Proteasome-ErB1 Impaired Interaction in Lung Hyperplasia

反应性氧化剂，如过氧化氢（H2 O2）和过氧亚硝酸根（ONOO-），强烈地 与肺部疾病有关。然而，将氧化剂暴露与肺部疾病发展联系起来的细胞和分子机制知之甚少。我们的研究方向之一是阐明氧化剂诱导神经酰胺介导的细胞凋亡的分子机制。另一个方向，这是本申请的重点，旨在阐明反应性氧化剂和ErbB 1，表皮生长因子受体（EGFR），驱动异常生长控制，导致气道上皮增生之间的分子相互作用。 我们建议，反应性氧化剂作为独立的参与者在“输入层”的信号ErbB 1受体，但与生理配体，如EGF不同的结果。我们已经表明，与配体诱导的二聚化和phn相反。当暴露于H2 O2导致EGFR的异常磷酸化时，暴露于H2 O2导致EGFR的异常磷酸化，暴露于ONOO产生共价交联的受体。此外，我们最近观察到H2 O2抑制泛素E3连接酶c-Cbl与EGFR的结合，从而阻止受体的泛素化。这些初步的研究使我们推测，氧化剂诱导的EGFR异常磷酸化阻止了c-Cbl和蛋白酶体对EGFR的泛素化。 (or溶酶体）下调。这可能导致持续的促生长信号。 为了测试这一点，我们将确定暴露于反应性氧化剂后EGFR的结构/功能。我们将首先绘制反应性氧化剂靶向的磷酸化或二聚化的特定位点。然后，我们将构建受影响位点的EGFR突变体，并阐明其在EGFR功能中的作用。我们将专门测试的假设，通过靶向异常磷酸化位点，反应性氧化剂阻止EGFR与蛋白酶体组分，c-Cbl的协会，并排除泛素化和下调的受体，从而导致气道上皮增生。 EGFR磷酸化（或二聚化）位点中氧化剂介导的改变的结构/功能表征，随后构建同源酪氨酸突变体，是将氧化剂特异性变化与肺增生在分子水平上联系起来的重要里程碑。从长远来看，这种方法应该为临床干预提供精确的靶点，以控制肺病中上皮细胞的增生。

Reactive oxidants, such as hydrogen peroxide (H202) and peroxynitrite (ONOO-), are strongly associated with lung disease. Yet, the cellular and molecular mechanisms that link oxidant exposure to the development of lung disease are poorly understood. One direction of our studies has been to unravel the molecular mechanisms of oxidant-induced ceramide mediated apoptosis. The other direction, which is the focus of this application, aims to elucidate the molecular interactions between reactive oxidants and ErbB1, the epidermal growth factor receptor (EGFR), that drive aberrant growth control leading to airway epithelial hyperplasia. We propose that reactive oxidants act as independent participants in the "input layer" of signals to the ErbB1 receptor but with a different outcome than that of physiologic ligands such as EGF. We have shown that in contrast to ligand-induced dimerization and phn._ohorylation of EGFR, H202 exposure results in an aberrantly phosphorylated EGFR, and ONOO- exposure generates a covalently cross-linked receptor. Moreover, we have recently observed that H202 inhibits the association of the ubiquitin E3 ligase, c-Cbl, with EGFR, and thus prevents the receptor's ubiquitination. These preliminary studies lead us to hypothesize that oxidant-induced aberrant phosphorylation of EGFR prevents its ubiquitination by c-Cbl and proteasomal (or lysosomal) down-regulation. This could result in continuous pro-growth signaling. To test this, we will determine the structure/function of EGFR following exposure to reactive oxidants. We will first map the specific sites of phosphorylation or dimerization targeted by reactive oxidants. Then, we will construct EGFR mutants of the affected sites and elucidate their roles in EGFR function. We will specifically test the hypothesis that by targeting aberrant phosphorylation sites, reactive oxidants prevent EGFR association with the proteasomal component, c-Cbl, and preclude ubiquitination and down-regulation of the receptor, thereby leading to airway epithelial hyperplasia. Structure/function characterization of oxidant-mediated alterations in EGFR sites of phosphorylation (or dimerization), followed by construction of the cognate tyrosine mutants, are important milestones that would link oxidant-specific changes to lung hyperplasia at a molecular level. In the long run, this approach should indicate precise targets for clinical intervention to control hyperplasia of epithelial cells in pulmonarydiseases.