Proteasome-ErB1 Impaired Interaction in Lung Hyperplasia

活性氧化剂，如过氧化氢(H202)和过氧亚硝酸根(ONOO-)，是强烈的 与肺部疾病有关。然而，将氧化剂暴露与肺部疾病的发展联系起来的细胞和分子机制还知之甚少。我们研究的一个方向是揭示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，也是本应用的重点，旨在阐明活性氧化剂和表皮生长因子受体(EGFR)ErbB1之间的分子相互作用，后者驱动异常生长控制，导致呼吸道上皮细胞增生。 我们认为，活性氧化剂在ErbB1受体信号的“输入层”中扮演着独立参与者的角色，但结果与EGF等生理配体不同。我们已经证明，与配体诱导的EGFR二聚化和PHN_ohor化不同，H202暴露导致EGFR异常磷酸化，而ONOO暴露产生共价交联的受体。此外，我们最近观察到，H202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。这些初步研究使我们假设，氧化剂诱导的egfr异常磷酸化可以阻止c-cbl和蛋白酶体泛素化。 (或溶酶体)下调。这可能会导致持续的促增长信号。 为了测试这一点，我们将确定暴露在活性氧化剂中的EGFR的结构/功能。我们将首先绘制出针对活性氧化剂的磷酸化或二聚化的特定位置。然后，我们将构建受影响部位的EGFR突变体，并阐明它们在EGFR功能中的作用。我们将专门测试这一假设，即通过靶向异常的磷酸化位点，活性氧化剂阻止EGFR与蛋白酶体成分c-Cbl结合，并阻止泛素化和受体下调，从而导致呼吸道上皮细胞增生。 氧化剂介导的EGFR磷酸化(或二聚化)位点的结构/功能变化的结构/功能表征，以及随后同源酪氨酸突变体的构建，是在分子水平上将氧化剂特异性变化与肺组织增生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控制肺部疾病中的上皮细胞增殖指明准确的目标。

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.