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: 8232001
• 负责人: Raquel L Lieberman
• 金额: $ 29.23万
• 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 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8232001
• 关键词: Age of Onset; Amyloid; Anterior; Aqueous Humor; Behavior; Binding; Biological Assay; Biology; Blindness; Calorimetry; Cell Death; Cells; Cellular Stress; Cessation of life; Circular Dichroism; Cystic Fibrosis; Defect; Detection; Disease; Endoplasmic Reticulum; Escherichia coli; Eukaryota; Event; Exhibits; Extracellular Matrix Proteins; Eye; Eye diseases; Eyedrops; Fiber; Fluorescence; Functional disorder; Gaucher Disease; Glaucoma; Goals; Human; In Vitro; Individual; Inherited; Kinetics; Knockout Mice; Knowledge; Lasers; Lead; Length; Libraries; Link; Missense Mutation; Molecular; Morphology; Mutation; Nerve Degeneration; Open-Angle Glaucoma; Operative Surgical Procedures; Ophthalmologist; Outcome; Pathogenesis; Pathway interactions; Patients; Physiologic Intraocular Pressure; Physiological; Property; Proteins; Protocols documentation; Quality Control; Recombinants; Retina; Retinal Degeneration; Retinal Ganglion Cells; Screening procedure; Solutions; Staining method; Stains; Structure; Symptoms; System; Techniques; Temperature; Therapeutic; Trabecular meshwork structure; Variant; Visual Fields; Work; X-Ray Crystallography; biological adaptation to stress; drug discovery; early onset; extracellular; gain of function; insight; monomer; mutant; myocilin; olfactomedin; pressure; prevent; protein aggregate; protein misfolding; public health relevance; small molecule; three dimensional structure

DESCRIPTION (provided by applicant): Glaucoma, the leading cause of blindness in the world, has no direct treatment. Eye drops, laser treatment, and/or surgery medically manage the symptom of increased intraocular pressure observed prior to retinal degeneration and loss of visual field, but do not constitute a therapy. Pressure is controlled in the anterior region of the eye, within the anatomical pathway for outflow of aqueous humor, called the trabecular extracellular meshwork (TEM). Of the ~70 million glaucoma cases, ~4% comprise an autosomal-dominant, inherited form closely linked to mutations in myocilin, a TEM component. Over 90% of the mutations in myocilin occur within its olfactomedin (OLF) domain, which is highly conserved among higher eukaryotes but is of unknown structure and function. Glaucoma-causing mutant myocilins are poorly secreted out of human trabecular meshwork (HTM) cells to the TEM. Instead, they accumulate in the endoplasmic reticulum (ER), resulting in abnormal TEM morphology, HTM cell death, and early-onset glaucoma. Interestingly, homozygous and heterozygous myocilin knock-out mice, and individuals with myocilin truncation mutations, exhibit no ocular abnormalities. Thus, pathogenesis is likely caused by a toxic gain-of-function mechanism, placing a significant number of glaucoma cases within the framework of a disease of protein misfolding and mistrafficking. The primary objective of this proposal is to characterize purified wild-type and mutant myocilins using solution biophysical techniques and X-ray crystallography to gain a better understanding of myocilin structure, function, and folding. Although difficulties in recombinant expression have limited in vitro characterization of myocilin thus far, we have recently filled this need for the key OLF domain. We have purified monomers, cytosolic aggregates, and insoluble inclusions from our recombinant system. The proposed work will lead to a molecular understanding of the pathogenesis in inherited and age-onset glaucoma, and thereby broaden our knowledge of protein conformational disorders and the function of OLF domains in biology. The proposed studies will also provide new opportunities for drug discovery for myocilin glaucoma, both "bottom-up" via a new stability assay, as well as "top-down" using the three dimensional structure. To date, our in vitro studies support the hypothesis that mutant myocilins retain near wild-type structure but exhibit compromised stability. We further hypothesize that these defects are recognized by ER quality control machinery, which prevents the mutant myocilins from being secreted to the TEM. The accumulation of mutant myocilin provokes cell stress responses that lead to HTM cell death. Thus, our long-term goal is to use the knowledge of myocilin structure, stability, and folding to identify small molecules that enable mutant myocilin to escape detection by ER quality control and prevent aggregation in the TEM. This treatment would reduce ER accumulation and pathogenic downstream events, and thereby directly delay the onset of intraocular pressure and subsequent vision loss for inherited glaucoma patients. PUBLIC HEALTH RELEVANCE: Our long-term goal is to develop a new therapy for glaucoma, a prevalent eye disease characterized by increased intraocular pressure, neurodegeneration of the retina, and vision loss. We are studying myocilin, an extracellular matrix protein involved in regulating eye pressure; mutations in myocilin lead to early-onset, inherited forms of glaucoma. We will study myocilin and disease-causing mutants in terms of their structure and stability, which will guide our efforts to develop an assay that will identify therapeutic compounds that bind to, and stabilize, mutant myocilin to delay the onset of inherited glaucoma.

描述（由申请人提供）：青光眼是世界上导致失明的主要原因，目前尚无直接治疗方法。滴眼剂、激光治疗和/或手术可通过医学手段控制视网膜变性和视野丧失之前观察到的眼压升高症状，但并不构成治疗。压力在眼睛前部区域控制，位于房水流出的解剖通路内，称为小梁细胞外网 (TEM)。在约 7000 万例青光眼病例中，约 4% 属于常染色体显性遗传形式，与肌纤蛋白（一种 TEM 成分）的突变密切相关。 myocilin 超过 90% 的突变发生在其 olfactomedin (OLF) 结构域内，该结构域在高等真核生物中高度保守，但结构和功能未知。导致青光眼的突变肌纤蛋白很少从人小梁网 (HTM) 细胞中分泌到 TEM。相反，它们在内质网 (ER) 中积累，导致 TEM 形态异常、HTM 细胞死亡和早发性青光眼。有趣的是，纯合和杂合肌纤蛋白敲除小鼠以及具有肌纤蛋白截短突变的个体没有表现出眼部异常。因此，发病机制可能是由有毒的功能获得机制引起的，将大量青光眼病例置于蛋白质错误折叠和错误运输疾病的框架内。 该提案的主要目的是使用溶液生物物理技术和 X 射线晶体学来表征纯化的野生型和突变型肌纤蛋白，以更好地了解肌纤蛋白的结构、功能和折叠。尽管迄今为止重组表达的困难限制了肌纤蛋白的体外表征，但我们最近满足了对关键 OLF 结构域的这一需求。我们从重组系统中纯化了单体、胞质聚集体和不溶性内含物。这项工作将有助于从分子角度理解遗传性青光眼和老年性青光眼的发病机制，从而拓宽我们对蛋白质构象紊乱和 OLF 结构域在生物学中的功能的了解。 拟议的研究还将为 myocilin 青光眼的药物发现提供新的机会，既通过新的稳定性测定进行“自下而上”，又使用三维结构进行“自上而下”。迄今为止，我们的体外研究支持这样的假设：突变型肌纤蛋白保留接近野生型的结构，但表现出稳定性受损。我们进一步假设这些缺陷被 ER 质量控制机制识别，从而阻止突变肌纤蛋白被分泌到 TEM。突变肌纤蛋白的积累会引发细胞应激反应，导致 HTM 细胞死亡。因此，我们的长期目标是利用肌纤蛋白结构、稳定性和折叠方面的知识来识别小分子，使突变肌纤蛋白能够逃避 ER 质量控制的检测并防止在 TEM 中聚集。这种治疗将减少 ER 积累和致病性下游事件，从而直接延迟遗传性青光眼患者的眼内压发作和随后的视力丧失。 公众健康相关性：我们的长期目标是开发一种治疗青光眼的新疗法，青光眼是一种常见的眼病，其特征是眼压升高、视网膜神经变性和视力丧失。我们正在研究肌纤蛋白，一种参与调节眼压的细胞外基质蛋白；肌纤蛋白突变会导致早发性遗传性青光眼。我们将研究肌纤蛋白和致病突变体的结构和稳定性，这将指导我们开发一种检测方法，识别与突变肌纤蛋白结合并稳定突变体肌纤蛋白以延缓遗传性青光眼发病的治疗化合物。