Calcium phosphate precipitation through matrix vesicles: comprehension of atomic scale mechanisms through biomimetic studies and multinuclear solid state NMR

• Grant number: 20/02026-0
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Start date: November 01, 2021
• End date: October 31, 2022
• Field of knowledge: Biological Sciences - Biophysics - Molecular Biophysics
• Principal Investigator: Antonio José da Costa Filho
• Grantee: Bruno Zoccaratto Favarin
• Supervisor: Thierry Azais
• Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: Université Paris-Sorbonne (Paris 4), France
• Associated to the scholarship: 18/12092-0 - Biophysical Studies of the Structure-function Correlation of TNAP in interaction with membrane models, BP.PD

Abstract

Alkaline phosphatases are a class of enzymes that catalyze the hydrolysis of phosphomonoesters, thus producing phosphate. In humans, four alkaline phosphatases have been identified so far. In mammals, this enzyme can be associated to genetic disorders leading to different levels of bone failure and pathological blood vessel mineralization. Due to its biological relevance, tissue non-specific alkaline phosphatase (TNAP) has attracted attention in different fields ranging from fundamental research - and molecular biophysics in particular - to public health. However, despite its vital importance, the detailed mechanism of TNAP during bone mineralization is still not understood. In this context, we propose an innovative project based on three complementary tasks. (I) First, we propose the design of biomimetic models of native matrix vesicles where TNAP are encapsulated in liposomes. The effect of lipid composition, using proteoliposomes as cell membrane models, on the anchoring mechanism of TNAP as well as on TNAP catalytic activity will be investigated. (II) Second, to progress on the comprehension of the mineralization action mechanism of TNAP at the atomic scale, advanced multinuclear (1H, 31P, 43Ca) solid state nuclear magnetic resonance (ssNMR) experiments will be undertaken to characterize calcium phosphate phases precipitated after hydrolysis of phosphorylated substrates. (III) Finally, the mineralization process of our biomimetic models will be compared to native matrix vesicles (MVs) extracted from osteoblasts and chondrocytes. Our approach is a first essential step in the study of enzymatic mutations associated with diseases, such as those found in hypophosphatasia that could be further investigated in membrane vesicles mimicking the biological environment of the enzyme in vivo. The tasks (I) and (II) will be developed at the laboratory "Chimie de la Matière Condensée de Paris" (LCMCP) at Sorbonne Université, Paris, under the supervision of Dr. Thierry Azaïs. First, in order to mimic the biomineralization process, proteoliposomes containing alkaline phosphatase mutants will be prepared and, second, their ability to precipitate calcium phosphates depending on the membranes composition, will be investigated by advanced multidimensional ssNMR experiments (1H-31P and 1H-13C HetCor, 13C-31P REDOR&) to reveal the organo-mineral interface underlying the mineralization process induced by TNAP. In parallel, the morphology of the proteoliposomes will be investigated by transmission electron microscopy in cryogenic mode (Cryo-TEM) in order to preserve their native hydration. The dynamic of proteoliposomes will be also studied by liquid-state NMR. Finally, the use of dynamic nuclear polarization (DNP), a new approach permitting a dramatic increase of the NMR signal (by several order of magnitude) both in the solid (DNP MAS) and liquid state (D-DNP) will be considered. The task (III), i.e. the isolation of MVs from chicken embryo osteoblasts and chondrocytes will be carried out at the Université Lyon 1, under the supervision of Dr Rene Buchet and Dra. Saida Mebark, and at the University of Rome Tor Vergata, under the supervision of Dr. Massimo Bottini. The originally of our project is linked to the design of artificial vesicles to regulate their composition and physical properties in order to separately investigate the influence and importance of each parameter in mineral formation. Moreover, the results acquired using proteoliposomes will be compared to those found with natural MVs to determine conditions that can lead to the best biomaterial for bone regeneration purposes help us to resolv anomalous/pathological problems of calcification. (AU)