Enamel-derived modulators of biomineralization

生物矿化是骨骼和牙齿等重要器官形成和维持的基础，但对这一过程的详细机制知之甚少。我们早些时候已经确定amelotin（AMTN）是一种牙釉质特异性蛋白质，并在之前的授权任期内证明了AMTN促进羟基磷灰石（HA）的形成，羟基磷灰石是骨骼和牙齿的矿物质。该提案的总体目标是优化和利用AMTN的矿物质诱导特性，用于釉质和其他矿化组织的矿化。背景资料：大部分牙釉质形成于主要（90%）由磷酸化蛋白釉原蛋白（AMEL）组成的无细胞有机基质中。该基质引导形成高度组织化的HA晶体棒交织网络，这赋予釉质其显著的机械性能。然而，釉质的表面结构独特，完全矿化且致密，类似于矿物“清漆”层，而不是矿化的网状结构。AMTN的定位与釉质表面层的形成完全一致。我们在最近的大量工作中已经证明，AMTN促进牙釉质中的HA，但也促进其他矿化组织中的HA。我们还表明，AMTN衍生肽的磷酸化可以增强其矿物质促进特性。在此基础上，本文提出了三个主要目标：1.评估AMTN丝氨酸磷酸化在促进矿化中的作用我们将优化重组生产在进化保守丝氨酸残基（pAMTN）处磷酸化的AMTN的方案。将在体外测定pAMTN的矿物质诱导特性，并与我们已经常规生产的未修饰的AMTN进行比较。2.生产包含AMTN蛋白或肽的HA纳米颗粒我们将生产小HA纳米颗粒作为AMTN和衍生肽的载体，包括未修饰和磷酸化版本，并对其进行详细表征。我们期望这些纳米颗粒增强AMTN和肽的矿物质促进特性，更容易整合到预先存在的矿物质中，并延长蛋白质和肽的保质期。3.在体外和体内测试AMTN-HA纳米颗粒的矿物质诱导性能HA-AMTN纳米颗粒将应用于体外从离体牙齿获得的釉质病变和脱矿牙本质。类似地，将用HA-AMTN纳米颗粒处理脱矿的大鼠臼齿釉质和颅骨以评估它们的体内效果。将确定矿化程度以及形成的矿物的范围和性质。重要性：如果成功的话，HA-AMTN纳米颗粒的确定的矿物质诱导特性将广泛适用于实现矿化和将修复和植入材料整合到矿化组织中。

Biomineralization is the basis for formation and maintenance of such important organs as bones and teeth, but the detailed mechanisms of this process are poorly understood. We have earlier identified amelotin (AMTN) as an enamel-specific protein and have demonstrated during the previous grant tenure that AMTN promotes the formation of hydroxyapatite (HA), the mineral of bones and teeth. The overarching goal of this proposal is to optimize and harness the mineral-inducing properties of AMTN for remineralization of enamel and other mineralized tissues. Background: The bulk of dental enamel is formed in an acellular organic matrix consisting predominantly (90%) of the phosphorylated protein amelogenin (AMEL). This matrix guides the formation of a highly organized interwoven network of HA crystal rods, which give enamel its remarkable mechanical properties. The surface of enamel, however, is structurally distinct, fully mineralized and compact, and resembles a mineral "varnish" layer rather than a mineralized mesh work. The localization of AMTN coincides perfectly with the formation of this enamel surface layer. We have demonstrated in extensive recent work that AMTN promotes HA in enamel, but also in other mineralized tissue. We have also shown that phosphorylation of AMTN-derived peptides can enhance their mineral-promoting properties. Based on this work, the current proposal has three main objectives as follows: 1. Evaluate the role of AMTN serine phosphorylation in promoting mineralization We will optimize protocols for the recombinant production of AMTN that is phosphorylated at evolutionary conserved serine residues (pAMTN). The mineral inducing properties of pAMTN will be determined in vitro and compared with those of unmodified AMTN, which we already produce routinely. 2. Produce HA nanoparticles that include AMTN protein or peptides We will produce small HA nanoparticles as carriers for AMTN and derived peptides, both in unmodified and phosphorylated versions, and characterize them in detail. We expect these nanoparticles to enhance the mineral-promoting properties of AMTN and peptides, integrate more readily into pre-existing mineral and prolong the shelf-life of proteins and peptides. 3. Test the mineral-inducing properties of AMTN-HA nanoparticles in vitro and in vivo HA-AMTN nanoparticles will be applied to enamel lesions and demineralized dentin obtained from extracted teeth in vitro. Similarly, demineralized rat molar enamel and calvarial bones will be treated with HA-AMTN nanoparticles to evaluate their effect in vivo. The degree of remineralization and the extent and nature of the mineral formed will be determined. Significance: If successful, the defined mineral-inducing properties of HA-AMTN nanoparticles will be widely applicable to achieve remineralization and integration of restorative and implant materials into mineralized tissues.