Inflammatory Gene Transcription in the Retina

视网膜中的炎症基因转录

基本信息

批准号：
10318582
负责人：
ALEXANDER G BASSUK
金额：
$ 38.32万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10318582
关键词：
Affect Age Amino Acids Animals Automobile Driving Binding Biochemical Biological Assay Blindness C-terminal Calcium Calpain Cell Death Cell Fractionation Cell Nucleus Cells Cyclic Peptides Cytoplasm DNA Data Disease Excision Extracellular Space Eye Eye diseases Fibrosis Gene Expression Genes Genetic Genetic Transcription Goals Histology Hyperactivity In Vitro Inflammation Inflammatory Inherited Injections Interleukin-6 Knockout Mice Knowledge Legal Blindness Mass Spectrum Analysis Modeling Molecular Molecular Genetics Mus Mutation N-terminal Nuclear Ocular Physiology Pathway interactions Peptide Hydrolases Peptides Pharmacology Phenocopy Phenotype Physiological Prevalence Proteins Reporter Research Resistance Retina Role Severities Site Testing Therapeutic Intervention Uveitis Work base cytokine experimental study functional genomics gene network genetic approach in vivo inhibitor innovation insight legal cases mouse model mutant neovascular new therapeutic target novel novel therapeutics promoter targeted treatment therapeutic target transcription factor

项目摘要

Project Summary Uveitis is a difficult-to-treat inflammatory eye disorder. Treatment options for uveitis are limited by an incomplete understanding of molecular causes of the disease. This proposal builds on our exciting discovery that Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy (ADNIV), an inherited uveitis, is caused by mutations to the gene CAPN5. The CAPN5 gene produces the Calpain-5 (CAPN5) protease, which cleaves off regions of other proteins to regulate their functions. Our data show that CAPN5 becomes hyperactive in ADNIV. We identified Iroquois-3 (IRX3) as a new cleavage target of CAPN5 in the retina. IRX3 is a potent transcription factor implicated in inflammatory disease states. Identification of CAPN5 uveitis and a new CAPN5 target make possible a highly innovative molecular-genetic approach to identifying factors responsible for uveitis. The long-term goal of our research is to identify new therapeutic targets for the treatment of uveitis. This proposal’s objective is to determine the role of IRX3 cleavage in ADNIV. Our central hypothesis is that hyperactive ADNIV CAPN5 hyper-cleaves regulatory domains from IRX3, over-activating IRX3 transcriptional activity at inflammatory genes and driving destructive inflammation in ADNIV. Our rationale is that by studying the newly-identified CAPN5-IRX3 pathway, we will identify new disease mechanisms and therapeutic targets. Our data indicate that CAPN5 cleaves IRX3 toward its C-terminus. Additionally, we generated peptide substrates and a potential inhibitor compound for CAPN5 based on IRX3. Our data also show that IRX3 regulates a subset of ADNIV inflammatory genes in vitro. When comparing wild-type IRX3 to CAPN5-cleaved (“cut”) IRX3, our assays found that cut-IRX3 shows greater nuclear localization and increased binding to the IL- 6 promoter (a key ADNIV inflammatory gene). Our specific aims are to use biochemical, molecular, and physiologic studies in cells and animals to test the hypotheses that: (1) CAPN5 targets a specific domain on IRX3 for cleavage and physical interaction which can be isolated and developed into peptide substrates; (2) CAPN5 cleavage of IRX3 increases IRX3 nuclear localization and activity, contributing to uveitis in mice; and (3) Irx3-null mice will show resistance to developing ADNIV and intraocular inflammation phenotypes. We expect this work to have a significant positive impact on identifying new molecular mechanisms for therapeutic intervention in uveitis. At the completion of this project, we expect to have: (1) Isolated the IRX3 cleavage site and developed peptide substrates and potential inhibitor compounds; (2) Determined the consequences of CAPN5 cleavage on IRX3, identifying new molecular pathways which might be targeted therapeutically; and (3) Determined the significance of the Irx3 pathway in intraocular inflammation. As ADNIV mimics other eye diseases, our work promises to have a broad impact beyond ADNIV and uveitis.

项目概要葡萄膜炎是一种难以治疗的炎症性眼部疾病，葡萄膜炎的治疗选择受到以下因素的限制。该提议建立在我们令人兴奋的发现之上。常染色体显性遗传性新生血管炎性玻璃体视网膜病变 (ADNIV)，一种遗传性葡萄膜炎，是由 CAPN5 基因突变产生 Calpain-5 (CAPN5) 蛋白酶，可裂解。我们的数据表明，CAPN5 会变得过度活跃。 ADNIV。我们确定 Iroquois-3 (IRX3) 是视网膜中 CAPN5 的新裂解靶标。与炎症性疾病状态有关的转录因子 CAPN5 葡萄膜炎的鉴定和新的 CAPN5 目标使高度创新的分子遗传学方法成为可能，以识别相关因素我们研究的长期目标是确定治疗葡萄膜炎的新治疗靶点。该提案的目的是确定 IRX3 裂解在 ADNIV 中的作用我们的中心假设是：过度活跃的 ADNIV CAPN5 过度切割 IRX3 的调节域，过度激活 IRX3 转录我们的理由是通过研究ADNIV中炎症基因的活性并驱动破坏性炎症。通过新发现的CAPN5-IRX3通路，我们将确定新的疾病机制和治疗靶点。我们的数据表明 CAPN5 将 IRX3 切割至其 C 末端此外，我们生成了肽。我们的数据还表明，IRX3 是 CAPN5 的底物和潜在抑制剂化合物。当比较野生型 IRX3 和 CAPN5 裂解时，它在体外调节 ADNIV 炎症基因的子集。（“cut”）IRX3，我们的分析发现 cut-IRX3 显示出更大的核定位以及与 IL-的结合增加 6 启动子（一个关键的 ADNIV 炎症基因）我们的具体目标是利用生物化学、分子和方法。在细胞和动物中进行生理学研究，以测试以下假设：(1) CAPN5 靶向特定的结构域 IRX3 用于裂解和物理相互作用，可分离并开发成肽底物 (2) CAPN5 对 IRX3 的裂解增加了 IRX3 的核定位和活性，导致小鼠葡萄膜炎； (3) Irx3缺失小鼠将表现出对ADNIV和眼内炎症表型的抵抗力。我们预计这项工作将对确定新的分子机制产生重大积极影响葡萄膜炎的治疗干预在该项目完成时，我们期望： (1) 分离IRX3。 (2)确定了裂解位点和开发的肽底物和潜在的抑制剂化合物； CAPN5 裂解对 IRX3 的影响，确定可能靶向的新分子途径 (3) 确定 Irx3 通路在眼内炎症中的重要性。与其他眼部疾病相似，我们的工作有望产生除 ADNIV 和葡萄膜炎之外的广泛影响。