ANCHORING FILAMENT ANALYSIS AND CORRECTION IN EPIDERMOLYSIS BULLOSA

大疱性表皮松解症的锚定丝分析和校正

• 批准号: 6470587
• 负责人: Matt Peter Marinkovich
• 金额: $ 17.11万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6470587
• 关键词: SCID mouse; animal genetic material tag; basal lamina; basement membrane; cell adhesion; cell adhesion molecules; clinical research; cytoskeleton; epidermolysis bullosa; gene mutation; gene therapy; human subject; intercellular connection; intermolecular interaction; keratinocyte; laboratory mouse; laminin; molecular cloning; nonhuman therapy evaluation; protein structure function; site directed mutagenesis; skin transplantation; transplantation immunology

This proposal aims to characterize novel basement membrane zone molecules associated with anchoring filaments and to determine the supramolecular structure and intermolecular interactions of anchoring filament components with lamina dense and hemidesmosomal components. The fundamental hypothesis of this project is that by elelucidating the structure and function of anchoring filaments we will be able to determine the molecular basis of junctional epidermolysis bullosa (JEB) and discover new genes which are affected in this disorder. Preliminary data are resented on three novel anchoring filaments proteins, LAD-1 (mAb 123 antigen), LH39 antigen and laminin 6, which are the three best candidates for new gene mutations in JEB. LAD-1 is a 120 kD upper anchoring filament protein which appears to be involved in insertion of anchoring filaments to the hemidesmosome and is absent in a subset of patients with generalized benign atrophic JEB (GABJEB). LH39 antigen is a lower anchoring filament protein which appears to be involved in insertion of anchoring filaments to the lamina densa and which is absent is a subset of patients with HBEB. Laminin-6 forms a disulfide complex with laminin-5 anchoring filaments, contains a novel a chain and could contain the primary defect in a subset of JEB patients. We propose to determine the entire cDNA sequence of the LH39 antigen and to propose additional experiments to determine its function and potential involvement in JEB. We similarly propose methods to determine the structure of the laminin 6 a chain. The interactions of anchoring filament components with hemidesmosome and lamina densa components will be analyzed by several methods, including solid state, chromatogrphic and centrifugation based ligand binding assays, cell binding assays and an in vitro basement membrane assembly model. Additionally, we propose to characterize the phenotypic features of GABJEB keratinocytes, to tranfect LAD-1 and BP180cDNA to effect phenotypic reversion in these cells and in conjunction with project 3, to study the effects of site directed mutagenesis in JEB keratinocytes using LAD-1, BP180 and laminin-5 cDNA. At the end of the proposed funding period, we hope to have significantly elucidated the basis of dermal-epidermal cohesion across the lamina lucida and to have demonstrated significant new mechanisms involving new candidate genes in JEB, setting the stage for further progress in gene therapy.

该建议旨在表征新颖的地下室膜区域 与锚定细丝相关的分子并确定 锚定的超分子结构和分子间相互作用 带有薄片密集和半底膜的细丝成分 成分。 该项目的基本假设是 启发锚定细丝的结构和功能，我们将 能够确定连接表皮溶解的分子基础 Bullosa（JEB）并发现了受影响的新基因 紊乱。 初步数据对三个新颖锚定 细丝蛋白，LAD-1（MAB 123抗原），LH39抗原和 层粘连蛋白6，这是新基因突变的三个最佳候选者 在杰布。 LAD-1是一种120 kD的上锚固丝蛋白，该蛋白 似乎参与将细丝插入到 半体体肌体，一部分有广义的患者 良性萎缩Jeb（Gabjeb）。 LH39抗原是较低的锚定 细丝蛋白似乎与锚定插入有关 lamina densa的细丝，不存在的是 HBEB患者。 层粘连蛋白6形成与 层粘连蛋白5锚固丝，包含一个小说A链，可以 在JEB患者子集中包含主要缺陷。 我们建议 确定LH39抗原的整个cDNA序列，然后 提出其他实验以确定其功能和潜力 参与JEB。 我们类似地提出了确定的方法 层粘连蛋白6 A链的结构。 锚定的相互作用 带有hemidesmosmom和lamina densa的细丝成分 组件将通过包括固态在内的几种方法分析 基于色谱和离心配体结合测定，细胞 结合测定和体外地下膜组装模型。 此外，我们建议表征 gabjeb角质形成细胞，转化LAD-1和BP180CDNA 这些细胞中的表型恢复并与项目3结合 研究位点定向诱变在JEB角质形成细胞中的影响 使用LAD-1，BP180和层粘连蛋白-5 cDNA。 在 拟议的资金期，我们希望大量阐明 在整个Lamina Lucida和To To的基础 已经显示出重要的新机制，涉及新的机制 JEB中的候选基因，为基因进一步进步奠定了基础 治疗。