Characterization of purified myocilin: glaucoma as a protein misfolding disease

项目总结 青光眼是导致失明的主要原因，可以通过治疗青光眼的致病风险因素来进行治疗。 眼压升高，通常在视网膜变性和视力丧失之前观察到 菲尔德。眼压控制在眼前区，包括小梁网(TM) 细胞外基质，房水引流的解剖途径。在约4500万起 全世界约3%的开角型青光眼与myocin基因突变有关，myocin是一种在 TM.尽管20多年来进行了大量的研究，但对其结构或功能知之甚少 麦考林。对肌球蛋白正常和疾病状态的分子理解的改进将改变 抗青光眼治疗的范例，使药物能够针对疾病过程而不是 间接控制眼压。 在其整个序列中都发现了与疾病相关的myocin突变。在之前的授权期内，我们 生物物理和结构特征的变异体聚集在其C-末端的嗅觉素(Molf) 领域，为主要的工作假说提供了关键的新细节和支持，其中突变 定位于Myoc-OLF导致毒性功能的获得：内质网(ER)相关的降解 被Myocin和内质网驻留的伴侣Grp94之间的异常相互作用所抑制，导致 突变的霉菌蛋白在TM细胞内沉积成淀粉样物质，具有细胞毒性。由此产生的TM细胞的积累 碎片被认为阻碍了液体从TM流出，导致眼压升高。持续性结构/功能障碍 对霉菌素的研究不仅有助于我们理解青光眼及其在TM中的作用，而且还将 也拓宽了我们对许多其他OLF结构域的理解，这些结构域在生理学中广泛涉及 和疾病。 这项提议的目的是扩大我们对结构和错误折叠的分子理解。 霉菌素相关的青光眼以及为功能研究和发现 减轻肌球蛋白异常行为的小分子。我们将(1)阐明Native Full的体系结构 长度为myocin，由N端卷曲的线圈决定，并表征生物物理和细胞特性 其中发现的疾病变体的特性，(2)阐明Myocin和Grp94之间的相互作用 分子水平，以及(3)实现了两种高通量分析。预期的结果是：(1)全面 青光眼相关肌球蛋白的错误折叠疾病机制，(2)扩大我们对 蛋白质构象紊乱，(3)对Grp94伴侣生物学的新见解，以及(4)新的配基分析 基于myoc-OLF结构和Molf/Grp94相互作用的治疗性小鼠的鉴定 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.