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

基本信息

批准号：
8420505
负责人：
Raquel L Lieberman
金额：
$ 27.77万
依托单位：
GEORGIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
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/8420505
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 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 screening small molecule three dimensional structure

项目摘要

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.

描述（由申请人提供）：青光眼是世界失明的主要原因，没有直接治疗。眼部滴，激光治疗和/或手术可以治疗视网膜变性和视野丧失之前观察到的眼内压增加的症状，但不构成治疗。压力在眼睛的前区域，在解剖学途径中，用于流出幽默的流出途径，称为小梁外细胞网（TEM）。在约有7000万个青光眼病例中，约有4％的构成常染色体主导者，其遗传形式与肌动蛋白（TEM成分）的突变密切相关。超过90％的肌动蛋白突变发生在其嗅觉素（OLF）结构域中，在较高的真核生物中，该结构域高度保守，但具有未知的结构和功能。引起青光眼的突变体肌动蛋白是从人的小梁网（HTM）细胞中分泌的很差的。取而代之的是，它们积聚在内质网（ER）中，导致TEM形态异常，HTM细胞死亡和早期发作的青光眼。有趣的是，纯合和杂合肌动蛋白敲除小鼠，以及肌动蛋白截断突变的个体没有眼异常。因此，发病机理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疾病错误折叠和贩运疾病的框架内。该建议的主要目的是使用溶液生物物理技术和X射线晶体学表征纯化的野生型和突变肌蛋白，以更好地了解肌动蛋白的结构，功能和折叠。尽管到目前为止，重组表达的困难限制了肌动蛋白的体外表征，但我们最近已经满足了对关键OLF结构域的需求。我们已经纯化了重组系统中纯化的单体，胞质聚集体和不溶性夹杂物。拟议的工作将导致对遗传和年龄衰老青光眼中发病机理的分子理解，从而扩大我们对蛋白质构象疾病的了解以及OLF结构域在生物学中的功能。拟议的研究还将为肌动蛋白青光眼的药物发现提供新的机会，既可以通过新的稳定性测定法，也可以使用三维结构来“自下而上”。迄今为止，我们的体外研究支持了以下假设：突变肌蛋白在野生型结构接近但表现出稳定性的稳定性上。我们进一步假设这些缺陷是通过ER质量控制机制认识到的，这可以防止突变的肌动蛋白分泌到TEM上。突变肌动蛋白的积累会引起导致HTM细胞死亡的细胞应激反应。因此，我们的长期目标是利用肌动蛋白结构，稳定性和折叠的知识来识别能够通过ER质量控制来逃避检测并防止TEM聚集的小分子。这种治疗方法将减少ER的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力和随后的视力丧失。