Investigating oncogenic mutations and regulatory mechanisms of HER2 by electron microscopy

项目摘要/摘要 这项建议的目标是解决我们对HER2受体酪氨酸理解上的根本差距 直接结构和生物物理研究中的蛋白激酶(RTK)激活机制及其调控 全长受体。通过扩增或癌基因突变而产生的HER2信号异常是 许多癌症，并且仍然是治疗的关键靶点。HER2与EGFR、HER3和HER4一起构成 人类表皮生长因子受体(HER)家族的RTK，是细胞内稳态不可或缺的。 这些受体通过同种和异种受体将细胞外信号转化为细胞内反应。 生长因子结合诱导的齐聚反应。HER2是一种孤儿受体，没有 已知的配体和信号通过与HER家族其他成员的异二聚化而产生。我们不明白 HER2如何在没有配体结合的辅助受体的情况下调节其催化活性，但许多HER2 致癌基因突变破坏了这些机制，并以不依赖于辅受体的方式赋予活性。 这些突变中有几个属于激酶结构域之外，但在缺乏对 生长因子如何结合在受体的胞外侧增加其催化活性 ，我们不能预测这些突变如何提高HER2信号，并且 最重要的是，改变HER2对已知疗法的脆弱性。我们假设孤儿受体 HER2具有内在的结构机制，在没有配体或辅基的情况下调节催化活性。 激活域以外的受体和致癌突变通过以下途径克服这些调节机制 寡聚或构象状态的改变以产生异常激活。 解决我们的假设依赖于对受体整体的生物物理分析。在目标1中，我们寻求 用低温电子显微镜(Cryo-EM)测定近全长HER2的高分辨结构。这个 缺乏全长RTK的任何高分辨率结构是由于在表达、纯化和 稳定均一受体样本。我们最近通过以下方式克服了HER2的这些挑战 设计一种几乎全长的HER2构建物，并以稳定的形式强健地表达和纯化。我们的 初步的负染电子显微镜成像显示高度的样品均一性，使得 由低温EM进行的结构调查。在目标2中，我们将利用我们分离HER2的能力来生物物理 用电子显微镜表征HER2癌基因突变对寡聚化状态和结构的影响。我们会 然后将体外观察与下游信号联系起来。完成这一多学科的 该项目将代表着重大的科学贡献，不仅是因为所需的技术进步 研究单程跨膜受体，但也要了解HER2如何在 配基或共受体。这些知识可以应用于开发新的药物，选择性地针对突变 HER2的各种形式，并对抗与抗HER2疗法常见的耐药性。

PROJECT SUMMARY/ABSTRACT The goal of this proposal is to address fundamental gaps in our understanding of HER2 receptor tyrosine kinase (RTK) activation mechanism and regulation through direct structural and biophysical studies on nearly full-length receptor. Aberrant HER2 signaling through amplification or oncogenic mutations is at the root of many cancers and remains a key target of therapies. HER2, together with EGFR, HER3, and HER4 comprise the Human Epidermal Growth Factor Receptor (HER) family of RTKs, indispensable for cellular homeostasis. These receptors convert extracellular cues into intracellular responses through homo- and hetero- oligomerization induced by growth factor binding. HER2 distinguishes itself as an orphan receptor with no known ligand and signals by heterodimerization with other members of the HER family. We do not understand how HER2 regulates its catalytic activity in the absence of ligand-bound co-receptors, but many HER2 oncogenic mutations compromise these mechanisms and confer activity in a co-receptor independent manner. Several of those mutations fall outside of the kinase domain, but in the absence of structural understanding of how growth factor binding on the extracellular side of the receptor increases the catalytic activity of the receptor’s intracellular kinase domain, we cannot predict how these mutations elevate HER2 signaling, and most importantly change HER2 vulnerability to known therapeutics. We hypothesize that the orphan receptor HER2 features intrinsic structural mechanisms to regulate catalytic activity in the absence of ligand or co- receptor and oncogenic mutations outside of the kinase domain overcome these regulatory mechanisms via alterations in oligomerization or conformational states to produce aberrant activation. Addressing our hypotheses relies on biophysical analyses of the receptor as a whole. In Aim 1 we seek to determine a high-resolution structure of near-full length HER2 by cryo-electron microscopy (cryo-EM). The lack of any high-resolution structure of a full-length RTK is attributed to challenges in expressing, purifying, and stabilizing a homogeneous receptor sample. We have recently overcome these challenges for HER2 by engineering a near-full length HER2 construct that is robustly expressed and purified in a stable form. Our preliminary negative stain-electron microscopy imaging demonstrates high sample homogeneity that permits structural investigations by cryo-EM. In Aim 2, we will leverage our abilities in isolating HER2 to biophysically characterize the influence of HER2 oncogenic mutations on oligomerization state and structure by EM. We will then correlate the in vitro observations with downstream signaling. The completion of this multidisciplinary project will represent a significant scientific contribution, not only due to the technological advances required to study single-pass transmembrane receptors but also in the light of learning how HER2 regulates itself without ligand or co-receptor. Such knowledge could be applied to developing new drugs, selectively targeting mutant forms of HER2, and counteracting drug resistance common with anti-HER2 therapies.