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Investigating Connexion Interactions in Skin

研究皮肤中的连接相互作用

基本信息

批准号：
8957997
负责人：
Ingrid Martha Skerrett
金额：
$ 29.4万
依托单位：
BUFFALO STATE COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8957997
关键词：
Binding Blood Vessels Cell Line Cells Clouston&apos s Syndrome Collaborations Communication Connexin 43 Connexins Coupling Dentistry Disease Docking Dominant-Negative Mutation Electrodes Epidermis Epithelial Gap Junctions Genes Germ-Line Mutation Gifts Goals Human Induced Mutation Inherited Ligands Link Literature London Mammalian Cell Modification Molecular Mutation Oocytes Outcome Pattern Peptides Positioning Attribute Property Protein Family Proteins Publishing Senter syndrome Skin Specificity Structure Surface Plasmon Resonance Syndrome System Technology Testing Time Work Xenopus oocyte deafness design disease-causing mutation expectation experience extracellular gap junction channel human GJB2 protein inhibitor/antagonist keratinocyte medical schools mimetics mutant novel strategies prevent public health relevance screening skin disorder tool trafficking vector voltage clamp

项目摘要

DESCRIPTION (provided by applicant): At least nine of the twenty-one or more human gap junction proteins are expressed in skin. These proteins, known as connexins, are expressed in specific and overlapping patterns and their interactions are likely to be very important. This proposal focuses on understanding interactions between gap junction proteins expressed in keratinocytes (Cx26, Cx30, Cx30.3, and Cx31). Mutations in several of the connexins expressed in skin cause hereditary skin diseases. These include erythrokeratadermia variabilis (EKV) Mendes Da Costa caused by mutations in Cx31, EKV Cram-Mevorah caused by mutations in Cx30.3, keratitis-ichthyosis-deafness (KID syndrome) caused by mutations in Cx26, Vohwinkel syndrome caused by mutations in Cx26 and Clouston syndrome caused by mutations in Cx30. This project has three aims including 1) expression of keratinocyte connexins in Xenopus oocytes, 2) analysis of disease-causing mutations likely to provide information about connexin interactions and 3) construction of a molecular toolkit for modifying connexin interactions. Our lab received genes encoding several keratinocyte connexins as a gift from Dr. David Kelsell (Barts and the London School of Medicine and Dentistry). As part of our preliminary studies we modified constructs for human Cx31 to optimize expression in oocytes. As far as we know this is the first time Cx31 has been expressed in oocytes. We also have previous experience analyzing deafness mutants in Cx26, and plan to create oocyte-expression constructs for Cx30 and Cx30.3, allowing characterization of gap junction channels resulting from these connexins. Our lab has successfully expressed and purified the extracellular loops of other gap junction proteins using the pET100 expression system. The extracellular loops will be individually expressed and used to screen a variety of connexin-mimetic peptides using surface Plasmon resonance. Peptides with the highest specificity will be further characterized using the oocyte expression system and mammalian cell lines. To the best of our knowledge, the four target human connexins have not been previously characterized in oocytes. Several modifications were required to optimize our hCx31 construct. The vector we identified has been associated with superior expression of other gap junction proteins in our lab. We believe we are in a unique position to accelerate understanding of Cx30 and Cx30.3 as well as many of the skin disease mutations using the oocyte expression system. We also propose to use novel approaches to identify connexin interactions. These include the use hereditary skin disease mutations and creation of interacting peptides. A subset of hereditary mutations with dominant-negative mechanisms of action will be identified and used to disrupt function of co-expressed connexins. In addition, mutants that produce functional channels when expressed alone in cells will be further assessed for their potential disruption of interactions. The third goal of this project involves the creation of connexin-specific inhibitors in the form of mimetic peptides. Connexin-mimetic peptides targeting Cx43 are effective in regulating epithelial and vascular function, and peptides that interact with amino terminus of Cx43 can influence interactions. This is the first application of peptide technology to manipulation and characterization of connexin interactions in skin. Identifying peptides that specifically target connexins expressed in skin is expected to present challenges due to the sequence similarity of these connexins. The extracellular loops represent excellent targets as their sequence and structure determine docking interactions and include regions with some of the lowest sequence similarity. In summary, the proposed work will contribute to an understanding of gap junctions in skin by characterizing interactions between connexins, investigating connexin mutations associated with disease and creating molecular tools that can be used manipulate connexin interactions.

描述（由申请人提供）：21种或多种人间隙连接蛋白中的至少9种在皮肤中表达。这些蛋白质被称为连接蛋白，以特定和重叠的模式表达，它们的相互作用可能非常重要。该提案的重点是了解角质形成细胞中表达的间隙连接蛋白（Cx 26、Cx 30、Cx30.3和Cx 31）之间的相互作用。皮肤中表达的几种连接蛋白的突变会导致遗传性皮肤病。这些包括由Cx 31突变引起的可变性红斑角化皮炎（EKV）门德斯达科斯塔、由Cx30.3突变引起的EKV Cram-Mevorah、由Cx 26突变引起的角膜炎-鱼鳞病-耳聋（KID综合征）、由Cx 26突变引起的Vohwinkel综合征和由Cx 30突变引起的Clouston综合征。该项目有三个目标，包括1）角质形成细胞连接蛋白在爪蟾卵母细胞中的表达，2）可能提供关于连接蛋白相互作用的信息的致病突变的分析和3）用于修饰连接蛋白相互作用的分子工具包的构建。我们的实验室从大卫凯尔塞尔博士（巴茨和伦敦医学和牙科学院）那里收到了编码几种角质细胞连接蛋白的基因。作为我们初步研究的一部分，我们修改了人Cx 31的构建体，以优化在卵母细胞中的表达。据我们所知，这是Cx 31首次在卵母细胞中表达。我们也有以前的经验，分析耳聋突变体在Cx 26，并计划创建Cx 30和Cx30.3的卵母细胞表达结构，允许从这些连接蛋白的间隙连接通道的特性。本实验室利用pET 100表达系统成功表达并纯化了其他间隙连接蛋白的胞外环。细胞外环将被单独表达，并用于使用表面等离子体共振筛选各种连接蛋白模拟肽。具有最高特异性的肽将使用卵母细胞表达系统和哺乳动物细胞系进一步表征。据我们所知，这四种靶向人类连接蛋白以前没有在卵母细胞中进行过表征。需要几个修改来优化我们的hCx 31构建体。我们鉴定的载体与我们实验室中其他间隙连接蛋白的上级表达相关。我们相信，我们处于一个独特的位置，以加速了解Cx 30和Cx30.3以及许多皮肤病突变使用卵母细胞表达系统。我们还建议使用新的方法来确定连接蛋白的相互作用。这些包括使用遗传性皮肤病突变和创造相互作用的肽。将鉴定具有显性负作用机制的遗传突变的子集，并用于破坏共表达的连接蛋白的功能。此外，将进一步评估当在细胞中单独表达时产生功能通道的突变体对相互作用的潜在破坏。该项目的第三个目标涉及以模拟肽的形式创建连接蛋白特异性抑制剂。针对Cx43的连接蛋白模拟肽在调节上皮和血管功能中是有效的，并且与Cx43的氨基末端相互作用的肽可以影响相互作用。这是肽技术首次应用于皮肤中连接蛋白相互作用的操纵和表征。由于这些连接蛋白的序列相似性，鉴定特异性靶向皮肤中表达的连接蛋白的肽预计会带来挑战。细胞外环代表了极好的靶标，因为它们的序列和结构决定了对接相互作用，并且包括具有一些最低序列相似性的区域。总之，拟议的工作将有助于理解皮肤间隙连接的特征连接蛋白之间的相互作用，调查与疾病相关的连接蛋白突变，并创建分子工具，可用于操纵连接蛋白的相互作用。