Identification of novel genes regulating metalloproteinase activity

调控金属蛋白酶活性的新基因的鉴定

基本信息

批准号：
8327862
负责人：
Andreas Herrlich
金额：
$ 24.25万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8327862
关键词：
Acute Adverse effects Alzheimer&apos s Disease Amyloid beta-Protein Precursor Angiotensin II Antibodies Biological Assay C-terminal Candidate Disease Gene Cell Count Cell physiology Cells Chronic Kidney Failure Cleaved cell Complementary DNA Coupled DTR gene Development Disease EGF gene Enhancers Epithelial Cells Epitopes Evaluation Fluorescence Fluorochrome Genes Goals Grant Heart Diseases Heart failure Human Infection Inflammation Injury Instruction Jurkat Cells Kidney Kidney Diseases Knockout Mice LacZ Genes Length Libraries Life Ligands Lung Malignant Neoplasms Measures Metalloproteases Modeling Mus Phase Phorbol Esters Phosphoric Monoester Hydrolases Phosphotransferases Physiological Polybrene Progress Reports Protein Isoforms Puromycin Regulation Resistance Signal Transduction Pathway Staining method Stains Transforming Growth Factor alpha Tubular formation Virus Diseases atypical protein kinase C base cDNA Library high throughput screening inhibitor/antagonist killings knock-down novel overexpression pro-EGF repaired response small hairpin RNA

项目摘要

Ectodomain (ECD) cleavage by metalloproteinases is involved in many diseases including kidney and cardiac disease, Alzheimer's disease, cancer and inflammation. EGF pro-ligands mature by metalloproteinase-cleavage of the ECD that represents the active ligand. They are important in a broad range of physiological and disease states. In the kidney, as examples, the EGF ligand TGFalpha is cleaved in response to Angiotensin II (Angll). TGFalpha knock-out mice or mice treated with a metalloprotease inhibitor are protected against Angll-induced chronic kidney disease. HB-EGF, another EGF ligand, is involved in tubular branching in the developing kidney and in tubular repair after kidney injury. Metalloproteinase-directed therapies in humans are limited by side effects, or non-existent. The signal transduction pathways regulating ECD cleavage are essentially unknown. The goal of our study is to identify novel genes that regulate ECD cleavage using a lentiviral shRNA gene knock-down approach. We have developed a high-throughput assay that detects cleavage of EGF-ligands in a FACS-based assay using cells stably expressing a ligand with an ECD epitope tag and a C-terminal GFP-fusion. The ECD can be tracked by staining with a fluorochrome-coupled antibody ("red"). In the uncleaved state any given cell has a 1:1 ratio of outside (ECD, "red") to inside (GFP, "green") fluorescence, as measured by FACS on life single cells. Stimulation of EGF ligand cleavage decreases, while inhibition increases this ratio. During the K99 phase of this grant we began examining the effect of knock-down of human kinases and phosphatases on EGF ligand cleavage. Here we present up-to-date results on the initial screen that identified several inhibitors and activators of phorbol ester-induced TGFalpha cleavage in Jurkat cells. Knock-down of PKCzeta, an atypical protein kinase C isoform not activated by phorbol ester is identified as an important inhibitor of cleavage in Jurkat cells and also in mouse lung epithelial cells and partipates in differential regulation of ECD cleavage of three different EGF ligands examined. We are outlining our plans for completion of the high-throughput screen and for further evaluation of candidate genes, including PKCzeta.

金属蛋白酶对胞外域 (ECD) 的裂解与许多疾病有关，包括肾脏和疾病心脏病、阿尔茨海默病、癌症和炎症。 EGF 前配体的成熟代表活性配体的 ECD 的金属蛋白酶裂解。它们在广泛的范围内都很重要生理和疾病状态的范围。例如，在肾脏中，EGF 配体 TGFalpha 被裂解响应血管紧张素 II (Angll)。 TGFα 敲除小鼠或用金属蛋白酶处理的小鼠抑制剂可预防 AngII 诱导的慢性肾病。 HB-EGF，另一种 EGF 配体，是参与发育中肾脏的肾小管分支和肾损伤后的肾小管修复。人类的金属蛋白酶导向疗法受到副作用的限制，或者根本不存在副作用。信号调节 ECD 裂解的转导途径基本上是未知的。我们研究的目标是确定使用慢病毒 shRNA 基因敲低方法调节 ECD 裂解的新基因。我们有开发了一种高通量测定法，可使用基于 FACS 的测定法检测 EGF 配体的裂解细胞稳定表达带有 ECD 表位标签和 C 端 GFP 融合的配体。 ECD 可以是通过荧光染料偶联抗体（“红色”）染色进行追踪。在未切割状态下，任何给定的细胞都具有通过 FACS 对生命进行测量，外部（ECD，“红色”）与内部（GFP，“绿色”）荧光的比例为 1:1 单细胞。 EGF 配体裂解的刺激会减少，而抑制会增加该比例。期间在本次资助的 K99 阶段，我们开始研究敲低人类激酶和磷酸酶的效果 EGF 配体裂解。在这里，我们在初始屏幕上展示了最新的结果，其中确定了几个 Jurkat 细胞中佛波酯诱导的 TGFα 裂解的抑制剂和激活剂。击倒的 PKCzeta 是一种不被佛波酯激活的非典型蛋白激酶 C 异构体，被认为是一种重要的 Jurkat 细胞和小鼠肺上皮细胞中的裂解抑制剂，并参与分化研究了三种不同 EGF 配体 ECD 裂解的调节。我们正在概述我们的计划完成高通量筛选并进一步评估候选基因，包括 PKCzeta。