Characterization of purified myocilin: glaucoma as a protein misfolding disease

纯化肌纤蛋白的表征：青光眼作为一种蛋白质错误折叠疾病

• 批准号: 9239535
• 负责人: Raquel L Lieberman
• 金额: $ 35.54万
• 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 2021-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9239535
• 关键词: Adhesions; Adopted; Amyloid; Anatomy; Anterior; Aqueous Humor; Architecture; Behavior; Binding; Biological; Biological Assay; Biology; Biomechanics; Biophysics; Blindness; C-terminal; Cell Death; Cell secretion; Cells; Chemicals; Communities; Comprehension; Computer Simulation; Defect; Deposition; Development; Disease; Drainage procedure; Endoplasmic Reticulum; Epitopes; Extracellular Matrix; Extracellular Matrix Proteins; Eye; Eye diseases; Functional disorder; Glaucoma; Goals; Grant; Inherited; Kinetics; Knowledge; Lead; Length; Libraries; Ligands; Link; Liquid substance; Mass Spectrum Analysis; Medical; Methods; Molecular; Molecular Chaperones; Molecular Conformation; Molecular Structure; Mutation; N-terminal; Nerve Degeneration; Open-Angle Glaucoma; Outcome; Pathogenesis; Pathway interactions; Phagocytosis; Physiologic Intraocular Pressure; Physiology; Population; Primary Cell Cultures; Process; Property; Protein Biochemistry; Protein Conformation; Proteins; Proteomics; Research; Research Personnel; Retina; Retinal Degeneration; Risk Factors; Role; Structure; Therapeutic; Tissues; Trabecular meshwork structure; Variant; Visual Fields; Work; base; c new; crosslink; cytotoxic; early onset; electric impedance; extracellular; high throughput screening; improved; insight; interest; mutant; myocilin; new therapeutic target; non-Native; novel; novel therapeutics; olfactomedin; pressure; prevent; protein misfolding; screening; small molecule; small molecule therapeutics; structural biology; targeted agent

PROJECT SUMMARY Glaucoma, a leading cause of blindness, is managed medically by treating the causal risk factor of increased intraocular pressure (IOP), which is typically observed prior to retina degeneration and loss of visual field. IOP is controlled in the anterior region of the eye, which contains the trabecular meshwork (TM) extracellular matrix, the anatomical pathway for drainage of aqueous humor fluid. Of the ~45 million cases of open angle glaucoma worldwide, ~3% are linked to mutations in myocilin, a protein highly expressed in the TM. Despite considerable research effort over ~20 years, little is known about the structure or function of myocilin. An improved molecular understanding of myocilin in its normal and disease states will change the paradigm for anti-glaucoma therapeutics by enabling agents that target the disease process instead of indirectly controlling IOP. Disease-associated mutations in myocilin are found throughout its sequence. In the prior grant period, we biophysically and structurally characterized the variants clustered in its C-terminal olfactomedin (mOLF) domain, lending critical new details and support for the predominant working hypothesis in which mutations localized to myoc-OLF lead to a gain of toxic function: Endoplasmic-reticulum (ER)-associated degradation is inhibited by an aberrant interaction between myocilin and the ER-resident chaperone Grp94, leading to amyloid deposits of mutant myocilin within TM cells, which are cytotoxic. The resulting accumulation of TM cell debris is thought to impede fluid outflow from the TM, causing IOP elevation. Continued structure/dysfunction studies of myocilin will not only contribute to our understanding of glaucoma and its role in the TM, but would also broaden our comprehension of the many other OLF domains, which are implicated broadly in physiology and diseases. The objectives of this proposal are to expand our molecular comprehension of structure and misfolding in myocilin-associated glaucoma as well as provide a path forward for functional studies and the discovery of small molecules that mitigate aberrant myocilin behavior. We will (1) elucidate the architecture of native full- length myocilin, which is dictated by N-terminal coiled-coils, and characterize biophysical and cellular properties of disease variants found therein, (2) clarify the interaction between myocilin and Grp94 at the molecular level, and (3) implement two high throughput assays. The expected outcomes are (1) the full scope of the misfolding disease mechanism for glaucoma-associated myocilin, (2) expansion of our knowledge of protein conformational disorders, (3) new insights into Grp94 chaperone biology, and (4) novel ligand assays based on the myoc-OLF structure and mOLF/Grp94 interaction for the identification of therapeutic small molecules.

项目摘要 青光眼是失明的主要原因，通过治疗因果风险因素来管理 人眼压（IOP）增加，通常在视网膜变性和视觉丧失之前观察到这一点 场地。 IOP在眼睛的前区域受控，其中包含小梁网（TM） 细胞外基质，这是流体流体排水的解剖途径。约有4500万箱 全球开放角度青光眼，约3％与肌动蛋白的突变有关，肌动蛋白是一种高度表达的蛋白 TM。尽管大量研究了约20年，但对 肌动蛋白。在正常状态和疾病状态下对肌动蛋白的分子理解的提高将改变 通过使针对疾病过程的药物而不是 间接控制IOP。 在整个序列中都发现了肌动蛋白中与疾病相关的突变。在以前的赠款期内，我们 在生物物理和结构上表征了聚集在其C末端嗅觉素（MOLF）中的变体 领域，贷款关键的新细节和对主要工作假设的支持，其中突变 本地将Myoc-folf局限于有毒功能：内质 - 逆转（ER）相关的降解是 由于肌动蛋白和er-er-ersent resident Grp94之间的异常相互作用所抑制，导致 TM细胞中突变肌动蛋白的淀粉样蛋白沉积物，其细胞毒性。 TM电池的产生积累 杂物被认为会阻碍TM的流体流出，从而导致IOP升高。持续结构/功能障碍 肌动蛋白的研究不仅会有助于我们对青光眼及其在TM中的作用的理解，而且还会有助于 还扩大了我们对许多其他OLF领域的理解，这些领域广泛涉及生理学 和疾病。 该提案的目标是扩大我们对结构和错误折叠的分子理解 与肌动蛋白相关的青光眼，并为功能研究提供了前进的路径和发现 减轻异常肌动蛋白行为的小分子。我们将（1）阐明本地全部的架构 长度肌动蛋白，由N末端卷曲螺旋决定，并表征生物物理和细胞 发现其中的疾病变异的特性，（2）阐明在 分子水平，（3）实施两个高通量测定。预期的结果是（1）完整范围 与青光眼相关的肌动蛋白的错误折叠疾病机制的疾病机制，（2）我们对 蛋白质构象疾病，（3）对GRP94伴侣生物学的新见解和（4）新型配体分析 基于MYOC-FOL结构和MOLF/GRP94相互作用，用于识别治疗性小的 分子。