411Y)-that exhibit near-optimal heterodimerization, without affecting IgG stability or function. We solved the crystal structures of these heterodimers and found that dynamic π-stacking interactions and polar contacts drive preferential heterodimeric interactions. Finally, we demonstrated the utility of our combYSelect heterodimers by engineering both a bispecific antibody and a cytokine trap for two unique therapeutic applications.

The Campylobacter jejuni Response Regulator and Cyclic-Di-GMP Binding CbrR Is a Novel Regulator of Flagellar Motility.

A leading cause of bacterial gastroenteritis, Campylobacter jejuni is also associated with broad sequelae, including extragastrointestinal conditions such as reactive arthritis and Guillain-Barré Syndrome (GBS). CbrR is a C. jejuni response regulator that is annotated as a diguanylate cyclase (DGC), an enzyme that catalyzes the synthesis of c-di-GMP, a universal bacterial second messenger, from GTP. In C. jejuni DRH212, we constructed an unmarked deletion mutant, cbrR-, and complemented mutant, cbrR+. Motility assays indicated a hyper-motile phenotype associated with cbrR-, whereas motility was deficient in cbrR+. The overexpression of CbrR in cbrR+ was accompanied by a reduction in expression of FlaA, the major flagellin. Biofilm assays and scanning electron microscopy demonstrated similarities between DRH212 and cbrR-; however, cbrR+ was unable to form significant biofilms. Transmission electron microscopy showed similar cell morphology between the three strains; however, cbrR+ cells lacked flagella. Differential radial capillary action of ligand assays (DRaCALA) showed that CbrR binds GTP and c-di-GMP. Liquid chromatography tandem mass spectrometry detected low levels of c-di-GMP in C. jejuni and in E. coli expressing CbrR. CbrR is therefore a negative regulator of FlaA expression and motility, a critical virulence factor in C. jejuni pathogenesis.

Binding of Campylobacter jejuni FliW Adjacent to the CsrA RNA-Binding Pockets Modulates CsrA Regulatory Activity.

Campylobacter jejuni CsrA is an mRNA-binding, post-transcriptional regulator that controls many metabolic- and virulence-related characteristics of this important pathogen. In contrast to E. coli CsrA, whose activity is modulated by binding to small non-coding RNAs (sRNAs), C. jejuni CsrA activity is controlled by binding to the CsrA antagonist FliW. In this study, we identified the FliW binding site on CsrA. Deletion of the C-terminus of C. jejuni CsrA, which is extended relative to sRNA-binding CsrA proteins, abrogated FliW binding. Bacterial two-hybrid experiments were used to assess the interaction of FliW with wild-type CsrA and mutants thereof, in which every amino acid was individually mutated. Two CsrA mutations (V51A and N55A) resulted in a significant decrease in FliW binding. The V51A and N55A mutants also showed a decrease in CsrA-FliW complex formation, as assessed by size-exclusion chromatography and surface plasmon resonance. These residues were highly conserved in bacterial species containing CsrA orthologs whose activities are predicted to be regulated by FliW. The location of FliW binding was immediately adjacent to the two RNA-binding sites of the CsrA homodimer, suggesting the model that FliW binding to CsrA modulates its ability to bind to its mRNA targets either by steric hindrance, electrostatic repulsion, or by altering the overall structure of the RNA-binding sites.

RNA Binding by the Campylobacter jejuni Post-transcriptional Regulator CsrA.

Campylobacter jejuni is a Gram-negative rod-shaped bacterium that commensally inhabits the intestinal tracts of livestock and birds, and which also persists in surface waters. C. jejuni is a leading cause of foodborne gastroenteritis, and these infections are sometimes associated with the development of post-infection sequelae such as Guillain-Barré Syndrome. Flagella are considered a primary virulence factor in C. jejuni, as these organelles are required for pathogenicity-related phenotypes including motility, biofilm formation, host cell interactions, and host colonization. The post-transcriptional regulator CsrA regulates the expression of the major flagellin FlaA by binding to flaA mRNA and repressing its translation. Additionally, CsrA has previously been shown to regulate 120-150 proteins involved in diverse cellular processes. The amino acid sequence of C. jejuni CsrA is significantly different from that of Escherichia coli CsrA, and no previous research has defined the amino acids of C. jejuni CsrA that are critical for RNA binding. In this study, we used in vitro SELEX to identify the consensus RNA sequence mAwGGAs to which C. jejuni CsrA binds with high affinity. We performed saturating site-directed mutagenesis on C. jejuni CsrA and assessed the regulatory activity of these mutant proteins, using a reporter system encoding the 5' untranslated region (5' UTR) upstream of flaA linked translationally to the C. jejuni astA gene. These assays allowed us to identify 19 amino acids that were involved in RNA binding by CsrA, with many but not all of these amino acids clustered in predicted beta strands that are involved in RNA binding by E. coli CsrA. Decreased flaA mRNA binding by mutant CsrA proteins L2A and A36V was confirmed by electrophoretic mobility shift assays. The majority of the amino acids implicated in RNA binding were conserved among diverse Campylobacter species.

FliW controls growth-phase expression of Campylobacter jejuni flagellar and non-flagellar proteins via the post-transcriptional regulator CsrA.

Campylobacter jejuni is an important human pathogen that causes 96 million cases of acute diarrheal disease worldwide each year. We have shown that C. jejuni CsrA is involved in the post-transcriptional regulation of more than 100 proteins, and altered expression of these proteins is presumably involved in the altered virulence-related phenotypes of a csrA mutant. Mutation of fliW results in C. jejuni cells that have greatly truncated flagella, are less motile, less able to form biofilms, and exhibit a reduced ability to colonize chicks. The loss of FliW results in the altered expression of 153 flagellar and non-flagellar proteins, the majority of which are members of the CsrA regulon. The number of proteins dysregulated in the fliW mutant was greater at mid-log phase (120 proteins) than at stationary phase (85 proteins); 52 proteins showed altered expression at both growth phases. Loss of FliW altered the growth-phase- and CsrA-mediated regulation of FlaA flagellin. FliW exerts these effects by binding to both FlaA and to CsrA, as evidenced by pull-down assays, protein-protein cross-linking, and size-exclusion chromatography. Taken together, these results show that CsrA-mediated regulation of both flagellar and non-flagellar proteins is modulated by direct binding of CsrA to the flagellar chaperone FliW. Changing FliW:CsrA stoichiometries at different growth phases allow C. jejuni to couple the expression of flagellar motility to metabolic and virulence characteristics.

Identification of new FGF1 binding partners-Implications for its intracellular function.

Besides its classical mode of action through activation of specific receptors at the cell surface, fibroblast growth factor 1 (FGF1) can also cross the cellular membrane and translocate into the cytosol and further to the nucleus. The mechanism of this translocation is described partially, but the role of FGF1 inside the cell remains unknown. The aim of our work was to identify novel binding partners of FGF1 to predict its intracellular functions. We combined three methods of identification of such partners based on different principles: yeast two-hybrid screen and mass spectrometry (MS) analysis of complexes obtained by Tandem Affinity Purification (TAP) or by co-precipitation from cell lysate using recombinant FGF1. Altogether, we identified twenty novel intracellular proteins interacting with FGF1. For selected proteins, their direct interaction with FGF1 was confirmed by pull-down assays and SPR measurements. Interestingly, half of the proteins found are involved in processes related to cell viability, such as apoptosis, cell proliferation, and cell cycle regulation. Thus, our study indicates that the role of intracellular FGF1 is to protect the cell against stress conditions by providing an additional signal for cell survival, independently of receptor-activated signaling cascades.

Reduction of T cell receptor diversity in NOD mice prevents development of type 1 diabetes but not Sjögren's syndrome.

Non-obese diabetic (NOD) mice are well-established models of independently developing spontaneous autoimmune diseases, Sjögren's syndrome (SS) and type 1 diabetes (T1D). The key determining factor for T1D is the strong association with particular MHCII molecule and recognition by diabetogenic T cell receptor (TCR) of an insulin peptide presented in the context of I-Ag7 molecule. For SS the association with MHCII polymorphism is weaker and TCR diversity involved in the onset of the autoimmune phase of SS remains poorly understood. To compare the impact of TCR diversity reduction on the development of both diseases we generated two lines of TCR transgenic NOD mice. One line expresses transgenic TCRß chain originated from a pathogenically irrelevant TCR, and the second line additionally expresses transgenic TCRαmini locus. Analysis of TCR sequences on NOD background reveals lower TCR diversity on Treg cells not only in the thymus, but also in the periphery. This reduction in diversity does not affect conventional CD4+ T cells, as compared to the TCRmini repertoire on B6 background. Interestingly, neither transgenic TCRß nor TCRmini mice develop diabetes, which we show is due to lack of insulin B:9-23 specific T cells in the periphery. Conversely SS develops in both lines, with full glandular infiltration, production of autoantibodies and hyposalivation. It shows that SS development is not as sensitive to limited availability of TCR specificities as T1D, which suggests wider range of possible TCR/peptide/MHC interactions driving autoimmunity in SS.