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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.

项目总结