First-in-class Deubiquitylase Inhibitors Reveal New Enzyme Conformations

Cells maintain protein homeostasis by adding a small protein, ubiquitin, to regulate a variety of cellular processes, dictating protein activity, localisation or degradation. The addition of ubiquitin, known as ubiquitylation, is a reversible process making it a versatile post-translational modification aptly suited for cell signalling. Removal of ubiquitin is catalysed by deubiquitylating enzymes, commonly referred to as DUBs. BRCC36 isopeptidase complex (BRISC) is a multi-protein DUB complex which hydrolyses lysine-63-linked ubiquitin chains on Type I interferon receptors (IFNAR1/2), thus regulating interferon-dependent signalling. Therefore, BRISC-mediated inflammatory signalling amplification is a promising target for autoimmune disease drug development. We performed a high-throughput screen to identify small molecules which inhibit BRISC enzymatic activity. Employing an integrative structural biology approach (cryo-electron microscopy, native mass spectrometry, hydrogen-deuterium exchange mass spectrometry), complemented with biochemical assays, we have uncovered new enzyme conformations, revealing a remarkable mode of action for BRISC inhibitors. Exploring these key mechanisms will expand current knowledge of inflammatory signalling pathways and establish the use of DUB inhibitors as therapeutics to combat autoimmune disease and hyperactive cytokine signalling.