The role of NBEAL2 in the cornea

NBEAL2 在角膜中的作用

基本信息

批准号：
10322135
负责人：
Shukti Chakravarti
金额：
$ 24.66万
依托单位：
NEW YORK UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-01 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10322135
关键词：
Actins Address Affect Alpha Granule Amino Acids Arginine Basement membrane Blood Platelets Capital Cell Culture Techniques Cell Line Cells Cellular Morphology Collagen Complex Concanavalin A Cornea Corneal Diseases Corneal Stroma Cytoskeleton DNA Data Debridement Development Disease Economic Burden Epithelial Epithelial Cells Exfoliation Syndrome Extracellular Matrix Extracellular Matrix Proteins Eye Eye diseases Family Foundations Future Genes Glutamates Health Hematological Disease Human Hypopigmentation Immunofluorescence Immunologic Impairment Injury Isoleucine Keratoconus Knowledge Lead Lectin Letters Location Maintenance Malignant Neoplasms Mediating Membrane Membrane Proteins Microscopic Molecular Target Mouse Strains Mus Mutation Nerve Degeneration Pathogenesis Pathogenicity Patients Persons Phenotype Production Protein Secretion Proteins Recombinants Reporting Role Scaffolding Protein Secretory Vesicles Signal Transduction Site Stains Stromal Cells Structure Syndrome Testing Therapeutic Thick Thinness Tissues Transcript Transfusion Transmission Electron Microscopy Valine Variant Vesicle Vision chediak-higashi syndrome corneal epithelial wound healing corneal epithelium health economics in vivo imaging insight light transmission mouse model mutant novel polymerization preservation protein transport repair function repaired response tissue injury wound healing

项目摘要

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.

我们在两个家族中发现了基因神经洞穴样2（NBEAL2）中与疾病相关的变体与角膜变性的变质性疾病有关角膜核（KC）。 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。该蛋白质存在于血小板中，而NBEAL2变体中存在于我们在KC中鉴定出的一种，引起一种极为罕见的血液学疾病，称为灰色血小板综合征（全球定位系统）。 GP患者的血小板低，在颗粒蛋白分泌中有缺陷。 nbeal2 - / - 小鼠，从系统上缺乏NBEAL2，也显示出血小板异常，但尚未检查它们的眼睛。这 NBEAL2蛋白在N末端附近有一个contanavalin-a凝集素样结构域，我们在其中检测到精氨酸到谷氨酸替代（R659Q）在一个KC家族中。拥有KC的第二个家庭带有Valine- 在一个高度保守的NBEAL2领域中的脱甲酸盐取代（V2118i），称为米色和chediak- Higashi（海滩）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。海滩蛋白的突变已经关联血液学疾病，异形，去角质综合征，神经变性，伤口愈合和癌症，但在眼部疾病中进行了研究。一项最近的一项研究报告说，NBEAL2被束缚在囊泡上血小板中的膜与Dock7，Sec16a和其他蛋白质相互作用以调节肌动蛋白聚合，蛋白质转运和分泌。我们的初步数据显示NBEAL2在上皮和角膜基质，提出了一个问题：NBEAL2在角膜中的作用是什么，以便它变体有助于KC？角膜居民细胞的主要功能是维护专业屏障通过有效分泌基底膜和基质外基质（ECM）蛋白的组织和胶原蛋白。我们假设在驻留角膜细胞中，NBEAL2调节了分泌的角膜完整性所需的ECM蛋白质和胶原蛋白。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后检查NBEAL2 - / - 小鼠的角膜清创伤。伤口愈合反应由修复的上皮和基质细胞精心策划通过ECM分泌的地下膜和基质，这种分泌机制可能需要 nbeal2。 AIM II将测试人角膜上皮和基质中的NBEAL2介导的分泌功能内源性NBEAL2扰动或NBEAL2 KC变体的表达后的细胞培养。我们的发现将阐明NBEAL2的功能，NBEAL2是一种海滩蛋白，是角膜的小说。从中的机械见解研究将确定可能被操纵的目标，以增加ECM的产生和角膜完整性 KC。