Characterization of purified myocilin: glaucoma as a protein misfolding disease

描述（由申请人提供）：青光眼是世界上最主要的致盲原因，没有直接的治疗方法。滴眼液、激光治疗和/或手术在医学上管理视网膜变性和视野丧失之前观察到的眼内压升高的症状，但不构成治疗。压力控制在眼睛的前部区域，在用于房水流出的解剖学通路内，称为小梁细胞外网（TEM）。在约7000万青光眼病例中，约4%包括常染色体显性遗传形式，与肌球蛋白（TEM组分）的突变密切相关。超过90%的突变发生在myocilin的嗅觉调节蛋白（olfactomedin，OLF）结构域，这是高度保守的高等真核生物，但未知的结构和功能。引起青光眼的突变型肌球蛋白很少从人小梁网（HTM）细胞分泌到TEM。相反，它们积聚在内质网（ER）中，导致异常TEM形态、HTM细胞死亡和早发性青光眼。有趣的是，纯合和杂合的肌球蛋白敲除小鼠，以及肌球蛋白截短突变的个体，没有表现出眼部异常。因此，发病机制可能是由毒性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调节的疾病框架内。 该提案的主要目标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球蛋白，以更好地了解肌球蛋白的结构、功能和折叠。虽然重组表达的困难限制了myocilin的体外表征，到目前为止，我们最近填补了关键OLF结构域的这一需求。我们已经从重组系统中纯化了单体、胞质聚集体和不溶性内含物。拟议的工作将导致遗传性和年龄发作性青光眼的发病机制的分子理解，从而拓宽我们的蛋白质构象障碍和生物学中的OLF结构域的功能的知识。 拟议的研究还将为肌球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进行“自下而上”，也可以使用三维结构进行“自上而下”。迄今为止，我们的体外研究支持突变型肌青霉素保持接近野生型结构但表现出受损的稳定性的假设。我们进一步假设这些缺陷被ER质量控制机制所识别，这阻止了突变型肌青霉素被分泌到TEM。突变型肌球蛋白的积累引起导致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球蛋白的结构，稳定性和折叠的知识，以确定小分子，使突变的肌球蛋白，以逃避检测ER质量控制和防止聚集在TEM。这种治疗将减少ER积聚和致病下游事件，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。

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.