The mechanism of deposition of physiologic hydroxyapatite crystals (mineralization) in collagen-based tissues (bone, dentin, cementum, calcified cartilage, etc.) is a poorly understood complex process. It is our belief, that during HA (hydroxyapatite) formation both collagen and noncollagenous proteins (NCPs) regulate (promote or inhibit) the growth and proliferation of HA. Many of the NCPs have structures that are predominately random coils, and are thus classified as """"""""Intrinsically Disordered Proteins"""""""" (IDPs). IDPs are abundant in mineralizing systems of vertebrates and non-vertebrates, and it is thought that their flexible structures allow them to interact with their respective """"""""partners"""""""". Fibrillar collagen is one of the """"""""partners"""""""" to which mineralized tissue IDPs or their subunits have been shown to bind. The driving force for this reaction and the reasons that this binding takes place are not known. Another """"""""partner"""""""" for the mineralized tissue IDPs is HA. Several NCPs (e.g. small leucine rich proteoglycans (SLRPS) and other glycoproteins) have well defined, non-flexible structures, and also interact with collagen and HA. It is our hypothesis that the small, flexible IDPs, bind to collagen, then to HA nuclei or nascent HA, and become more ordered in consequence. The energetics of conformational change in the IDPs are postulated to be more favorable than the binding energetics of more ordered NCPs to collagen and HA, due to a lesser need for energy in the interaction process. The purpose of this R21 application is to develop and optimize analytical methods to determine the conformation of IDPs, and the interaction energetics needed for the regulation of HA growth, supporting or refuting this hypothesis. There are three specific aims:
Aim 1) :Demonstrate the conformational changes that occur in the binding of milk osteopontin (mOPN) and decorin (DCN), both referred to herein as """"""""study proteins"""""""", to HA using FTIR spectroscopy. Along with these data we will measure the binding affinities of the """"""""study proteins"""""""" to HA and collagen and also measure the kinetics of HA growth and replication in the presence of (a) collagen alone, (b) collagen coated with either """"""""study protein"""""""", (c) collagen formed in the presence of each """"""""study protein"""""""" separately, and (d) """"""""study protein"""""""" alone. These studies will determine the optimal """"""""study protein"""""""" to collagen ratio for use in aims 2 &3.
Aim 2) : Develop surface plasmon resonance for studies of collagen-NCP-HA interactions using """"""""study proteins"""""""".
Aim 3) : Develop a group of Fluorescence methods to study collagen-NCP- HA interaction and apply these to the """"""""study proteins"""""""". The development of these methods will enable us to begin to validate our hypotheses, while providing both new techniques for the study of IDP interaction with collagen and HA and new insight into the mechanisms of collagen-mediated biomineralization.
The goal of this study is to develop analytical methods for investigating the importance of collagen and collagen-associated proteins in the process of mineralization. Successful completion of this exploratory study will provide information on many techniques, previously unused, in the field, generating data that has the potential to change our understanding of the biomineralization process. Furthermore, these studies can lead to the development of treatments for common bone and tooth diseases like osteomalacia and osteoporosis as well as rarer diseases such as osteogenesis- and dentinogenesis- imperfecta and skeletal dysplasias, along with new tissue engineering strategies.
| Villarreal-Ramirez, Eduardo; Eliezer, David; Garduño-Juarez, Ramon et al. (2017) Phosphorylation regulates the secondary structure and function of dentin phosphoprotein peptides. Bone 95:65-75 |
| Boskey, Adele L; Villarreal-Ramirez, Eduardo (2016) Intrinsically disordered proteins and biomineralization. Matrix Biol 52-54:43-59 |
| Villarreal-Ramirez, Eduardo; Garduño-Juarez, Ramón; Gericke, Arne et al. (2014) The role of phosphorylation in dentin phosphoprotein peptide absorption to hydroxyapatite surfaces: a molecular dynamics study. Connect Tissue Res 55 Suppl 1:134-7 |
