Alan Fincham and the era of enamel protein Biochemistry

Enamel research experienced an unprecedented period of growth during the latter part of the 20th century until today. This growth is in part due to the contributions of a number of iconic scientists such as Alan G. Fincham, the focus of the present review. Alan was involved in many of the seminal discoveries of this time, including the identification of the critical amelogenin peptides TRAP and LRAP, the determination of the amelogenin amino acid sequence, the identification of the sole serin-16 phosphorylation site, and the amelogenin nanosphere theory. Alan was also a superb mentor to graduate students and others. His experience and leadership related to problem-based learning greatly affected predoctoral dental education at the University of Southern California and in the United States

Highly acidic pH facilitates enamel protein self-assembly, apatite crystal growth and enamel protein interactions in the early enamel matrix

Tooth enamel develops within a pH sensitive amelogenin-rich protein matrix. The purpose of the present study is to shed light on the intimate relationship between enamel matrix pH, enamel protein self-assembly, and enamel crystal growth during early amelogenesis. Universal indicator dye staining revealed highly acidic pH values (pH 3–4) at the exocytosis site of secretory ameloblasts. When increasing the pH of an amelogenin solution from pH 5 to pH 7, there was a gradual increase in subunit compartment size from 2 nm diameter subunits at pH 5 to a stretched configuration at pH6 and to 20 nm subunits at pH 7. HSQC NMR spectra revealed that the formation of the insoluble amelogenin self-assembly structure at pH6 was critically mediated by at least seven of the 11 histidine residues of the amelogenin coil domain (AA 46–117). Comparing calcium crystal growth on polystyrene plates, crystal length was more than 20-fold elevated at pH 4 when compared to crystals grown at pH 6 or pH 7. To illustrate the effect of pH on enamel protein self-assembly at the site of initial enamel formation, molar teeth were immersed in phosphate buffer at pH4 and pH7, resulting in the formation of intricate berry tree-like assemblies surrounding initial enamel crystal assemblies at pH4 that were not evident at pH7 nor in citrate buffer. Amelogenin and ameloblastin enamel proteins interacted at the secretory ameloblast pole and in the initial enamel layer, and co-immunoprecipitation studies revealed that this amelogenin/ameloblastin interaction preferentially takes place at pH 4—pH 4.5. Together, these studies highlight the highly acidic pH of the very early enamel matrix as an essential contributing factor for enamel protein structure and self-assembly, apatite crystal growth, and enamel protein interactions

Elongated Polyproline Motifs Facilitate Enamel Evolution through Matrix Subunit Compaction

Vertebrate body designs rely on hydroxyapatite as the principal mineral component of relatively light-weight, articulated endoskeletons and sophisticated tooth-bearing jaws, facilitating rapid movement and efficient predation. Biological mineralization and skeletal growth are frequently accomplished through proteins containing polyproline repeat elements. Through their well-defined yet mobile and flexible structure polyproline-rich proteins control mineral shape and contribute many other biological functions including Alzheimer's amyloid aggregation and prolamine plant storage. In the present study we have hypothesized that polyproline repeat proteins exert their control over biological events such as mineral growth, plaque aggregation, or viscous adhesion by altering the length of their central repeat domain, resulting in dramatic changes in supramolecular assembly dimensions. In order to test our hypothesis, we have used the vertebrate mineralization protein amelogenin as an exemplar and determined the biological effect of the four-fold increased polyproline tandem repeat length in the amphibian/mammalian transition. To study the effect of polyproline repeat length on matrix assembly, protein structure, and apatite crystal growth, we have measured supramolecular assembly dimensions in various vertebrates using atomic force microscopy, tested the effect of protein assemblies on crystal growth by electron microscopy, generated a transgenic mouse model to examine the effect of an abbreviated polyproline sequence on crystal growth, and determined the structure of polyproline repeat elements using 3D NMR. Our study shows that an increase in PXX/PXQ tandem repeat motif length results (i) in a compaction of protein matrix subunit dimensions, (ii) reduced conformational variability, (iii) an increase in polyproline II helices, and (iv) promotion of apatite crystal length. Together, these findings establish a direct relationship between polyproline tandem repeat fragment assemblies and the evolution and the design of vertebrate mineralized tissue microstructures. Our findings reveal that in the greater context of chordate evolution, the biological control of apatite growth by polyproline-based matrix assemblies provides a molecular basis for the evolution of the vertebrate body plan.</p

Elongated Polyproline Motifs Facilitate Enamel Evolution through Matrix Subunit Compaction

How does proline-repeat motif length in the proteins of teeth and bones relate to the evolution of vertebrates? Counterintuitively, longer repeat stretches are associated with smaller aggregated subunits within a supramolecular matrix, resulting in enhanced crystal length in mammalian versus amphibian tooth enamel

Amelogenin Supramolecular Assembly in Nanospheres Defined by a Complex Helix-Coil-PPII Helix 3D-Structure

Tooth enamel, the hardest material in the human body, is formed within a self-assembled matrix consisting mostly of amelogenin proteins. Here we have determined the complete mouse amelogenin structure under physiological conditions and defined interactions between individual domains. NMR spectroscopy revealed four major amelogenin structural motifs, including an N-terminal assembly of four α-helical segments (S9-V19, T21-P33, Y39-W45, V53-Q56), an elongated random coil region interrupted by two 310 helices (∼P60-Q117), an extended proline-rich PPII-helical region (P118-L165), and a charged hydrophilic C-terminus (L165-D180). HSQC experiments demonstrated ipsilateral interactions between terminal domains of individual amelogenin molecules, i.e. N-terminal interactions with corresponding N-termini and C-terminal interactions with corresponding C-termini, while the central random coil domain did not engage in interactions. Our HSQC spectra of the full-length amelogenin central domain region completely overlapped with spectra of the monomeric Amel-M fragment, suggesting that the central amelogenin coil region did not involve in assembly, even in assembled nanospheres. This finding was confirmed by analytical ultracentrifugation experiments. We conclude that under conditions resembling those found in the developing enamel protein matrix, amelogenin molecules form complex 3D-structures with N-terminal α-helix-like segments and C-terminal PPII-helices, which self-assemble through ipsilateral interactions at the N-terminus of the molecule

Epigenetics and Early Development

The epigenome controls all aspect of eukaryotic development as the packaging of DNA greatly affects gene expression. Epigenetic changes are reversible and do not affect the DNA sequence itself but rather control levels of gene expression. As a result, the science of epigenetics focuses on the physical configuration of chromatin in the proximity of gene promoters rather than on the mechanistic effects of gene sequences on transcription and translation. In the present review we discuss three prominent epigenetic modifications, DNA methylation, histone methylation/acetylation, and the effects of chromatin remodeling complexes. Specifically, we introduce changes to the methylated state of DNA through DNA methyltransferases and DNA demethylases, discuss the effects of histone tail modifications such as histone acetylation and methylation on gene expression and present the functions of major ATPase subunit containing chromatin remodeling complexes. We also introduce examples of how changes in these epigenetic factors affect early development in humans and mice. In summary, this review provides an overview over the most important epigenetic mechanisms and provides examples of the dramatic effects of epigenetic changes in early mammalian development

Epigenetic Repression of <i>RUNX2</i> and <i>OSX</i> Promoters Controls the Nonmineralized State of the Periodontal Ligament

The nonmineralized state of the mammalian periodontal ligament is one of the hallmarks of vertebrate evolution as it provides resilient and nontraumatic tooth anchorage for effective predation. Here we sought to determine how the chromatin state of key mineralization gene promoters contributes to the nonmineralized periodontal ligament in the midst of fully mineralized alveolar bone and cementum anchor tissues. In developing mouse periodontal tissues, RUNX2 was localized to alveolar bone–lining cells, while OSX was localized throughout the periodontal ligament’s soft tissue. Matching RT-PCR amplification data and western blot comparisons demonstrated that the expression of RUNX2 and OSX bone mineralization transcription factors was at least 2.5-fold elevated in alveolar bone osteoblasts versus periodontal ligament fibroblasts. ChIP enrichment data along the RUNX2 and OSX promoters revealed increased H3K4me3 marks in alveolar bone osteoblasts, while H3K9me3 and H3K27me3 marks were elevated in periodontal ligament fibroblasts. In support of an epigenetic mechanism responsible for the inhibition of mineralization gene expression in periodontal progenitors, histone methylation inhibitors DZNep and Chaetocin reactivated RUNX2 and OSX expression in periodontal progenitors and increased alkaline phosphatase and Alizarin Red, while the in vivo application of DZNep in rat maxillae resulted in aberrant mineralization in the periodontal ligament and a narrowing of the nonmineralized periodontal space. Together, these studies demonstrate that the nonmineralized state of the mammalian periodontal ligament is controlled by an epigenetic regulation of the RUNX2 and OSX key mineralization gene promoters

Changes in <i>Hox</i> Gene Chromatin Organization during Odontogenic Lineage Specification

Craniofacial tissues comprise highly evolved organs characterized by a relative lack of expression in the HOX family transcription factors. In the present study, we sought to define the epigenetic events that limit HOX gene expression from undifferentiated neural crest cells to semi-differentiated odontogenic progenitors and to explore the effects of elevated levels of HOX. The ChIP-chip data demonstrated high levels of repressive H3K27me3 marks on the HOX gene promoters in ES and cranial neural crest cells when compared to the H3K4me3 marks, while the K4/K27 ratio was less repressive in the odontogenic progenitors, dental follicle, dental pulp, periodontal ligament fibroblasts, alveolar bone osteoblasts, and cementoblasts. The gene expression of multiple HOX genes, especially those from the HOXA and HOXB clusters, was significantly elevated and many times higher in alveolar bone cells than in the dental follicle cells. In addition, the HOX levels in the skeletal osteoblasts were many times higher in the trunk osteoblasts compared to the alveolar bone osteoblasts, and the repressive mark H3K27me3 promoter occupancy was substantially and significantly elevated in the alveolar bone osteoblasts when compared to the trunk osteoblasts. To explore the effect of elevated HOX levels in craniofacial neural crest cells, HOX expression was induced by transfecting cells with the Cdx4 transcription factor, resulting in a significant decrease in the mineralization markers, RUNX2, OSX, and OCN upon HOX elevation. Promoting HOX gene expression in developing teeth using the small molecule EZH2 inhibitor GSK126 resulted in an increased number of patterning events, supernumerary cusp formation, and increased Hoxa4 and Hoxb6 gene expression when compared to the controls. Together, these studies illustrate the profound effects of epigenetic regulatory events at all stages of the differentiation of craniofacial peripheral tissues from the neural crest, including lineage specification, tissue differentiation, and patterning

Posttranslational Amelogenin Processing and Changes in Matrix Assembly during Enamel Development

The extracellular tooth enamel matrix is a unique, protein-rich environment that provides the structural basis for the growth of long and parallel oriented enamel crystals. Here we have conducted a series of in vivo and in vitro studies to characterize the changes in matrix shape and organization that take place during the transition from ameloblast intravesicular matrices to extracellular subunit compartments and pericrystalline sheath proteins, and correlated these changes with stages of amelogenin matrix protein posttranslational processing. Our transmission electron microscopic studies revealed a 2.5-fold difference in matrix subunit compartment dimensions between secretory vesicle and extracellular enamel protein matrix as well as conformational changes in matrix structure between vesicles, stippled materials, and pericrystalline matrix. Enamel crystal growth in organ culture demonstrated granular mineral deposits associated with the enamel matrix framework, dot-like mineral deposits along elongating initial enamel crystallites, and dramatic changes in enamel matrix configuration following the onset of enamel crystal formation. Atomic force micrographs provided evidence for the presence of both linear and hexagonal/ring-shaped full-length recombinant amelogenin protein assemblies on mica surfaces, while nickel-staining of the N-terminal amelogenin N92 His-tag revealed 20 nm diameter oval and globular amelogenin assemblies in N92 amelogenin matrices. Western blot analysis comparing loosely bound and mineral-associated protein fractions of developing porcine enamel organs, superficial and deep enamel layers demonstrated (i) a single, full-length amelogenin band in the enamel organ followed by 3 kDa cleavage upon entry into the enamel layer, (ii) a close association of 8–16 kDa C-terminal amelogenin cleavage products with the growing enamel apatite crystal surface, and (iii) a remaining pool of N-terminal amelogenin fragments loosely retained between the crystalline phases of the deep enamel layer. Together, our data establish a temporo-spatial correlation between amelogenin protein processing and the changes in enamel matrix configuration that take place during the transition from intracellular vesicle compartments to extracellular matrix assemblies and the formation of protein coats along elongating apatite crystal surfaces. In conclusion, our study suggests that enzymatic cleavage of the amelogenin enamel matrix protein plays a key role in the patterning of the organic matrix framework as it affects enamel apatite crystal growth and habit