Molecular Characterization of Key Proteins Directing Otoconial Mineralization

描述（由申请人提供）：耳石是微小大小的生物矿物颗粒，对感知重力和维持平衡至关重要。数以百万计的美国老年人在他们的行动能力，生活质量和他们的健康受到影响的进行性耳蜗脱矿。目前，没有有效的手段来防止或抵消这一过程。由于解剖学和生物学的限制，耳蜗的研究远远落后于其他系统，如骨和牙齿。通过生成主要基质蛋白Otoconin 90和Otoconin 22以及Otolin 1的重组版本，我们已经克服了严格耳锥体研究的主要障碍。在体外评估了蛋白质对方解石晶体生长参数的影响，包括成核、生长速率和形态，并结合生物物理溶液状态研究，提供了必要的，但有限的结构和机制见解。到目前为止，我们的研究结果与其他研究人员的研究结果相结合，提出了三个关键的概念：1)三级结构很重要，因为它将远处的残基并列在一起，从而在蛋白质和矿化物种之间建立了一种立体化学关系，这种关系不能从线性链或随机线圈（即一级结构）中产生。2)耳膜是中晶，因此有必要了解它们在稳定纳米颗粒（可能是无定形的）和/或介导聚集方面的作用。3)耳膜纤维状Otolin - 1核表明不溶性有机基质在矿化初期起指导作用。因此，我们现在已经到了一个阶段，需要更先进的工具，能够对生长条件进行精确的定量控制，并在分子水平上评估生长动力学和潜在机制。为此，我们与J. DeYoreo合作，J. DeYoreo是晶体生长科学领域的领先专家，他建立了最强大的分子尺度技术。该团队将进行先进的分子水平研究，我们将提供必要的高质量正常和突变蛋白质和构建体。在提交给分子水平研究之前，这些将通过生化和生物物理研究进行彻底验证，并辅以标准晶体生长测定。联合小组有望获得基本的机制见解，使我们能够设计和实施预防、减缓或改善耳锥体变性的措施。

DESCRIPTION (provided by applicant): Otoconia are biomineral particles of microscopic size essential for perception of gravity and maintenance of balance. Millions of older Americans are affected in their mobility, quality of life, and in their health by progressive demineralization of otoconia. Currently, no effective means to prevent or counteract this process are available. Because of prohibitive anatomical and biological constraints, otoconial research is lagging far behind other systems, such as bone and teeth. We have overcome the major obstacles to rigorous otoconial research by generating recombinant versions of the principal matrix proteins, Otoconin 90 and 22, as well as Otolin 1. The proteins were evaluated in vitro for their effects upon calcite crystal growth parameters, including nucleation, growth rates and morphology, and combined with biophysical solution state studies, which provided essential, but limited structural and mechanistic insights. Our results to date combined with those of other researchers suggest three key concepts to be pursued: 1) Tertiary structure matters because it juxtaposes distant residues to create a stereochemical relationship between protein and mineralizing species that could not come about from a linear chain or a random coil (i.e. primary structure). 2) Otoconia are mesocrystals and therefore it is necessary to understand their role in stabilizing nanoparticles (that may be amorphous) and/or mediating aggregation. 3) The fibrillar Otolin 1 core of otoconia indicates a role for an insoluble organic matrix in directing the initial stages of mineralization. Thus we have now arrived at a stage that demands more advanced tools capable of precise quantitative control over growth conditions and evaluation of growth kinetics and underlying mechanisms at a molecular level. For this purpose we joined forces with J. DeYoreo, a leading expert in crystal growth science who has established the most powerful molecular scale technology. This team will perform the advanced molecular level studies for which we will provide the necessary high quality normal and mutated protein and constructs. These will be validated exhaustively by biochemical and biophysical studies, complemented by standard crystal growth determinations, prior to submitting them to molecular level studies. The joint team is expected to arrive at essential mechanistic insights, enabling us to design and implement measures to prevent, slow down or ameliorate otoconial degeneration.