Identification of a novel KRIT1 interactor involved in the control of actin cytoskeleton dynamics and cell resistance to oxidative stress

Loss-of-function mutations of the KRIT1 gene (CCM1) have been associated with the pathogenesis of Cerebral Cavernous Malformations (CCM), a major vascular disease characterized by the presence of abnormally enlarged and leaky capillaries in the brain. Although the presence of multiple protein-protein interaction domains has suggested that the KRIT1 protein acts as a molecular scaffold for the assembly of signaling complexes, KRIT1 molecular functions and CCM pathogenesis mechanisms remain incompletely understood. Recently, we defined structure-function relationships underlying KRIT1 intramolecular and intermolecular interactions and nucleocytoplasmic shuttling, and found that KRIT1 plays an important role in molecular mechanisms involved in the maintenance of the intracellular Reactive Oxygen Species (ROS) homeostasis to prevent oxidative cellular damage. To further elucidate KRIT1 molecular and cellular functions, we sought to identify new protein interaction partners. Using a yeast two-hybrid screening of a mouse embryo cDNA library, we identified a novel KRIT1 binding protein, Nd1-L, a kelch family protein previously involved in the regulation of the actin cytoskeleton and Rho GTPases, and suggested to play a role in cell protection against oxidative stress. The novel interaction was confirmed by pull-down and co-immunoprecipitation assays of recombinant proteins, as well as by co-immunoprecipitation of endogenous Nd1 and KRIT1 proteins in human endothelial cells. Furthermore, using KRIT1A and KRIT1B isoforms and distinct KRIT1 mutated constructs, we defined the contribute of the different KRIT1 domains to the Nd1-L/KRIT1 interaction. Finally, fluorescence microscopy studies showed that the overexpression of Nd1-L induces a nucleus-to-cytoplasm translocation of KRIT1, indicating that the novel identified interacting protein may contribute to the regulation of KRIT1 nucleocytoplasmic shuttling. As both KRIT1 and Nd1 have been involved in the regulation of Rho GTPases and cell resistance to oxidative stress, our findings provide a novel piece of the molecular puzzle involving KRIT1, expanding the knowledge of molecular complexes and mechanisms that may underlie CCM disease.