LAUSR

Elevated intestinal permeability has long been appreciated as a pathophysiological marker of gut inflammatory disease, in particular in inflammatory bowel disease (IBD). In addition to the association of IBD risk genetic variants with multiple aspects of intestinal permeability and barrier dysfunction, increased permeability has been identified as a predictor of IBD onset in healthy firstdegree relatives of patients with Crohn’s disease, and of relapse in patients with Crohn’s disease. However, despite extensive investigation of the role of inflammation in diseaseassociated permeability, development of barrierrestoring agents beyond broadacting antiinflammatory agents has proven extremely challenging. In GUT, Zuo et al present an innovative study identifying that the tacrolimusbinding protein FKBP8 is a specific binding partner for the tight junction regulatory mediator, myosin lightchain kinase 1 (MLCK1). MLCK1 is one of two splice variants of the canonical tight junction regulatory protein, MLCK. MLCK phosphorylates myosin II regulatory light chain (MLC) within the perijunctional actomyosin ring to increase tight junction permeability to molecules with diameters up to ~100 Å. This route of paracellular permeability is often referred to as the ‘leak’ pathway. In studies using geneticallymodified mice and enzymatic inhibitors, MLCK has been identified as a driver of acute cytokineinduced diarrhoea as well as chronic immunemediated experimental IBD. Moreover, MLCK acts as a critical downstream mediator of the inflammatory cytokines tumour necrosis factor (TNF)-α and interferon (IFN)-γ, culminating in alterations of epithelial tight junctions the key protein aggregate structures that regulate epithelial permeability. 8 9 However, targeting of MLCK for therapeutic intervention to restore barrier function has been greatly hampered by the difficulty in avoiding unwanted side effects on MLCK expressed in cardiac, smooth and skeletal muscle. This paper addresses a significant problem by determining a route to circumvent the nonspecific effects of broad MLCK inhibition, by identifying FKBP8 as an additional factor that can be targeted to reduce accumulation of MLCK1 at tight junctions. In their prior work, the authors elucidated that TNFinduced recruitment of MLCK1—but not the MLCK2 splice variant—to the perijunctional actomyosin ring was sensitive to small molecule blockade of an aminoterminal immunoglobulinlike domain, IgCAM3, that is unique to MLCK1. They hypothesised that the small molecule that blocked MLCK1 recruitment acted by interfering with IgCAM3 binding to another—as yet unidentified—protein. To identify this putative mediator, they used a yeast twohybrid screen to probe a human intestinal epithelial complementary DNA library for MLCK1specific binding proteins. They identified FK506binding protein FKBP8, also known as FKBP38, a protein that has been linked to autophagy, mitophagy and the unfolded protein response but had not previously been functionally characterised in intestinal epithelial cells. Using protein binding and proximity ligation approaches, they showed that MLCK1 binds directly to FKBP8 and that these interactions are essential for MLCK1 recruitment, MLC phosphorylation and TNFinduced barrier loss in intestinal epithelial cell lines and human intestinal organoid cultures (see figure 1). Notably, while TNF increases MLCK1 expression, the effect of TNF on FKBP8 was specific in promoting MLCKFKBP8 interactions as no change in FKBP8 expression or distribution levels was observed in response to TNF treatment. The translational importance of MLCK1FKBP8 interactions was indicated by examination of biopsies from Crohn’s disease patients that also showed increased MLCK1FKBP8 interactions relative to control subjects. In intervention studies, MLCK1FKBP8 binding was inhibited by the immunosuppressive agent,