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部疾病。葡萄膜炎的治疗选择受到 对疾病的分子原因的不完全理解。这个提议建立在我们激动人心的发现上 常染色体显性遗传性新生血管炎性玻璃体视网膜病变（ADNIV）是一种遗传性葡萄膜炎， 是由CAPN 5基因突变引起的。CAPN 5基因产生钙蛋白酶-5（CAPN 5）蛋白酶，其切割 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，CAPN 5在 ADNIV.我们鉴定Iroquois-3（IRX3）为视网膜中CAPN 5的新切割靶标。IRX3是一种有效的 转录因子参与炎症性疾病状态。CAPN 5葡萄膜炎的鉴定和一种新的 CAPN 5靶点使高度创新的分子遗传学方法成为可能，以确定相关因素 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的治疗靶点。 该提案的目的是确定IRX3切割在ADNIV中的作用。我们的核心假设是， 超活性ADNIV CAPN 5超切割IRX3的调节结构域，过度激活IRX3转录 在ADNIV中，炎症基因的活性和驱动破坏性炎症。我们的理论是通过研究 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，CAPN 5切割IRX3向其C-末端。此外，我们还生成了 底物和基于IRX3的CAPN 5的潜在抑制剂化合物。我们的数据还表明，IRX3 在体外调节ADNIV炎性基因的子集。当比较野生型IRX3与CAPN 5切割的 对于切割的IRX3，我们的测定发现切割的IRX3显示出更大的核定位和增加的与IL-10的结合。 6启动子（一个关键的ADNIV炎性基因）。我们的具体目标是利用生物化学，分子， 在细胞和动物中的生理学研究以测试以下假设：（1）CAPN 5靶向细胞上的特定结构域。 用于切割和物理相互作用的IRX3，其可以被分离并开发成肽底物;（2） IRX3的CAPN 5切割增加IRX3核定位和活性，导致小鼠的葡萄膜炎;和 (3)Irx3缺失小鼠将显示出对发展中的ADNIV和眼内炎症表型的抗性。 我们希望这项工作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。在本项目完成后，我们期望：（1）隔离IRX3 切割位点和开发的肽底物和潜在的抑制剂化合物;（2）确定了 CAPN 5切割对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意义。作为ADNIV 类似于其他眼部疾病，我们的工作有望产生广泛的影响，超越ADNIV和葡萄膜炎。

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.