Study of PHOSPHO1 and nSMase2 interaction with model membranes: a possible correlation in matrix vesicle secretion

Abstract

Bone tissue mineralization is a highly ordered process that begins with the release of matrix vesicles (MVs) from the plasma membrane of osteogenic cells. Mvs are responsible for providing adequate conditions for both nucleation and growth of the first mineral particles that are released into bone's organic matrix, making possible the process to continue. Little is known about the factors that regulate the budding of MVs from the cells however, the presence of nSMase2, a key enzyme on the biogenesis of extracelular vesicles, also present in the proteome of MVs, allows some questioning. Thus, this proposal regards on a comprehensive study of nSMase2 interaction with membrane models (mono- and bilayers) mimicking the lipid composition of MVs (high levels of cholesterol and sphingomyelin). Also, a possible synergic mechanism of nSMase2 and PHOSPHO1 (which its product acts as substrate to nSMase2) will be investigated regarding its involvement in MVs' secretion. Monolayers will be characterized using a Langmuir trough by means of surface pressure measurements. In addition, microscopic (BAM and fluorescence microscopy), spectroscopic (PM-IRRAS) and rheologic analysis will be carried out. Interactions in bilayer models will be evaluated using classical techniques for nanoparticles characterization (such as DLS, NTA, AFM and zeta potential) as well as calorimetric assays. Finally, the effect of the inhibition of these enzymes on the secretion of MVs by osteoblasts using in vitro cultures will be evaluated both qualitative- and quantitatively (DLS, NTA, zeta potential) alongside to in vitro mineralization assays. The results obtained in this proposal will allow opening new paths for elucidating and controlling the pathways of MVs' biogenesis, which is essential for a better understanding of the bone formation process and towards more efficient bone repair/regeneration therapeutic approaches. (AU)