Protein kinase R (PKR) is an interferon-induced kinase which plays a pivotal role in the innate immunity response to viral infection. PKR is activated upon binding to dsRNA. Our previous analysis of PKR binding to dsRNAs ranging from 20-40 bp supports a dimerization model for activation where 30 bp represents the minimal length required to bind two PKR monomers and activate PKR via autophosphorylation. These studies were complicated by the formation of protein-RNA aggregates, particularly at low salt concentrations using longer dsRNAs. Here, we have taken advantage of the enhanced sensitivity afforded using fluorescence-detected analytical ultracentrifugation to reduce the RNA concentrations from micromolar to nanomolar. Under these conditions, we are able to characterize high affinity binding of PKR to longer dsRNAs in 75 mM NaCl. The PKR binding stoichiometries are increased at lower salt but remain lower than those previously obtained for the dsRNA binding domain. The dependence of the limiting PKR binding stoichiometries on dsRNA length does not conform to standard models for nonspecific binding and suggests that binding to longer sequences occurs via a different binding mode with a larger site size. Although dimerization plays a key role in the PKR activation mechanism, the ability of shorter dsRNAs to bind two PKR monomers is not sufficient to induce autophosphorylation. We propose that activation of PKR by longer RNAs is correlated with an alternative binding mode where both of the dsRNA binding motifs contact the RNA, inducing PKR to dimerize via a direct interaction of the kinase domains.
Bispecific antibodies have moved from being an academic curiosity with therapeutic promise to reality, with two molecules being currently commercialized (Hemlibra® and Blincyto®) and many more in clinical trials. The success of bispecific antibodies is mainly due to the continuously growing number of mechanisms of actions (MOA) they enable that are not accessible to monoclonal antibodies. One of the earliest MOA of bispecific antibodies and currently the one with the largest number of clinical trials is the redirecting of the cytotoxic activity of T-cells for oncology applications, now extending its use in infective diseases. The use of bispecific antibodies for crossing the blood–brain barrier is another important application because of its potential to advance the therapeutic options for neurological diseases. Another noteworthy application due to its growing trend is enabling a more tissue-specific delivery or activity of antibodies. The different molecular solutions to the initial hurdles that limited the development of bispecific antibodies have led to the current diverse set of bispecific or multispecific antibody formats that can be grouped into three main categories: IgG-like formats, antibody fragment-based formats, or appended IgG formats. The expanded applications of bispecific antibodies come at the price of additional challenges for clinical development. The rising complexity in their structure may increase the risk of immunogenicity and the multiple antigen specificity complicates the selection of relevant species for safety assessment.
Highlights d The immunoglobulin superfamily (IgSF) interactome identifies over 500 receptor pairs d The IgSF interactome reveals receptor-ligand communities dysregulated in cancer d More than 60 receptor-ligand pairs confirmed using orthogonal methods d Interacting protein signatures are correlated with clinical outcome
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