The biomineralization process consists of the accumulation of a mineral consisting mainly of phosphate and calcium ions that form a calcium phosphate salt, whose structure is transformed into hydroxyapatite. This process is mediated by osteoblasts, cells that are responsible for the initiation of the biomineralization process, mediated by the release of vesicles from the matrix (VMs). These vesicles arise by budding from cell surfaces and are secreted at the specific location of the beginning of biomineralization in the bone tissue matrix. MVs contain high concentrations of Ca2 + ions and inorganic phosphate (Pi), providing a suitable microenvironment for the initial formation and propagation of hydroxyapatite crystals. Particular attention should be paid to some proteins present in MVs such as annexins. Our laboratory has been studying the performance of PHOSPHO1, an enzyme found inside the MVs responsible mainly for the hydrolysis of phosphocholine and phosphoethanolamine. Phosphocholine is generated by the action of a sphingomyelinase (Smpd3) and, therefore, such proteins can also regulate the formation of apatites in the VM lumen, thus acting directly on the bone mineralization process. However, there are still many doubts about the possible association and performance in the interface, as well as its real function. We intend to study, in this project, specifically the effect of pH and calcium ions in the process of incorporation of PHOSPHO1 and Smpd3 in lipid monolayers and bilayers. For that, the methods for preparing monolayers will be optimized in a way that protein / protein and protein / lipid interactions regulate and modulate the formation process of the first minerals at the interface. This information will be essential for the construction of the best biomimetic models of MVs (i.e., composition optimized for enzymatic activity and formation of minerals).
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