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 Cerebral Cavernous Malformations disease, which is characterized by serious alterations of brain capillary architecture. The KRIT1 protein contains multiple interaction domains and motifs, suggesting that it might act as a scaffold for the assembly of functional protein complexes involved in signalling networks. In previous work, 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 homeostasis to prevent oxidative cellular damage. Here we report the identification of the Kelch family protein Nd1-L as a novel molecular interactor of KRIT1. This interaction was discovered through yeast two-hybrid screening of a mouse embryo cDNA library, and confirmed by pull-down and co-immunoprecipitation assays of recombinant proteins, as well as by co-immunoprecipitation of endogenous proteins in human endothelial cells. Furthermore, using distinct KRIT1 isoforms and mutants, we defined the role of KRIT1 domains in the Nd1-L/KRIT1 interaction. Finally, fluorescence microscopy studies showed that 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-L have been involved in the regulation of Rho GTPases, cytoskeleton dynamics and cellular responses to oxidative stress, our findings provide a novel and important piece of the molecular puzzle involving KRIT1, thus expanding the knowledge of molecular complexes and mechanisms that may underlie CCM disease.