Understanding the involvement of Poly (ADP-ribose) in physiological and pathological calcification processes
Lead Research Organisation:
University of Cambridge
Department Name: Chemistry
Abstract
This PhD project will explore poly(ADP-Ribosyl)ation (PARylation), and PAR, which is the free molecule that results upon the removal of this post-translational modification of proteins. The PARylation reaction is mediated by PARP enzymes, which add PAR chains of heterogeneous length and branching to various amino acids residues (and even to DNA itself) depending on the required biological function. The first discovered and best characterised activator is DNA damage.
The main focus will be on understanding how Poly (ADP-ribose) is involved in mediating biomineralisation processes. Duer group proved that Poly (ADP-ribose) is found in bone and calcified arterial tissue, that it has affinity for calcium. The PAR-calcium complexes are electron dense spherical structures under TEM, change size depending on calcium concentration and were shown to bind preferentially to the gap zones of collagen where mineralisation is initiated. My research focuses on studying Poly (ADP-ribosylation) in two in vitro models for calcification: the MC3T3-E1 cell line (osteoblast like, for the study of bone) and bovine vascular smooth muscle cells (model for pathological calcification of arteries).
The roles played by PAR are dependent on its structure (size, branching pattern) which lead to distinct physico-chemical properties, such as affinity for calcium and the binding to specific proteins. This project aims to develop novel methods to characterise poly(ADP-ribose) in situ. The current approaches to determine properties such as extent of branching, conformation, size, involve isolating PAR fractions from cell lysates, but this is not always straightforward. NMR is poorly referenced in literature as a tool for studying PAR/ PAR-protein complexes, probably because of the heterogeneity in chain length. Very few protocols for isotopic labeling of the polymer exist and the labeling happens in a reaction tube, rather than living cells. Thus, this project will develop a methodology to add 13C and 15N at convenient positions in the poly (ADP-ribose) chains as they are produced in cells. This would allow studying the extent of branching, by comparing intensity of speci?fic peaks, and give insight about conformation, by means of multidimensional experiments. Using solid state NMR can overcome the need to extract PAR from the cells/ extracellular matrix and might be a promising technique for the study in situ. Signal enhancement technique, such as DNP, will be used to characterise the poly (ADP-ribose) deposited by the VSCM/ MC3T3 cells in the matrix.
The main focus will be on understanding how Poly (ADP-ribose) is involved in mediating biomineralisation processes. Duer group proved that Poly (ADP-ribose) is found in bone and calcified arterial tissue, that it has affinity for calcium. The PAR-calcium complexes are electron dense spherical structures under TEM, change size depending on calcium concentration and were shown to bind preferentially to the gap zones of collagen where mineralisation is initiated. My research focuses on studying Poly (ADP-ribosylation) in two in vitro models for calcification: the MC3T3-E1 cell line (osteoblast like, for the study of bone) and bovine vascular smooth muscle cells (model for pathological calcification of arteries).
The roles played by PAR are dependent on its structure (size, branching pattern) which lead to distinct physico-chemical properties, such as affinity for calcium and the binding to specific proteins. This project aims to develop novel methods to characterise poly(ADP-ribose) in situ. The current approaches to determine properties such as extent of branching, conformation, size, involve isolating PAR fractions from cell lysates, but this is not always straightforward. NMR is poorly referenced in literature as a tool for studying PAR/ PAR-protein complexes, probably because of the heterogeneity in chain length. Very few protocols for isotopic labeling of the polymer exist and the labeling happens in a reaction tube, rather than living cells. Thus, this project will develop a methodology to add 13C and 15N at convenient positions in the poly (ADP-ribose) chains as they are produced in cells. This would allow studying the extent of branching, by comparing intensity of speci?fic peaks, and give insight about conformation, by means of multidimensional experiments. Using solid state NMR can overcome the need to extract PAR from the cells/ extracellular matrix and might be a promising technique for the study in situ. Signal enhancement technique, such as DNP, will be used to characterise the poly (ADP-ribose) deposited by the VSCM/ MC3T3 cells in the matrix.
Organisations
People |
ORCID iD |
| Melinda Duer (Primary Supervisor) | |
| Adrian Murgoci (Student) |
| Description | The project is still in progress. So far the main focus of the project was to understand key interactions at molecular level between collagen residues and a molecule called poly (ADP-ribose), which is believed to play an important role in biomineralisation processes, via solid state NMR experiments. Using isotopic enrichment in 13C of these molecules ( and also amino acid residues), the structure of poly (ADP-ribose) was partially characterised so far, which can eliminate the requirement to isolate it from tissue samples via damaging treatments, for MS analysis. Moreover, new data gathered via enzymatic assays brought new insight on PARP enzymes producing PAR, the activity of some members of the PARP family in different compartments of cells, which could be relevant for bone development. |
| Exploitation Route | If the structure of poly (ADP-ribose) is fully characterised via solid state NMR, this new methodology might provide tools to study structure-dependent biological roles of PAR in various in vitro models. Moreover, a better understanding of bone development by the end of this award could open up the possibility of new research directions in the area (even outside of my research group) and the development of new treatments for bone/ calcification related diseases. |
| Sectors | Healthcare,Pharmaceuticals and Medical Biotechnology |