Intracellular MLCK1 diversion reverses barrier loss to restore mucosal homeostasis

Abstract

Epithelial barrier loss is a driver of intestinal and systemic diseases. Myosin light chain kinase (MLCK) is a key effector of barrier dysfunction and a potential therapeutic target, but enzymatic inhibition has unacceptable toxicities. Here, we show that a unique domain within the MLCK splice-variant MLCK1 directs perijunctional actomyosin ring (PAMR) recruitment. Using the domain structure and multiple screens, we identified a domain-binding small molecule (Divertin) that blocks MLCK1 recruitment without inhibiting enzymatic function. Divertin blocks acute, TNF-induced MLCK1 recruitment as well as downstream MLC phosphorylation, barrier loss, and diarrhea in vitro and in vivo . Divertin corrects barrier dysfunction and prevents disease development and progression in experimental inflammatory bowel disease. Beyond applications of Divertin in gastrointestinal disease, this general approach to enzymatic inhibition by preventing access to specific subcellular sites provides a new paradigm for safely and precisely targeting individual properties of enzymes with multiple functions.

Contributions of Myosin Light Chain Kinase to Regulation of Epithelial Paracellular Permeability and Mucosal Homeostasis

“…Moreover, targeted knockdown of long MLCK1 within Caco-2 monolayers reduced paracellular permeability [125]. Further study showed that, in addition to increasing long MLCK expression, TNF specifically induced long MLCK1 recruitment to the perijunctional actomyosin ring [128]. This selective effect on MLCK1 was only post-translational, as intestinal epithelial MLCK1 and MLCK2 transcripts were comparably increased by TNF (unpublished data, Graham and Turner).…”

“…Structural analysis indicated that the 69 amino acids unique to long MLCK1 completed the immunoglobulin-cell adhesion molecule (IgCAM) domain 3, one of nine IgCAM domains within long MLCK1 [128]. Because this is the only difference between MLCK1 and MLCK2, it stands to reason that the key features responsible for TNF-induced MLCK 1 recruitment to the perijunctional actomyosin ring reside within IgCAM3.…”

“…The observation that an enzymatically inactive region, i.e., IgCAM3, was responsible for long MLCK1 recruitment to the perijunctional actomyosin ring presented an opportunity for blocking MLCK-dependent tight junction regulation in disease. After solving the IgCAM3 crystal structure [128,129], a hydrophobic drug-binding pocket within IgCAM3, but not the other long MLCK1 IgCAM domains, was identified. Importantly, this drug binding pocket was conserved between human and Structural analysis indicated that the 69 amino acids unique to long MLCK1 completed the immunoglobulin-cell adhesion molecule (IgCAM) domain 3, one of nine IgCAM domains within long MLCK1 [128].…”

“…After solving the IgCAM3 crystal structure [128,129], a hydrophobic drug-binding pocket within IgCAM3, but not the other long MLCK1 IgCAM domains, was identified. Importantly, this drug binding pocket was conserved between human and Structural analysis indicated that the 69 amino acids unique to long MLCK1 completed the immunoglobulin-cell adhesion molecule (IgCAM) domain 3, one of nine IgCAM domains within long MLCK1 [128]. Because this is the only difference between MLCK1 and MLCK2, it stands to reason that the key features responsible for TNF-induced MLCK 1 recruitment to the perijunctional actomyosin ring reside within IgCAM3.…”

Claudin-12 deficiency causes nerve barrier breakdown, mechanical hypersensitivity and painfulness in polyneuropathy

“…This raises the question to the extent of which other tight junction proteins and specifically claudin-1, claudin-5, claudin-12, claudin-19, ZO-1 or occludin are expressed in the BNB and the myelin barrier in CIDP patients and, if so, whether their expression is associated with painfulness. Different signaling elements contribute to barrier stabilization including the wnt pathway (6,31), myosin light chain kinase (32) and neuronal guidance molecules (33). The morphogen sonic hedgehog (SHH) on the one hand functions to regulate cell division, vertebra development and differentiation of neurons in the development of the nervous system.…”