Characterization of purified myocilin: glaucoma as a protein misfolding disease

描述(申请人提供)：青光眼是世界上导致失明的主要原因，没有直接的治疗方法。滴眼液、激光治疗和/或手术在医学上处理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。压力控制在眼的前部区域，在房水流出的解剖路径内，称为小梁细胞外网络(TEM)。在约7000万青光眼患者中，约4%的人是常染色体显性遗传性青光眼，与透射电子显微镜的一种成分--肌球蛋白的突变密切相关。超过90%的myoclin突变发生在其olfactomedin(OLF)结构域中，该结构域在高等真核生物中高度保守，但结构和功能未知。从人的小梁细胞(HTM)向透射电子显微镜观察，导致青光眼的突变型肌菌素分泌能力很差。相反，它们聚集在内质网(ER)中，导致透射电子显微镜形态异常、HTM细胞死亡和早发性青光眼。有趣的是，纯合子和杂合子的myocin基因敲除小鼠，以及带有myocin截短突变的个体，都没有表现出眼部异常。因此，发病机制可能是由一种有毒的功能获得机制引起的，将相当数量的青光眼病例置于蛋白质错误折叠和错误提取的疾病框架内。这项建议的主要目的是利用溶液生物物理技术和X射线结晶学对纯化的野生型和突变型myocilin进行表征，以更好地了解myocin的结构、功能和折叠。虽然到目前为止，重组表达的困难限制了myoclin的体外特性，但我们最近已经满足了关键的OLF结构域的这一需求。我们已经从我们的重组系统中提纯了单体、胞浆聚集体和不溶性包裹体。这项工作将有助于从分子水平上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认识。拟议中的研究还将为Myoclin青光眼的药物开发提供新的机会，既可以通过新的稳定性分析来“自下而上”，也可以使用三维结构来“自上而下”。到目前为止，我们的体外研究支持这样的假设，即突变的肌丝蛋白保持了接近野生型的结构，但表现出折衷的稳定性。我们进一步假设，这些缺陷是由ER质量控制机制识别的，这阻止了突变的肌菌素分泌到透射电子显微镜。突变的myocin的积累引发细胞应激反应，导致HTM细胞死亡。因此，我们的长期目标是利用myocin的结构、稳定性和折叠的知识来识别能够使突变的myoclin逃脱ER质量控制的检测并防止在透射电子显微镜中聚集的小分子。这种治疗将减少内质网积聚和致病的下游事件，从而直接延迟遗传性青光眼患者的眼压和随后的视力丧失的发生。 公共卫生意义：我们的长期目标是开发一种治疗青光眼的新方法，青光眼是一种以眼压升高、视网膜神经变性和视力丧失为特征的常见眼病。我们正在研究肌球蛋白，一种参与调节眼压的细胞外基质蛋白；肌球蛋白的突变会导致早发性遗传性青光眼。我们将从结构和稳定性的角度研究myoclin和致病突变，这将指导我们努力开发一种检测方法，识别与myoclin结合并稳定突变的治疗化合物，以延缓遗传性青光眼的发生。

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.