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兼临床科学家，曾在约翰霍普金斯大学和哈佛大学接受过内科和肾脏病方面的广泛培训。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裂解的EGF配体参与了G蛋白信号转导到MAPK，也参与了渗透胁迫信号转导水通道。在肾脏中，EGF配体HB(肝素结合)-EGF参与了损伤后的肾小管修复。在心脏中，它参与了心肌肥厚的产生。胞外区域(ECD)裂解也与许多其他疾病有关，如阿尔茨海默病、癌症和炎症。调控卵裂的信号转导途径基本上是未知的。金属蛋白酶导向的治疗受到副作用的限制，或者根本不存在。有了怀特黑德研究所和哈佛大学的肾病学项目，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，并作为一名独立的肾脏病学临床科学家发展我的职业生涯。 我这项研究的目标是使用高通量表达克隆策略来识别调控ECD切割的新基因。我们使用稳定表达带有ECD表位标签的配体和C端的EGFP融合的细胞，在基于FACS的检测中检测到EGF配体的切割。ECD可以用荧光偶联的抗表位抗体检测，C末端用绿色荧光检测。在未被切割的状态下，任何给定的细胞都有1：1的外部和内部荧光比率，如FACS对LIFE单个细胞的测量。对卵裂的刺激减少了，而抑制基础或诱导的分裂则增加了这一比例。感染逆转录病毒文库或条形码shRNAi文库，然后对显示裂解比改变的细胞进行FACS分选，可以识别新的ECD裂解正调控因子或负调控因子。原理验证实验已经完成。 我们的长期目标是开发对普通人群有广泛影响的医学上重要疾病的新候选疗法。例如，尽管进行了多年的临床和基础研究，急性肾衰竭和心力衰竭的死亡率仍然很高。我们的研究产生的知识也可能扩展到常见疾病，如癌症、阿尔茨海默氏症或炎症性疾病。

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.