Identification of novel genes regulating metalloproteinase activity

描述（由申请人提供）： 我是一名德国医学博士/博士。在约翰霍普金斯和哈佛接受过内科和肾脏病学广泛培训的临床医生兼科学家。我的研究重点是表皮生长因子受体（EGFR）信号通路。在我早期的工作中，我发现某些金属蛋白酶切割的EGF配体参与G蛋白信号转导到MAP激酶，也参与渗透压应激信号转导到水通道蛋白水通道。在肾脏中，EGF配体HB（肝素结合）-EGF参与损伤后的肾小管修复。在心脏中，它参与心脏肥大的产生。胞外结构域（ECD）裂解还涉及许多其他疾病，如阿尔茨海默病、癌症和炎症。调节切割的信号转导途径基本上是未知的。金属蛋白酶导向疗法受到副作用的限制，或不存在。随着怀特黑德研究所和哈佛的肾脏病学项目，我选择了理想的研究和临床环境，继续在该领域做出重大贡献，并发展我作为肾脏病学独立临床医生科学家的职业生涯。 本研究的目的是利用高通量表达克隆策略来鉴定调节ECD切割的新基因。我们在基于FACS的测定中使用稳定表达具有ECD表位标签和C-末端EGFP融合的配体的细胞检测EGF配体的裂解。ECD可以用荧光染料偶联的抗表位抗体检测，C-末端通过绿色荧光检测。在未切割状态下，任何给定的细胞具有1：1的外部与内部荧光的比率，如通过FACS对生命单细胞测量的。刺激卵裂降低，而抑制基础或诱导卵裂增加这个比例。感染逆转录病毒cDNA文库或条形码shRNAi文库，然后对显示改变的切割比率的细胞进行FACS分选，可以鉴定ECD切割的新的阳性或阴性调节剂。原理性实验已经完成。 我们的长期目标是开发新的候选疗法，用于治疗对普通人群有广泛影响的医学重要疾病。例如，尽管经过多年的临床和基础研究，急性肾衰竭和心力衰竭仍然具有非常高的死亡率。我们的研究所产生的知识也可能扩展到常见疾病，如癌症，阿尔茨海默病或炎症性疾病。

DESCRIPTION (provided by applicant): I am a German MD/Ph.D. clinician-scientist with extensive training in Internal Medicine and Nephrology at Johns Hopkins and Harvard. My research focuses on the epidermal growth factor receptor (EGFR) signaling pathway. In my earlier work I discovered that certain metalloproteinase-cleaved EGF ligands are involved in G-protein signal transduction to MAPkinase and also in osmotic stress signaling to aquaporin water channels. In the kidney, the EGF ligand HB (heparin-binding)-EGF is involved in tubular repair after injury. In the heart it is involved in the generation of cardiac hypertrophy. Ectodomain (ECD) cleavage is also involved in a number of other diseases, like Alzheimer's disease, cancer and inflammation. The signal transduction pathways regulating cleavage are essentially unknown. Metalloproteinase-directed therapies are limited by side effects, or non-existent. With the Whitehead Institute and the Nephrology program at Harvard I have chosen the ideal research and clinical environment to continue to make significant contributions in the field and develop my career as an independent clinician-scientist in Nephrology. The goal of my study is to identify novel genes that regulate ECD cleavage using a high-throughput expression cloning strategy. We detect cleavage of EGF-ligands in a FACS-based assay using cells stably expressing a ligand with an ECD epitope tag and a C-terminal EGFP-fusion. The ECD can be detected with a fluorochrome-coupled anti-epitope antibody, the C-terminus by green fluorescence. In the uncleaved state any given cell has a 1:1 ratio of outside to inside fluorescence, as measured by FACS on life single cells. Stimulation of cleavage decreases, while inhibition of basal or induced cleavage increases this ratio. Infection of a retroviral cDNA library or of a bar-coded shRNAi library, followed by FACS sorting of cells which show altered cleavage ratio allows the identification of novel positive or negative regulators of ECD cleavage. Proof of principle experiments have already been completed. Our long-term goal is the development of new candidate therapies for medically important diseases with a wide impact on the general population. As example, acute kidney failure and heart failure still have a very high mortality despite years of clinical and basic research. Knowledge generated by our research will likely also be extendable to common diseases like cancer, alzheimer's disease or inflammatory disorders.