The yeast three-hybrid system as a tool to study caspases

Caspases are cysteine proteases that play an essential role during apoptotic cell death and inflammation. They are synthesized as catalytically dormant proenzymes, containing an N-terminal prodomain, a large subunit (p20) containing the active site cysteine, and a small subunit (p10). The active enzymes function as tetramers, consisting of two p20/p10 subunit heterodimers. Both subunits contribute residues that are essential for substrate recognition. Activation of caspases culminates in the cleavage of a set of cellular proteins, resulting in disassembly of the cell or proinflammatory cytokine production. Inappropriate caspase activation contributes to or accounts for several diseases. The identification of caspase-interacting proteins that might act as activators, substrates, or inhibitors is therefore an attractive step in the development of novel therapeutics. However, caspase substrates and other proteins that bind specifically with the active heterodimeric p20/p10 form of caspases will escape detection in a classical two-hybrid approach with an unprocessed caspase precursor as bait. Alternatively, a number of so-called three-hybrid systems to analyze more complex macromolecular interactions have been developed. We describe the use of a three-hybrid approach adapted to the needs of caspases to detect and analyze the interaction of mature heteromeric caspases with protein substrates or inhibitors

TIM3+ TRBV11-2 T cells and IFNγ signature in patrolling monocytes and CD16+ NK cells delineate MIS-C

In rare instances, pediatric SARS-CoV-2 infection results in a novel immunodysregulation syndrome termed multisystem inflammatory syndrome in children (MIS-C). We compared MIS-C immunopathology with severe COVID-19 in adults. MIS-C does not result in pneumocyte damage but is associated with vascular endotheliitis and gastrointestinal epithelial injury. In MIS-C, the cytokine release syndrome is characterized by IFN gamma and not type I interferon. Persistence of patrolling monocytes differentiates MIS-C from severe COVID-19, which is dominated by HLA-DRlo classical monocytes. IFN gamma levels correlate with granzyme B production in CD16(+) NK cells and TIM3 expression on CD38(+)/HLA-DR+ T cells. Single-cell TCR profiling reveals a skewed TCR beta repertoire enriched for TRBV11-2 and a superantigenic signature in TIM3(+)/CD38(+)/HLA-DR+ T cells. Using NicheNet, we confirm IFN gamma as a central cytokine in the communication between TIM3(+)/CD38(+)/HLA-DR+ T cells, CD16(+) NK cells, and patrolling monocytes. Normalization of IFN gamma, loss of TIM3, quiescence of CD16(+) NK cells, and contraction of patrolling monocytes upon clinical resolution highlight their potential role in MIS-C immunopathogenesis. MIS-C is a novel immunodysregulation syndrome in children with a history of SARS-CoV-2 infection. This study employs a multi-omics approach to explore its immunopathogenesis. The authors show that IFN gamma-mediated interactions between T cells, monocytes, and NK cells reside at the heart of the disease