Molecular Characterization of Key Proteins Directing Otoconial Mineralization

指导耳锥矿化的关键蛋白的分子表征

• 批准号: 8263376
• 负责人: RUEDIGER R THALMANN
• 金额: $ 39.54万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-09 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8263376
• 关键词: Affect; Age; Aging-Related Process; American; Amphibia; Area; Biochemical; Biological; Biological Models; Biomimetics; C-terminal; Calcite; Calculi; Cessation of life; Characteristics; Collaborations; Collagen Type X; Complement; Complement 1q; Deletion Mutagenesis; Disease; Distant; Ear; Elderly; Equilibrium; Evaluation; Fiber; Fracture; Functional disorder; Generations; Globular Protein Foldings; Gravity Perception; Growth; Health; Human; Hydrogen Bonding; In Situ; In Vitro; Invertebrates; Joints; Kinetics; Maintenance; Measurement; Measures; Mediating; Methods; Microscopic; Modification; Molecular; Morphology; Mutagenesis; Mutate; Pathologic; Peptides; Play; Post-Translational Protein Processing; Process; Proteins; Quality of life; Recombinants; Research; Research Personnel; Resolution; Role; Scaffolding Protein; Science; Site; Solutions; Staging; Structure; System; Techniques; Technology; Testing; Tooth structure; Wheat; base; biomineralization; bone; demineralization; design; falls; glycosylation; insight; knowledge base; mineralization; molecular scale; nanoparticle; neutrophil; normal aging; otoconia; otoconin 90; otolin-1; particle; prevent; public health relevance; synthetic peptide; tool; two-dimensional

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.

描述(申请人提供)：耳石是微小尺寸的生物矿物颗粒，对感知重力和保持平衡是必不可少的。数以百万计的美国老年人的行动能力、生活质量和健康都受到耳石渐进性脱矿的影响。目前，还没有有效的手段来防止或抵消这一进程。由于令人望而却步的解剖学和生物学限制，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，通过产生主要基质蛋白Otoconin 90和22以及Otolin 1的重组版本。体外评估了这些蛋白对方解石晶体生长参数的影响，包括成核、生长速度和形态，并结合生物物理溶液状态研究，提供了基本但有限的结构和机制方面的见解。到目前为止，我们的结果与其他研究人员的结果相结合，提出了三个关键的概念：1)三级结构很重要，因为它并列在远距离残基之间，在蛋白质和矿化物种之间建立了一种立体化学关系，而这种关系不可能来自线性链或随机卷曲(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/或调节聚集方面的作用。3)耳石的纤维状奥氏石-1芯指示不溶有机基质在成矿初期的导向作用。因此，我们现在已经到了一个阶段，需要更先进的工具，能够精确地定量控制生长条件，并在分子水平上评估生长动力学和潜在的机制。为此，我们与晶体生长科学领域的领先专家J.DeYoreo联手，他建立了最强大的分子尺度技术。该团队将进行高级分子水平的研究，我们将为其提供必要的高质量正常和突变蛋白质和结构。在提交给分子水平的研究之前，将通过生化和生物物理研究，并辅之以标准的晶体生长测定，对这些研究进行详尽的验证。预计联合小组将达成基本的机械性见解，使我们能够设计和实施预防、减缓或改善耳锥变性的措施。 与公共卫生相关：我们打算利用最先进的生化/生物物理技术，研究耳石的形成、维持和易损性。耳石是耳朵中的“结石”，在维持平衡方面发挥着重要作用。在正常的衰老过程和多种疾病的作用下，耳锥会退化，导致失去平衡、灵活性、跌倒和骨折--这是导致老年人意外死亡的主要原因。