Characterization of purified myocilin: glaucoma as a protein misfolding disease

青光眼是全球范围内导致失明的主要原因（7000万患者）， 眼内压（IOP）升高的症状，但仍有10%的患者失明。眼压控制在 眼前节，其中含有小梁网（TM）细胞外基质，解剖 水状体液的排出途径。在大多数形式的青光眼中，TM组织是患病的; TM组织的丧失导致青光眼。 TM稳态导致IOP升高。遗传性开角型青光眼，影响约300万年轻患者， 是由肌球蛋白突变引起的，肌球蛋白是一种在TM中高度表达的蛋白质。自2011年3月以来， R 01 EY 021205通过奠定分子基础改变了抗青光眼治疗的范式 目前学术界和工业界都在寻求针对疾病过程的方法。 我们阐明了毒性功能获得性致病机制的分子细节， 肌球蛋白在TM细胞的内质网（ER）中积累，导致TM细胞死亡， 视力丧失的加速时间轴。R 01 EY 021205（a）中的研究提供了以下基本知识 肌球蛋白结构，（B）发现肌球蛋白和ER-驻留之间的反作用相互作用 Hsp 90伴侣蛋白Grp 94，和（c）表征为淀粉样蛋白的肌球蛋白错误折叠。野生型和许多不同的 肌球蛋白变异体具有错误折叠的倾向;因此，在家族性肌球蛋白- 相关的青光眼可能在更多的患者中起作用。 由肌球蛋白引起的淀粉样蛋白形成将青光眼与淀粉样蛋白疾病并列， 阿尔茨海默病和SOD-1依赖性肌萎缩侧索硬化症，但我们对淀粉样蛋白在 青光眼还处于婴儿期。我们目前的目标是更好地了解myocilin的分子方面 原纤维化，集中在相关的嗅觉调节蛋白（OLF）结构域。我们的多学科团队将（a）澄清 通过研究野生型和选择的OLF变体的溶液结构引发聚集，以及 相应的多长度尺度动力学，使用氢-氘交换质谱， 核磁共振（NMR）结构和弛豫方法（Wade范霍恩，Co-I）（B）比较了 通过固态NMR（AnantParavastu，Co-Ltd.， I）和评估中间聚集体的细胞毒性和（c）评估常见等位基因全长肌细胞素 致病性的实验标志的变体。预期的结果是更好地了解 myocilin在分子水平上的错误折叠过程，包括致病性的分子决定因素， 研究、诊断和治疗肌球蛋白相关性青光眼的新方法。更广泛地说，继续 肌球蛋白结构/功能研究不仅有助于我们理解青光眼及其作用， 在TM中，但也将扩展我们对许多其他OLF领域的理解，这些领域涉及 广泛地应用于生理学和疾病。

Glaucoma, a leading cause of blindness worldwide (70 million patients), is managed medically by treating the symptom of increased intraocular pressure (IOP), but 10% of patients still go blind. IOP is controlled in the anterior segment of the eye, which contains the trabecular meshwork (TM) extracellular matrix, the anatomical pathway for drainage of aqueous humor fluid. The TM tissue is diseased in most forms of glaucoma; loss of TM homeostasis leads to elevated IOP. Hereditary open angle glaucoma, affecting ~3 million young patients, is caused by mutations in myocilin, a protein highly expressed in the TM. Since 3/2011, studies funded from R01EY021205 have changed the paradigm for anti-glaucoma therapeutics by laying the molecular foundation for approaches that target the disease process, which are now being pursued in academia and industry. We clarified molecular details of the toxic gain-of-function pathogenic mechanism in which mutant myocilin accumulates in the endoplasmic reticulum (ER) of TM cells, leading to TM cell death and an accelerated timeline for vision loss. Studies from R01EY021205 (a) contributed fundamental knowledge of myocilin structure, (b) discovered a counter-productive interaction between myocilin and the ER-resident Hsp90 chaperone Grp94, and (c) characterized myocilin misfolding as amyloid. Wild-type and many different myocilin variants harbor a misfolding propensity; thus, proteostasis issues identified in familial myocilin- associated glaucoma are likely at play in many more patients. Amyloid formation by myocilin places glaucoma alongside more well-studied amyloid diseases like Alzheimer and SOD-1 dependent amyotrophic lateral sclerosis, yet our comprehension of the role of amyloid in glaucoma is in its infancy. Our current objective is to better understand molecular aspects of myocilin fibrilization, focused on the relevant olfactomedin (OLF) domain. Our multidisciplinary team will (a) clarify initiation of aggregation by studying solution structures of wild-type and selected OLF variants, as well as corresponding multi-length scale dynamics, using hydrogen-deuterium exchange mass spectrometry and nuclear magnetic resonance (NMR) structure and relaxation methods (Wade Van Horn, Co-I) (b) compare the end-point structures of selected OLF aggregates to known amyloids by solid state NMR (Anant Paravastu, Co- I) and evaluate cytotoxicity of intermediate aggregates and (c) evaluate common allele full-length myocilin variants for experimental hallmarks of pathogenicity. The expected outcome is a better understanding of the myocilin misfolding process at the molecular level, including molecular determinants of pathogenicity, to enable novel modalities for studying, diagnosing, and treating myocilin-associated glaucoma. More broadly, continued structure/dysfunction studies of myocilin will not only contribute to our understanding of glaucoma and its role in the TM, but will also extend our comprehension of the many other OLF domains, which are implicated broadly in physiology and diseases.