Identification of the Kelch Family Protein Nd1-L as a Novel Molecular Interactor of KRIT1

Abstract

Loss-of-function mutations of the KRIT1 gene (CCM1) have been associated with the Cerebral Cavernous Malformation (CCM) 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 signaling 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 (ROS) 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, functional assays showed that Nd1-L may contribute to the regulation of KRIT1 nucleocytoplasmic shuttling and cooperate with KRIT1 in modulating the expression levels of the antioxidant protein SOD2, opening a novel avenue for future mechanistic studies. The identification of Nd1-L as a novel KRIT1 interacting protein provides a novel piece of the molecular puzzle involving KRIT1 and suggests a potential functional cooperation in cellular responses to oxidative stress, thus expanding the framework of molecular complexes and mechanisms that may underlie the pathogenesis of CCM disease.

Strategy for Identifying Repurposed Drugs for the Treatment of Cerebral Cavernous Malformation

“…Indeed, a role of oxidative stress in the pathophysiology of CCM disease due to two other causative genes, KRIT1 and PDCD10 , has been reported 48, 59–62 . In cell culture, loss of CCM2 induced increased ROS and decreased FOX01 expression, suggesting a potential common mechanism of CCM pathophysiology as has been proposed for KRIT1 and PDCD10 (Fig.…”

Oxidative stress and inflammation in cerebral cavernous malformation disease pathogenesis: Two sides of the same coin

“…In this intricate mechanistic scenario, a further level of complexity is added by the discovery that CCM proteins modulate distinct redox-sensitive signaling pathways and mechanisms, including pro-oxidant and antioxidant pathways and autophagy (Gibson et al, 2015, Goitre et al, 2010, Goitre et al, 2014, Guazzi et al, 2012, Marchi et al, 2015), and are implicated in molecular and cellular responses to oxidative stress and inflammatory stimuli (Corr et al, 2012, Goitre et al, 2014). However, besides adding mechanistic complexity, these innovative research findings have also provided a potential integrative explanation for the pleiotropic effects of CCM protein dysfunctions, shedding new light on the mechanisms of CCM pathogenesis and opening new perspectives for disease prevention and treatment ( see Sections below for details ).…”

Detection of Novel Mutation in Ccm3 Causes Familial Cerebral Cavernous Malformations

“…While genotype-phenotype correlations for CCM1 and CCM2 mutations are still unclear, a great aggressiveness and an early onset were observed in CCM3 mutations carriers (Shenkar et al 2014). The three CCM genes encode for proteins involved in regulation of angiogenesis (Fischer et al 2013) and stress response (Guazzi et al 2012;Uhlik et al 2003).…”