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）：青光眼是世界上致盲的主要原因，目前尚无直接治疗方法。滴眼液、激光治疗和/或手术可在医学上控制在视网膜变性和视野丧失之前观察到的眼压升高的症状，但不构成治疗。眼压控制在眼前区，在房水流出的解剖通路内，称为小梁细胞外网（TEM）。在约7000万青光眼病例中，约4%为常染色体显性遗传形式，与TEM成分心肌蛋白的突变密切相关。超过90%的心肌突变发生在其olfactomedin （OLF）结构域，该结构域在高等真核生物中高度保守，但结构和功能未知。引起青光眼的突变型心肌蛋白很少从人小梁网（HTM）细胞分泌到TEM。相反，它们在内质网（ER）中积累，导致TEM形态异常、热媒细胞死亡和早发性青光眼。有趣的是，纯合子和杂合子心肌蛋白敲除小鼠，以及心肌蛋白截断突变的个体，没有表现出眼部异常。因此，发病机制可能是由毒性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的疾病框架内。本提案的主要目的是利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心肌蛋白，以更好地了解心肌蛋白的结构、功能和折叠。尽管到目前为止，重组表达的困难限制了心肌蛋白的体外表征，但我们最近填补了对关键黄韧带黄韧带结构域的需求。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。这项工作将导致对遗传性和年龄性青光眼发病机制的分子理解，从而拓宽我们对蛋白质构象障碍和生物学中黄韧带黄韧带结构域功能的认识。拟议的研究也将为心肌性青光眼的药物发现提供新的机会，无论是通过新的稳定性测定的“自下而上”，还是使用三维结构的“自上而下”。迄今为止，我们的体外研究支持突变心肌蛋白保持接近野生型结构的假设，但表现出受损的稳定性。我们进一步假设这些缺陷被内质网质量控制机制识别，从而阻止突变心肌蛋白分泌到TEM。突变心肌蛋白的积累引起细胞应激反应，导致HTM细胞死亡。因此，我们的长期目标是利用对心肌蛋白结构、稳定性和折叠的了解来识别小分子，使突变心肌蛋白能够逃避ER质量控制的检测，并防止在TEM中聚集。这种治疗可以减少内质网积聚和致病性下游事件，从而直接延缓遗传性青光眼患者眼压和视力下降的发生。