ABSTRACT
The proinflammatory enzyme caspase-1 plays an important role in the innate immune system and is involved in a variety of inflammatory conditions. Rare naturally occurring human variants of the caspase-1 gene (CASP1) lead to different protein expression and structure and to decreased or absent enzymatic activity. Paradoxically, a significant number of patients with such variants suffer from febrile episodes despite decreased IL-1ß production and secretion. In this study, we investigate how variant (pro)caspase-1 can possibly contribute to inflammation. In a transfection model, such variant procaspase-1 binds receptor interacting protein kinase 2 (RIP2) via Caspase activation and recruitment domain (CARD)/CARD interaction and thereby activates NF-κB, whereas wild-type procaspase-1 reduces intracellular RIP2 levels by enzymatic cleavage and release into the supernatant. We approach the protein interactions by coimmunoprecipitation and confocal microscopy and show that NF-κB activation is inhibited by anti-RIP2-short hairpin RNA and by the expression of a RIP2 CARD-only protein. In conclusion, variant procaspase-1 binds RIP2 and thereby activates NF-κB. This pathway could possibly contribute to proinflammatory signaling.
Subject(s)
Caspase 1/genetics , Fever/genetics , Inflammation/genetics , NF-kappa B/metabolism , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , Blotting, Western , Caspase 1/metabolism , Fever/enzymology , Fluorescent Antibody Technique , Gene Knockdown Techniques , Genetic Variation , HEK293 Cells , Humans , Immunoprecipitation , Inflammation/immunology , Inflammation/metabolism , Signal Transduction/physiology , Transduction, Genetic , TransfectionABSTRACT
Caspase-1 (Interleukin-1 Converting Enzyme, ICE) is a proinflammatory enzyme that plays pivotal roles in innate immunity and many inflammatory conditions such as periodic fever syndromes and gout. Inflammation is often mediated by enzymatic activation of interleukin (IL)-1ß and IL-18. We detected seven naturally occurring human CASP1 variants with different effects on protein structure, expression, and enzymatic activity. Most mutations destabilized the caspase-1 dimer interface as revealed by crystal structure analysis and homology modeling followed by molecular dynamics simulations. All variants demonstrated decreased or absent enzymatic and IL-1ß releasing activity in vitro, in a cell transfection model, and as low as 25% of normal ex vivo in a whole blood assay of samples taken from subjects with variant CASP1, a subset of whom suffered from unclassified autoinflammation. We conclude that decreased enzymatic activity of caspase-1 is compatible with normal life and does not prevent moderate and severe autoinflammation.
Subject(s)
Caspase 1/genetics , Caspase 1/metabolism , Genetic Variation , Interleukin-1beta/metabolism , Biocatalysis , Caspase 1/chemistry , Cell Line , Crystallography, X-Ray , Cytokines/blood , Cytokines/metabolism , DNA Mutational Analysis , Genetic Predisposition to Disease/genetics , HEK293 Cells , Humans , Inflammation/enzymology , Inflammation/genetics , Models, Molecular , Mutation , Protein Multimerization , Protein Structure, TertiarySubject(s)
Bacterial Proteins/chemistry , Chlorine/chemistry , Halogenation , Oxidoreductases/chemistry , Tryptophan/chemistry , Amino Acid Substitution , Bacterial Proteins/genetics , Catalysis , Catalytic Domain , Glutamic Acid/chemistry , Glutamic Acid/genetics , Lysine/chemistry , Lysine/genetics , Mutation , Oxidoreductases/genetics , Protein Structure, Secondary , Pseudomonas fluorescens/enzymologyABSTRACT
Chlorinated natural products include vancomycin and cryptophycin A. Their biosynthesis involves regioselective chlorination by flavin-dependent halogenases. We report the structural characterization of tryptophan 7-halogenase (PrnA), which regioselectively chlorinates tryptophan. Tryptophan and flavin adenine dinucleotide (FAD) are separated by a 10 angstrom-long tunnel and bound by distinct enzyme modules. The FAD module is conserved in halogenases and is related to flavin-dependent monooxygenases. On the basis of biochemical studies, crystal structures, and by analogy with monooxygenases, we predict that FADH2 reacts with O2 to make peroxyflavin, which is decomposed by Cl-. The resulting HOCl is guided through the tunnel to tryptophan, where it is activated to participate in electrophilic aromatic substitution.
