The role of NBEAL2 in the cornea

我们在两个家族的神经海滩蛋白样 2 (NBEAL2) 基因中发现了与疾病相关的变异 圆锥角膜 (KC)，一种角膜退行性变薄疾病。 NBEAL2 编码细胞支架 蛋白质（2,750 个氨基酸，302 kDa），其在角膜中的功能尚不清楚。我们建议阐明 NBEAL2 在角膜中的作用。该蛋白存在于血小板和 NBEAL2 变体中，除了 我们在堪萨斯城发现的那些，会导致一种极其罕见的血液疾病，称为灰血小板综合症 （全球定位系统）。 GPS 患者的血小板含量较低，颗粒蛋白分泌有缺陷。 Nbeal2-/- 小鼠， 全身缺乏 Nbeal2 的人也表现出血小板异常，但尚未对他们的眼睛进行检查。的 NBEAL2 蛋白在 N 末端附近有一个刀豆球蛋白 A 凝集素样结构域，在该结构域中我们检测到 一个具有 KC 的家族中的精氨酸到谷氨酸取代 (R659Q)。第二个患有 KC 的家庭携带缬氨酸- NBEAL2 高度保守的结构域（称为 Beige 和 Chediak-）中的 to-异亮氨酸取代 (V2118I) Higashi (BEACH) 结构域，仅存在于其他八种蛋白质中。 BEACH 蛋白调节囊泡分泌， 溶酶体功能、膜动力学和信号传导。 BEACH 蛋白突变与 血液系统疾病、色素沉着不足、剥脱综合征、神经退行性变、伤口愈合和 癌症，但对眼部疾病的研究还不够。最近的一项研究报告称 NBEAL2 与囊泡相连 血小板膜上与 DOCK7、SEC16A 和其他蛋白质相互作用以调节肌动蛋白 聚合、蛋白质运输和分泌。我们的初步数据显示 NBEAL2 存在于 上皮和角膜基质，提出了一个问题：NBEAL2 在角膜中的作用是什么？ 变体对 KC 有贡献吗？角膜驻留细胞的主要功能是维持专门的屏障 通过有效分泌基底膜和基质细胞外基质 (ECM) 蛋白来形成组织 和胶原蛋白。我们假设，在常驻角膜细胞中，NBEAL2 调节 ECM 蛋白质和胶原蛋白是角膜完整性所必需的。我们将检验我们的中心假设 两个目标。目的 我将在稳态条件下和上皮细胞移植后检查 Nbeal2-/- 小鼠的角膜 清创损伤。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 干扰内源 NBEAL2 或 NBEAL2 KC 变体表达后的细胞培养物。我们的发现 将阐明 NBEAl2 的功能，NBEA12 是一种对角膜来说新颖的 BEACH 蛋白。由此得到的机理见解 研究将确定可能被操纵以增加 ECM 产生和角膜完整性的目标 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.