Molecular basis of congenital disorder of glycosylation type 1N

1N型先天性糖基化障碍的分子基础

基本信息

批准号：
10510784
负责人：
ANANT K MENON
金额：
$ 25.43万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-08 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10510784
关键词：
Binding Binding Proteins Blindness Cadherins Categories Cell Adhesion Molecules Cell Surface Receptors Cell-Free System Cells Chemicals Child Clinical Collaborations Congenital disorders of glycosylation Cryoelectron Microscopy Defect Development Developmental Delay Disorders Disease Dolichol Endoplasmic Reticulum Enzymes Epitopes Europe Extracellular Matrix Proteins Face Failure to Thrive Family Fibroblasts Genetic Diseases Glycoproteins Goals Human Genetics India Integrins Laminin Lateral Life Link Lipids Location Maps Membrane Membrane Biology Membrane Proteins Metabolic Diseases Microcephaly Microsomes Middle East Missense Mutation Modeling Molecular Morphology Muscle hypotonia National Institute of Child Health and Human Development Nature North America Oligosaccharides Organogenesis Pathway interactions Patients Phenotype Polysaccharides Property Proteins Role Seizures Sensorineural Hearing Loss Side Site Speech Delay Structure Symptoms Testing Therapeutic Tissues United States National Institutes of Health Vesicle X-Ray Crystallography cognitive disability experience experimental study glycosylation glycosyltransferase improved interest isoprenoid novel strategies physically handicapped prevent programs seal stroke-like episode sugar therapy development three dimensional structure treatment strategy

项目摘要

N-glycosylation is essential for life. Glycoproteins such as cadherins, integrins and laminins are key to development, organogenesis, and tissue organization. Defects in N-glycosylation underlie numerous human genetic disorders including a heterogeneous group of autosomal-recessive, metabolic diseases termed Congenital Disorders of Glycosylation (CDGs). CDGs present with a range of devastating symptoms, including failure to thrive, developmental and speech delays, vision loss, hypotonia, microcephaly, seizures, and stroke- like episodes. Children with CDGs suffer cognitive and physical disabilities. While some of these symptoms can be treated, there is no cure for CDGs. Our goal in this R21 application is to explore the molecular basis of CDG type 1N, a poorly understood disease caused by missense mutations in the endoplasmic reticulum membrane protein RFT1. Patients, identified thus far in North America, U.K./Europe, the Middle East, and India, often present with a unique sensorineural deafness in addition to typical severe CDG symptoms. There is no therapy for CDG1N. Unlike most CDGs for which the underlying molecular defect is well understood, e.g., deficiency of a glycosyltransferase or sugar-processing enzyme, CDG1N is an enigma because the function of RFT1 is not known. The pathway of N-glycosylation is blocked at a critical stage in CDG1N cells, resulting in the build-up of a lipid intermediate (termed M5-DLO) that cannot be utilized by the glycosylation machinery. We hypothesize that the critical role of RFT1 in cells is to catalyze utilization of M5-DLO either by overcoming an impediment posed by endoplasmic reticulum structure/morphology to facilitate its handoff to downstream machinery, or by preventing its mis-localization. We will test this hypothesis in two specific aims. The first aim will focus on the role of RFT1 in M5-DLO utilization, making use of RFT1-deficient cells (including CDG1N patient fibroblasts), cell-free systems, and a novel approach to sub-fractionating the endoplasmic reticulum. We will also test whether RFT1 is an M5-DLO binding protein. Recognizing that structural information is critical to understand the function of RFT1 at a molecular level, the second aim will develop a structure-function model of RFT1. Here we will define its membrane topology, identify key functional residues, and initiate a program to elucidate its 3-dimensional structure. In the long term, our results will illuminate why RFT1 deficiency results in M5-DLO accumulation and consequently CDG1N, thereby paving the way for the development of treatment strategies and therapeutics that could subserve RFT1's function to restore glycosylation and improve clinical symptoms.

N-糖基化对于生命至关重要。糖蛋白如钙粘蛋白、整合素和层粘连蛋白是发育、器官发生和组织组织。 N-糖基化缺陷是许多人类疾病的基础遗传性疾病，包括一组异质性常染色体隐性代谢疾病，称为先天性糖基化障碍 (CDG)。 CDG 表现出一系列破坏性症状，包括发育迟缓、发育和言语迟缓、视力丧失、肌张力减退、小头畸形、癫痫发作和中风就像剧集一样。患有 CDG 的儿童患有认知和身体残疾。虽然其中一些症状可能会 CDG 无法治愈。我们在此 R21 应用中的目标是探索 CDG 1N 型的分子基础，这是一种人们知之甚少的药物。由内质网膜蛋白 RFT1 错义突变引起的疾病。患者，迄今为止在北美、英国/欧洲、中东和印度发现的，通常具有独特的特征除了典型的严重 CDG 症状外，还有感音神经性耳聋。 CDG1N 没有治疗方法。不像大多数 CDG 的潜在分子缺陷已被充分了解，例如，缺乏糖基转移酶或糖加工酶，CDG1N 是一个谜，因为 RFT1 的功能并不已知。 N-糖基化途径在 CDG1N 细胞的关键阶段被阻断，导致一种不能被糖基化机制利用的脂质中间体（称为 M5-DLO）。我们假设 RFT1 在细胞中的关键作用是通过克服障碍来催化 M5-DLO 的利用由内质网结构/形态构成，以促进其向下游机器的传递，或通过防止其错误定位。我们将在两个具体目标中检验这一假设。第一个目标将重点关注 RFT1 在 M5-DLO 中的作用利用，利用 RFT1 缺陷细胞（包括 CDG1N 患者成纤维细胞）、无细胞系统和细分内质网的新方法。我们还将测试 RFT1 是否是 M5-DLO 结合蛋白。认识到结构信息对于理解 RFT1 的功能至关重要分子水平上，第二个目标是开发 RFT1 的结构功能模型。这里我们将定义它的膜拓扑结构，识别关键功能残基，并启动一个程序来阐明其 3 维结构结构。从长远来看，我们的结果将阐明为什么 RFT1 缺陷会导致 M5-DLO 积累和因此，CDG1N，从而为治疗策略和疗法的开发铺平了道路，可以促进RFT1恢复糖基化并改善临床症状的功能。