Cerebral cavernous malformations (CCMs) are sporadically acquired or inherited vascular lesions of the central nervous system consisting of clusters of dilated, thin-walled blood vessels that predispose individuals to seizures and stroke. CCMs have a prevalence of 0.1-0.5% in the human population and are associated with inherited or sporadic mutations in three genes and their resulting proteins: KRIT1 (Krev/Rap1 Interacting Trapped 1, CCM1), CCM2 (malcavernin) and CCM3 (programmed cell death 10, PDCD10). Early work suggests the direct interaction of the CCM proteins with one another forms a signaling platform around a ternary KRIT1:CCM2:CCM3: complex termed the CCM complex. In this model, CCM2 acts as the central hub using independent binding sites to simultaneously interact with KRIT1 and CCM3. It is believed the KRIT1 binding partner ICAP1 (integrin cytoplasmic associated protein 1) is also part of this complex. However, the CCM complex independent and dependent roles of the CCM proteins is yet to be determined. Using knockdown studies, I will by evaluate how the loss of one CCM protein affects the mRNA expression, protein levels and cellular localization of the other CCM-complex members. Mutants which interrupt CCM protein interactions or change CCM protein localization will be used in knockdown/rescue experiments to determine the importance CCM complex formation and proper localization of CCM proteins. Tube formation, cell migration, response to shear stress, integrin activation, Rho/ROCK activation and monolayer permeability assays will determine the functional consequences of these mutations for endothelial processes. Additionally, endogenous CCM protein levels and localization in endothelial cells will be assessed after activating pathways previously connected to CCM proteins. The functional focus of the studies proposed will provide a significant enhancement to the molecular-level understanding of the proteins involved in CCM, and a consequent improvement in understanding the molecular basis for CCM acquisition and CCM-associated hemorrhagic stroke.
Cerebral cavernous malformations (CCMs), a dysplasia of the vasculature, are associated with loss-of- function mutations in any one of the three CCM genes: KRIT1, CCM2 or CCM3. The protein products of these genes (KRIT, CCM2 and CCM3) can bind one another to form what is known as the CCM- complex. The studies proposed in the application will determine the complex-dependent and complex- independent roles of these proteins in regards to endothelial tube formation, cell migration, barrier integrity and other vascular biological processes.
|Draheim, Kyle M; Huet-Calderwood, Clotilde; Simon, Bertrand et al. (2017) Nuclear Localization of Integrin Cytoplasmic Domain-associated Protein-1 (ICAP1) Influences ?1 Integrin Activation and Recruits Krev/Interaction Trapped-1 (KRIT1) to the Nucleus. J Biol Chem 292:1884-1898|