Gerodermia osteodysplastica is a hereditary segmental progeroid disorder affecting skin, connective tissues, and bone that is caused by loss-of-function mutations in GORAB. The golgin, RAB6-interacting (GORAB) protein localizes to the Golgi apparatus and interacts with the small GTPase RAB6. In this study, we used different approaches to shed more light on the recruitment of GORAB to this compartment. We show that GORAB best colocalizes with trans-Golgi markers and is rapidly displaced upon Brefeldin A exposition, indicating a loose association with Golgi membranes. A yeast two-hybrid screening revealed a specific interaction with the small GTPase ADP-ribosylation factor (ARF5) in its active, GTP-bound form. ARF5 and RAB6 bind to GORAB via the same internal Golgi-targeting RAB6 and ARF5 binding (IGRAB) domain. Two GORAB missense mutations identified in gerodermia osteodysplastica patients fall within this IGRAB domain. GORAB carrying the mutation p.Ala220Pro had a cytoplasmic distribution and failed to interact with both RAB6 and ARF5. In contrast, the p.Ser175Phe mutation displaced GORAB from the Golgi compartment to vesicular structures and selectively impaired ARF5 binding. Our findings indicate that the IGRAB domain is crucial for the Golgi localization of GORAB and that loss of this localization impairs its physiological function.
Bone fragility due to osteopenia, osteoporosis or debilitating focal skeletal dysplasias is a frequent observation in the Mendelian disease Neurofibromatosis type 1 (NF1). To determine the mechanisms underlying bone fragility in NF1 we analyzed two conditional mouse models, Nf1Prx1 (limb knock-out) and Nf1Col1 (osteoblast specific knock-out), as well as cortical bone samples from individuals with NF1. We examined mouse bone tissue with micro-computed tomography, qualitative and quantitative histology, mechanical tensile analysis, small-angle X-ray scattering (SAXS), energy dispersive X-ray spectroscopy (EDX), and scanning acoustic microscopy (SAM). In cortical bone of Nf1Prx1 mice we detected ectopic blood vessels that were associated with diaphyseal mineralization defects. Defective mineral binding in the proximity of blood vessels was most likely due to impaired bone collagen formation, as these areas were completely devoid of acidic matrix proteins and contained thin collagen fibers. Additionally, we found significantly reduced mechanical strength of the bone material, which was partially caused by increased osteocyte volume. Consistent with these observations, bone samples from individuals with NF1 and tibial dysplasia showed increased osteocyte lacuna volume. Reduced mechanical properties were associated with diminished matrix stiffness, as determined by SAM. In line with these observations, bone tissue from individuals with NF1 and tibial dysplasia showed heterogeneous mineralization and reduced collagen fiber thickness and packaging. Collectively, the data indicate that bone fragility in NF1 tibial dysplasia is partly due to an increased osteocyte-related micro-porosity, hypomineralization, a generalized defect of organic matrix formation, exacerbated in the regions of tensional and bending force integration, and finally persistence of ectopic blood vessels associated with localized macro-porotic bone lesions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.