Inflammatory Gene Transcription in the Retina

项目摘要 葡萄膜炎是一种难以治疗的炎症性眼病。葡萄膜炎的治疗选择受到 对这种疾病的分子病因的了解不完全。这项建议是建立在我们令人兴奋的发现基础上的 常染色体显性遗传性新生血管炎性玻璃体视网膜病变(ADNIV)是一种遗传性葡萄膜炎 通过CAPN5基因的突变。CAPN5基因产生钙蛋白酶-5(CAPN5)蛋白酶，该酶可裂解 关闭其他蛋白质的区域来调节它们的功能。我们的数据显示CAPN5在 ADNIV.我们发现Iroquis-3(IRX3)是CAPN5在视网膜中的一个新的切割靶点。IRX3是一种强大的 转录因子与炎症性疾病状态有关。CAPN5葡萄膜炎的鉴定和一种新的 CAPN5靶标使识别相关因素的高度创新的分子遗传学方法成为可能 治疗葡萄膜炎。我们研究的长期目标是确定治疗葡萄膜炎的新靶点。 这项建议的目的是确定IRX3裂解在ADNIV中的作用。我们的中心假设是 高度活跃的ADNIV CAPN5过度切割IRX3的调节域，过度激活IRX3转录 在ADNIV中，炎症基因的活性和推动破坏性炎症。我们的理论基础是通过研究 对于新发现的CAPN5-IRX3通路，我们将发现新的发病机制和治疗靶点。 我们的数据表明，CAPN5将IRX3切割到其C末端。此外，我们还生成了多肽 底物和潜在的基于IRX3的CAPN5抑制剂化合物。我们的数据还表明，IRX3 在体外调节ADNIV炎症基因的一个子集。比较野生型IRX3和CAPN5裂解 (“Cut”)IRX3，我们的检测发现Cut-IRX3表现出更大的核定位和与IL-3的结合。 6启动子(ADNIV的关键炎症基因)。我们的具体目标是使用生化、分子和 在细胞和动物中进行的生理学研究，以检验以下假设：(1)CAPN5靶向于 IRX3用于切割和物理相互作用，可以分离和发展成多肽底物；(2) IRX3的CAPN5裂解增加了IRX3的核定位和活性，导致小鼠葡萄膜炎；以及 (3)IRX3基因缺失的小鼠将表现出对ADNIV和眼内炎症表型的抵抗。 我们预计这项工作将对确定新的分子机制产生重大的积极影响 葡萄膜炎的治疗干预。在这个项目完成后，我们希望：(1)分离出IRX3 裂解位点和形成的多肽底物和潜在的抑制剂化合物；(2)测定 IRX3上CAPN5裂解的后果，确定可能成为靶点的新的分子途径 (3)确定IRX3通路在眼内炎症中的意义。作为ADNIV 模仿其他眼病，我们的工作有望产生超越ADNIV和葡萄膜炎的广泛影响。

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.