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Biogenesis of matrix vesicles (MV) during physiological mineralization and ectopic calcification.

Grant number: 23/14655-0
Support Opportunities:Scholarships abroad - Research Internship - Doctorate
Effective date (Start): April 01, 2024
Effective date (End): March 31, 2025
Field of knowledge:Biological Sciences - Biochemistry - Enzymology
Principal Investigator:Pietro Ciancaglini
Grantee:Luiz Henrique da Silva Andrilli
Supervisor: Jose Luis Millan
Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Research place: Sanford Burnham Prebys Medical Discovery Institute, La Jolla, United States  
Associated to the scholarship:21/13140-1 - Study of PHOSPHO1 and nSMase2 interaction with model membranes: a possible correlation in matrix vesicle secretion, BP.DR

Abstract

Mineralization of bone tissue is a highly ordered process that begins with the release of matrix vesicles (MV) from the plasma membrane of osteogenic cells, which are responsible for providing the necessary conditions for the nucleation and growth of the first calcium phosphate crystals, which are subsequently released into the organic matrix to propagate mineralization. Little is known about the factors that regulate the sprouting of MV from progenitor cells, however, the presence of Sphingomyelinase (nSMase2), a key enzyme in the biogenesis of extracellular vesicles and present in the proteome of MV, points to its potential involvement. In this context, this project proposes a comprehensive study of the interaction of nSMase2 with membrane models (monolayers and bilayers) that mimic the lipid composition of MV, abundant in cholesterol and sphingomyelin. Furthermore, a possible mechanism of synergy between nSMase2 and phosphoethanolamine/phosphocholine Phosphatase 1 (PHOSPHO1), that uses phosphocholine (the product of nSMase2 cleavage of sphingomyelin) as substrate to generate Pi, will be investigated about its involvement in MV secretion, since PHOSPHO1 knockout osteogenic cells show greatly reduced MV biogenesis. Understanding the mechanism of MV biogenesis is of paramount importance to better understand the mechanism of initiation of skeletal dental mineralization and the pathophysiology of ectopic calcification disorders and to enable the development of novel therapeutic approaches. Thus, the aims of my research project are, I) to use mimetic models made up of lipids from native murine MV, incorporating them with nSMase2 and PHOSPHO1 to observe the formation of vesicles with the aid of microscopic techniques such as Cryo-TEM, optical microscope and fluorescence microscope; II) to use native PHOSPHO1 and catalytically inactive mutants of PHOSPHO1 to understand if the catalytic activity of this enzyme is necessary for its role in MV biogenesis, or if that function is independent of its Pi-generating catalytic activity and III) to use inhibitors of nSMase2 and PHOSPHO1 in cell cultures of osteoblasts and chondrocytes to observe the biogenesis of MV by these cells and IV) study the disruption of MV biogenesis in vivo and in primary cultures of knockout osteogenic cells obtained from PHOSPHO1, nSMase2 and PHOSPHO1/nSMase2 double knockout mice. All these tasks will be performed during year 2024 at the Sanford Burnham Prebys Medical Discovery Institute, La Jolla, San Diego, CA, USA, in the laboratory, and under the supervision, of Prof. Dr. José Luis Millan where he has all the infrastructure for this project, including the knock-out animal models, primary cell cultures, mutagenesis and protein expression and necessary instrumentation such as a Cryo-TEM facility and other microscopy modalities to characterize MV biogenesis.

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