The role of NBEAL2 in the cornea

我们在两个家族中发现了与疾病相关的神经信号蛋白样蛋白2(NBEAL2)基因变异 圆锥角膜(KC)是一种角膜退行性变薄的疾病。NBEAL2编码一种细胞支架 蛋白质(2750个氨基酸，302 kDa)，其在角膜中的功能尚不清楚。我们建议澄清 NBEAL2在角膜中的作用这种蛋白质存在于血小板和NBEAL2变体中，而不是 我们在KC中发现的这种物质会导致一种极其罕见的血液疾病，称为灰色血小板综合征。 (GPS)。患有GPS的患者有低血小板，颗粒蛋白分泌有缺陷。Nbeal2-/-老鼠， 系统性缺乏Nbeal2的人，也显示出血小板异常，但他们的眼睛没有接受检查。这个 NBEAL2蛋白在N端附近有一个刀豆蛋白-A凝集素样结构域，我们在其中检测到一个 1个KC家系的精氨酸-谷氨酸替代(R659Q)。患有KC的第二个家庭携带一种Valine- -异亮氨酸取代(V2118I)在NBEAL2的一个高度保守的结构域，称为米色和切迪亚克- Higashi(海滩)结构域，只在其他八种蛋白质中发现。海滩蛋白调节囊泡分泌， 溶酶体功能、膜动力学和信号转导。海滩蛋白的突变与 患有血液病、色素减退、剥脱综合征、神经变性、伤口愈合和 癌症，但在眼科疾病方面研究不足。最近的一项研究报道，NBEAL2与囊泡相连 与DOCK7、SEC16A和其他蛋白质相互作用调节肌动蛋白的血小板膜 聚合、蛋白质运输和分泌。我们的初步数据显示NBEAL2在 上皮细胞和角膜基质，提出了一个问题：NBEAL2在角膜中的作用是什么，以至于它的 变异对KC有贡献吗？常驻角膜细胞的主要功能是维持一种特殊的屏障。 通过有效分泌基底膜和基质细胞外基质(ECM)蛋白 还有胶原蛋白。我们假设，在常驻角膜细胞中，NBEAL2调节 角膜完整性所需的ECM蛋白质和胶原蛋白。我们将在以下位置测试我们的中心假设 两个目标。目的观察Nbeal2-/-小鼠在动态平衡条件下和上皮下角膜。 清创伤。伤口愈合反应是由修复的上皮细胞和基质细胞协调的 基底膜和间质通过ECM分泌，这种分泌机制可能需要 NBEAL2。AIM II将测试NBEAL2介导的人角膜上皮和基质的分泌功能 内源性NBEAL2或表达NBEAL2 KC变异体后的细胞培养。我们的发现 将阐明NBEAl2的功能，这是一种海滩蛋白，对角膜来说是新的。从这一点上的机械论见解 研究将确定可能被操纵以增加ECM产量和角膜完整性的靶点 KC。

We discovered disease-associated variants in the gene neurobeachin-like 2 (NBEAL2) in two families with keratoconus (KC), a degenerative, thinning disease of the cornea. NBEAL2 encodes a cellular scaffold protein (2,750 amino acids, 302 kDa) whose function in the cornea is unknown. We propose to elucidate the role of NBEAL2 in the cornea. The protein is present in platelets, and NBEAL2 variants, other than the ones we identified in KC, cause an extremely rare hematological disease known as gray platelet syndrome (GPS). Patients with GPS have low platelets that are defective in granular protein secretion. Nbeal2-/- mice, which systemically lack Nbeal2, also show platelet anomalies, but their eyes have not been examined. The NBEAL2 protein has a concanavalin-A lectin-like domain near the N-terminus, within which we detected an arginine-to-glutamate substitution (R659Q) in one family with KC. The second family with KC carries a valine- to-isoleucine substitution (V2118I) in a highly conserved domain of NBEAL2 called the Beige and Chediak- Higashi (BEACH) domain, found in only eight other proteins. BEACH proteins regulate vesicular secretion, lysosomal functions, membrane dynamics, and signaling. Mutations in BEACH proteins have been associated with hematologic diseases, hypopigmentation, exfoliation syndrome, neurodegeneration, wound healing, and cancer, but are understudied in eye diseases. A single recent study reported that NBEAL2 is tethered to vesicle membranes in platelets where it interacts with DOCK7, SEC16A, and other proteins to modulate actin polymerization, protein transport, and secretion. Our preliminary data show the presence of NBEAL2 in the epithelium and the corneal stroma, raising the question: what is the role of NBEAL2 in the cornea, such that its variants contribute to KC? A major function of cornea-resident cells is the maintenance of a specialized barrier tissue through effective secretion of basement membrane and stromal extracellular matrix (ECM) proteins and collagens. We hypothesize that, in resident corneal cells, NBEAL2 regulates secretion of ECM proteins and collagens necessary for corneal integrity. We will test our central hypothesis in two aims. Aim I will examine corneas of Nbeal2-/- mice under homeostatic conditions and after epithelial debridement injuries. The wound healing response is orchestrated by epithelial and stromal cells that repair the basement membrane and the stroma through ECM secretion, and this secretion mechanism may require NBEAL2. Aim II will test NBEAL2-mediated secretory functions in human corneal epithelial and stromal cell cultures after perturbation of endogenous NBEAL2 or expression of NBEAL2 KC variants. Our findings will elucidate functions of NBEAl2, a BEACH protein, novel to the cornea. Mechanistic insights from this study will identify targets that might be manipulated for increased ECM production and corneal integrity in KC.