The role of NBEAL2 in the cornea

我们在两个家族中发现了神经海滩蛋白样2（NBEAL 2）基因的疾病相关变异。 圆锥角膜（KC），一种角膜的退行性变薄疾病。NBEAL 2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。我们建议澄清 NBEAL 2在角膜中的作用。该蛋白存在于血小板和NBEAL 2变体中，除了 我们在KC中鉴定出的那些，引起一种极其罕见的血液病，称为灰色血小板综合征 （GPS）。GPS患者血小板减少，颗粒蛋白分泌缺陷。Nbeal 2-/-小鼠， 全身缺乏Nbeal 2的人也显示血小板异常，但他们的眼睛没有被检查。的 NBEAL 2蛋白在N端附近有一个伴刀豆球蛋白-A凝集素样结构域，在该结构域内我们检测到一个与NBEAL 2蛋白相似的结构域。 一个KC家族中的精氨酸-谷氨酸取代（R659 Q）。第二个家族携带缬氨酸 在NBEAL 2的高度保守的结构域中被称为Beige和Chediak的异亮氨酸取代（V2118 I）， Higashi（海滩）结构域，仅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、膜动力学和信号传导。海滩蛋白的突变与 血液病、色素减退、剥脱综合征、神经变性、伤口愈合， 癌症，但在眼科疾病中研究不足。最近的一项研究报告说，NBEAL 2被拴在囊泡上， 血小板膜，在那里它与DOCK 7，SEC 16 A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，NBEAL 2的存在， 上皮和角膜基质，提出了一个问题：NBEAL 2在角膜中的作用是什么，以至于它的 变异有助于KC？角膜驻留细胞的一个主要功能是维持一个专门的屏障 组织通过有效分泌基底膜和基质细胞外基质（ECM）蛋白 和胶原蛋白。我们假设，在常驻角膜细胞中，NBEAL 2调节角膜上皮细胞分泌。 角膜完整性所必需的ECM蛋白和胶原蛋白。我们将测试我们的中心假设， 两个目标。目的：我将在稳态条件下和上皮细胞移植后检查Nbeal 2-/-小鼠的角膜。 清创术损伤。伤口愈合反应由上皮细胞和基质细胞协调， 基底膜和基质通过ECM分泌，这种分泌机制可能需要 NBA 2.目的II检测NBEAL 2介导的人角膜上皮和基质细胞分泌功能 干扰内源性NBEAL 2或表达NBEAL 2 KC变体后的细胞培养物。我们的研究结果 将阐明NBEA 12，一种新的角膜海滩蛋白的功能。机械论的观点 这项研究将确定可能被操纵的目标，以增加ECM的产生和角膜的完整性， 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.