CHARACTERIZATION OF PURIFIED MYOCILIN: INSIGHT INTO GLAUCOMA

纯化肌青蛋白的表征：洞察青光眼

• 批准号: 10622963
• 负责人: Raquel L Lieberman
• 金额: $ 7.67万
• 依托单位: GEORGIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10622963
• 关键词: 3-Dimensional; Academia; Affect; Age of Onset; Alleles; Alzheimer' s Disease; Amyloid; Amyloid Fibrils; Amyloidosis; Amyotrophic Lateral Sclerosis; Anatomy; Anterior eyeball segment structure; Aqueous Humor; Benign; Blindness; Cell Death; Cells; Chemicals; Clinic; Comprehension; Data; Databases; Defect; Deuterium; Development; Diagnosis; Disease; Drainage procedure; Endoplasmic Reticulum; Exhibits; Extracellular Matrix; Extracellular Matrix Proteins; Eye; Eye diseases; Fingerprint; Foundations; Functional disorder; Funding; Glaucoma; Glean; Goals; Grant; Growth; High temperature of physical object; Homeostasis; Horns; Hydrogen; Industry; Inherited; Knowledge; Lead; Length; Liquid substance; Mass Spectrum Analysis; Measures; Medical; Medicine; Methods; Missense Mutation; Modality; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Structure; Mutagenesis; Mutation; Nerve Degeneration; Nuclear Magnetic Resonance; Open-Angle Glaucoma; Outcome; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Peptides; Physiologic Intraocular Pressure; Physiology; Play; Process; Protein Biochemistry; Proteins; Relaxation; Research Personnel; Retina; Risk Factors; Role; Sampling; Seeds; Structure; Susceptibility Gene; Testing; Therapeutic; Time; TimeLine; Tissues; Toxic effect; Trabecular meshwork structure; Variant; Vertebral column; amyloid formation; biophysical properties; blind; cytotoxic; cytotoxicity; early onset; experimental study; gain of function; genome wide association study; infancy; insight; multidisciplinary; mutant; myocilin; non-Native; novel; novel therapeutics; olfactomedin; patient population; precision medicine; pressure; protein folding; proteostasis; rare variant; solid state nuclear magnetic resonance; structural biology; superoxide dismutase 1; symptom treatment

Glaucoma, a leading cause of blindness worldwide (70 million patients), is managed medically by treating the symptom of increased intraocular pressure (IOP), but 10% of patients still go blind. IOP is controlled in the anterior segment of the eye, which contains the trabecular meshwork (TM) extracellular matrix, the anatomical pathway for drainage of aqueous humor fluid. The TM tissue is diseased in most forms of glaucoma; loss of TM homeostasis leads to elevated IOP. Hereditary open angle glaucoma, affecting ~3 million young patients, is caused by mutations in myocilin, a protein highly expressed in the TM. Since 3/2011, studies funded from R01EY021205 have changed the paradigm for anti-glaucoma therapeutics by laying the molecular foundation for approaches that target the disease process, which are now being pursued in academia and industry. Studies from R01EY021205 have clarified molecular details of the toxic gain-of-function pathogenic mechanism in which mutant myocilin accumulates in the endoplasmic reticulum (ER) of TM cells, leading to TM cell death and an accelerated timeline for vision loss. Studies from R01EY021205 (a) contributed fundamental knowledge of myocilin structure, (b) discovered a counter-productive interaction between myocilin and the ER-resident Hsp90 chaperone Grp94, and (c) characterized myocilin misfolding as amyloid. Wild-type and many different myocilin variants harbor a misfolding propensity; thus, proteostasis issues identified in familial myocilin-associated glaucoma are likely at play in many more patients. Amyloid formation by myocilin places glaucoma alongside more well-studied amyloid diseases like Alzheimer and SOD-1 dependent amyotrophic lateral sclerosis, yet comprehension of the role of amyloid in glaucoma is in its infancy. The current objective is to better understand molecular aspects of myocilin fibrilization, focused on the relevant olfactomedin (OLF) domain. The multidisciplinary team led by Raquel Lieberman will (a) clarify initiation of aggregation by studying solution structures of wild-type and selected OLF variants, as well as corresponding multi-length scale dynamics, using hydrogen-deuterium exchange mass spectrometry and nuclear magnetic resonance (NMR) structure and relaxation methods (Wade Van Horn, Co- I) (b) compare the end-point structures of selected OLF aggregates to known amyloids by solid state NMR (Anant Paravastu, Co-I) and evaluate cytotoxicity of intermediate aggregates and (c) evaluate common allele full-length myocilin variants for experimental hallmarks of pathogenicity. The expected outcome is a better understanding of the myocilin misfolding process at the molecular level, including molecular determinants of pathogenicity, to enable novel modalities for studying, diagnosing, and treating myocilin-associated glaucoma. More broadly, continued structure/dysfunction studies of myocilin will not only contribute to an understanding of glaucoma and its role in the TM, but will also extend comprehension of the many other OLF domains, which are implicated broadly in physiology and diseases.

青光眼是世界范围内导致失明的主要原因之一(7000万患者)，通过治疗进行医学治疗。 眼压升高的症状，但仍有10%的患者失明。眼压控制在 眼前段，其中包含小梁网络(TM)细胞外基质，解剖 房水引流通道。TM组织在大多数形式的青光眼中都有病变； TM动态平衡导致眼压升高。遗传性开角型青光眼，影响约300万年轻患者， 是由TM中高度表达的一种蛋白质myoclin的突变引起的。自2011年3月起，资助的研究 R01EY021205通过奠定分子基础改变了抗青光眼治疗的范式 针对疾病过程的方法，目前学术界和工业界正在探索这一方法。 来自R01EY021205的研究已经阐明了毒性功能获得致病的分子细节 突变的霉菌素在TM细胞的内质网(ER)中积聚的机制，导致 TM细胞死亡和视力丧失的加速时间线。来自R01EY021205(A)的研究贡献 Myocin结构的基本知识，(B)发现Myocin与Myocin之间的相互作用适得其反 和内质网驻留的Hsp90伴侣Grp94，和(C)将myocin错误折叠表征为淀粉样蛋白。野生型 许多不同的myocin变体都有错误折叠的倾向；因此，在 家族性肌球蛋白相关性青光眼可能在更多的患者中起作用。 由霉菌蛋白形成的淀粉样蛋白使青光眼与更多研究较好的淀粉样蛋白疾病并存，如 阿尔茨海默病和SOD-1依赖性肌萎缩侧索硬化症，但对淀粉样蛋白在 青光眼还处于婴儿期。目前的目标是更好地了解肌球蛋白的分子方面 纤维化，侧重于相关的嗅觉素(OLF)领域。拉克尔领导的多学科团队 利伯曼将(A)通过研究野生型和选定的OLF的溶液结构来阐明聚集的启动 变种，以及相应的多长度标度动力学，使用氢-氚交换质量 光谱和核磁共振(核磁共振)结构和松弛方法(Wade Van Horn，Co- I)(B)通过固体核磁共振将选定的OLF聚集体的终点结构与已知的淀粉样蛋白进行比较 (Anant Paravastu，Co-I)和评估中间聚集体的细胞毒性以及(C)评估常见的等位基因 作为致病性实验标志的全长myocin变异体。预期的结果是更好的 在分子水平上对myocin错误折叠过程的理解，包括 致病性，使研究、诊断和治疗肌球蛋白相关青光眼的新方法成为可能。 更广泛地说，对肌球蛋白的持续结构/功能研究不仅有助于理解 青光眼及其在TM中的作用，但也将扩大对许多其他OLF领域的理解，这些领域 与生理学和疾病有广泛的关系。