Identification of novel genes regulating metalloproteinase activity

金属蛋白酶对胞外结构域(ECD)的裂解与许多疾病有关，包括肾脏和 心脏病、阿尔茨海默病、癌症和炎症。EGF前体通过以下途径成熟 金属蛋白酶-代表活性配体的ECD的裂解。它们在一个广泛的领域中很重要 生理和疾病状态的范围。例如，在肾脏中，EGF配体TGFα被切割 对血管紧张素II(Ang11)的反应。TGFα基因敲除小鼠或用金属蛋白酶处理的小鼠 抑制剂对血管紧张素转换酶诱导的慢性肾脏疾病具有保护作用。Hb-EGF，另一种EGF配体，是 参与发育中的肾脏的肾小管分支和肾损伤后的肾小管修复。 金属蛋白酶在人类中的直接治疗受到副作用的限制，或者根本不存在。该信号 调控ECD裂解的转导通路基本上是未知的。我们研究的目标是确定 用慢病毒shRNA基因敲除方法调节ECD裂解的新基因。我们有 开发了一种高通量检测方法，在基于FACS的检测中检测EGF配体的切割 稳定表达带有ECD表位标签和C端GFP融合的配体的细胞。ECD可以是 用一种荧光偶联抗体(“红色”)染色进行追踪。在未分裂状态下，任何给定的细胞都具有 外(ECD，“红”)与内(GFP，“绿”)荧光的比例为1：1，由FACS对生命进行测量 单细胞。刺激对EGF配体的切割减少，而抑制增加这一比率。在.期间 K99阶段的这笔赠款，我们开始研究敲除人类激酶和磷酸酶的效果 关于EGF配体的切割。在这里，我们在初始屏幕上显示最新结果，其中标识了几个 佛波酯的抑制剂和激活剂诱导Jurkat细胞中的转化生长因子α裂解。推倒 PKcheeta是一种不被佛波酯激活的非典型蛋白激酶C亚型，被认为是一种重要的 Jurkat细胞和小鼠肺上皮细胞及颗粒中的裂解抑制物 研究了三种不同的EGF配体对ECD裂解的调节。我们正在概述我们的计划 完成高通量筛选，并对候选基因进行进一步评估，包括PKcheeta。

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.