Characterization of purified myocilin: glaucoma as a protein misfolding disease

项目摘要 青光眼是致盲的主要原因，通过治疗青光眼的因果风险因素来进行医学管理。 增加的眼内压（IOP），其通常在视网膜变性和视力丧失之前观察到。 领域眼压控制在眼睛的前部区域，该区域包含小梁网（TM） 细胞外基质是房水引流的解剖学途径。在约4500万例 全世界约3%的开角型青光眼与肌球蛋白突变有关，肌球蛋白是一种在青光眼组织中高度表达的蛋白质。 TM.尽管在过去的20年里进行了大量的研究工作，但对它的结构或功能知之甚少。 myocilin对正常和疾病状态下肌球蛋白分子水平的进一步了解将改变 通过使药剂能够靶向疾病过程而不是 间接控制IOP。 在myocilin的整个序列中发现了疾病相关的突变。在过去的一段时间里，我们 在生物学和结构上表征了聚集在其C-末端嗅觉调节蛋白（mOLF）中的变体 领域，贷款的关键新的细节和支持的主要工作假设，其中突变 定位于肌-OLF导致毒性功能的获得：内质网（ER）相关降解是 抑制肌球蛋白和ER-居民伴侣Grp 94之间的异常相互作用，导致 TM细胞内突变型肌球蛋白的淀粉样沉积物，具有细胞毒性。由此产生的TM细胞的积累 碎片被认为阻碍流体从TM流出，导致IOP升高。持续结构/功能障碍 肌钙蛋白的研究不仅有助于我们理解青光眼及其在TM中的作用， 也拓宽了我们对许多其他OLF结构域的理解，这些结构域与生理学有着广泛的联系 和疾病。 这项建议的目的是扩大我们的分子理解的结构和错误折叠， 肌球蛋白相关性青光眼，并为功能研究和发现 减轻异常肌细胞素行为的小分子。我们将（1）阐明本地完整的架构- 长度肌球蛋白，其由N-末端卷曲螺旋决定，并表征生物物理和细胞 其中发现的疾病变体的性质，（2）澄清肌球蛋白和Grp 94之间的相互作用， 分子水平，和（3）实施两个高通量测定。预期成果是：（1）全面 （2）扩大我们对肿瘤相关性肌球蛋白的认识， 蛋白质构象紊乱，（3）对Grp 94分子伴侣生物学的新认识，和（4）新的配体分析 基于myoc-OLF结构和mOLF/Grp 94相互作用，用于鉴定治疗性小 分子。

PROJECT SUMMARY Glaucoma, a leading cause of blindness, is managed medically by treating the causal risk factor of increased intraocular pressure (IOP), which is typically observed prior to retina degeneration and loss of visual field. IOP is controlled in the anterior region of the eye, which contains the trabecular meshwork (TM) extracellular matrix, the anatomical pathway for drainage of aqueous humor fluid. Of the ~45 million cases of open angle glaucoma worldwide, ~3% are linked to mutations in myocilin, a protein highly expressed in the TM. Despite considerable research effort over ~20 years, little is known about the structure or function of myocilin. An improved molecular understanding of myocilin in its normal and disease states will change the paradigm for anti-glaucoma therapeutics by enabling agents that target the disease process instead of indirectly controlling IOP. Disease-associated mutations in myocilin are found throughout its sequence. In the prior grant period, we biophysically and structurally characterized the variants clustered in its C-terminal olfactomedin (mOLF) domain, lending critical new details and support for the predominant working hypothesis in which mutations localized to myoc-OLF lead to a gain of toxic function: Endoplasmic-reticulum (ER)-associated degradation is inhibited by an aberrant interaction between myocilin and the ER-resident chaperone Grp94, leading to amyloid deposits of mutant myocilin within TM cells, which are cytotoxic. The resulting accumulation of TM cell debris is thought to impede fluid outflow from the TM, causing IOP elevation. Continued structure/dysfunction studies of myocilin will not only contribute to our understanding of glaucoma and its role in the TM, but would also broaden our comprehension of the many other OLF domains, which are implicated broadly in physiology and diseases. The objectives of this proposal are to expand our molecular comprehension of structure and misfolding in myocilin-associated glaucoma as well as provide a path forward for functional studies and the discovery of small molecules that mitigate aberrant myocilin behavior. We will (1) elucidate the architecture of native full- length myocilin, which is dictated by N-terminal coiled-coils, and characterize biophysical and cellular properties of disease variants found therein, (2) clarify the interaction between myocilin and Grp94 at the molecular level, and (3) implement two high throughput assays. The expected outcomes are (1) the full scope of the misfolding disease mechanism for glaucoma-associated myocilin, (2) expansion of our knowledge of protein conformational disorders, (3) new insights into Grp94 chaperone biology, and (4) novel ligand assays based on the myoc-OLF structure and mOLF/Grp94 interaction for the identification of therapeutic small molecules.