Molecular Characterization of Key Proteins Directing Otoconial Mineralization

描述（由申请人提供）：耳石是对重力感知和维持平衡至关重要的微观尺寸的生物矿物颗粒。数以百万计的美国老年人的活动能力、生活质量和健康受到耳石进行性脱矿的影响。目前，没有有效的手段来防止或抵制这一进程。由于解剖学和生物学的限制，耳锥研究远远落后于其他系统，如骨骼和牙齿。我们已经克服了严格的耳锥研究的主要障碍，产生重组版本的主要基质蛋白，耳锥蛋白90和22，以及耳锥蛋白1。蛋白质在体外评估其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的研究，这提供了必要的，但有限的结构和机制的见解。我们迄今为止的研究结果与其他研究人员的研究结果相结合，提出了三个关键概念：1）三级结构很重要，因为它将遥远的残基并置，在蛋白质和矿化物质之间建立了一种立体化学关系，这种关系不能来自线性链或无规卷曲（即一级结构）。2)耳石是介晶，因此有必要了解它们在稳定纳米颗粒（可能是无定形的）和/或介导聚集中的作用。3)耳石的纤维状Otolin 1核心表明不溶性有机基质在指导矿化的初始阶段中的作用。因此，我们现在已经达到了一个阶段，需要更先进的工具，能够精确的定量控制生长条件和生长动力学的评价和潜在的机制在分子水平上。为此，我们与晶体生长科学领域的领先专家J. DeYoreo合作，他建立了最强大的分子级技术。该团队将进行先进的分子水平研究，我们将为此提供必要的高质量正常和突变蛋白质和构建体。在将其提交分子水平研究之前，将通过生物化学和生物物理研究进行彻底验证，并辅以标准晶体生长测定。联合团队有望获得重要的机制见解，使我们能够设计和实施预防，减缓或改善耳石退化的措施。 公共卫生相关性：我们打算使用最先进的生物化学/生物物理技术研究耳石的形成、维持和脆弱性，耳石是耳朵中的“石头”，在维持平衡方面起着至关重要的作用。在正常的老化过程和许多疾病耳石退化，导致失去平衡，敏捷，福尔斯和骨质疏松症的主要原因意外死亡的老年人。

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. PUBLIC HEALTH RELEVANCE: We intend to study the formation, maintenance and vulnerability of otoconia, the "stones" in the ear that play an essential role in the maintenance of balance, using state-of-the art biochemical/biophysical techniques. In the normal aging process and numerous diseases otoconia degenerate, resulting in loss of equilibrium, agility, falls, and bone fracture-the leading cause of accidental death in the elderly.