Investigating oncogenic mutations and regulatory mechanisms of HER2 by electron microscopy

通过电子显微镜研究 HER2 的致癌突变和调控机制

• 批准号: 10297848
• 负责人: Devan Diwanji
• 金额: $ 5.18万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-11 至 2023-12-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10297848
• 关键词: Address; Adopted; Alternative Therapies; Architecture; Binding; Biological Assay; Biophysics; Bypass; C-terminal; Cell Survival; Cell physiology; Cells; Chromatography; Complement; Cryoelectron Microscopy; Crystallization; Cues; Detergents; Drug Design; Drug resistance; ERBB2 gene; ERBB3 gene; Electron Microscopy; Engineering; Epidermal Growth Factor Receptor; ErbB4 gene; Family; Gene Amplification; Goals; Growth Factor; Heterodimerization; Homeostasis; Homo; Human; Image; Immunotherapeutic agent; In Vitro; Individual; Investigation; Knowledge; Learning; Length; Ligands; Lipids; Malignant Neoplasms; Medical; Membrane; Modeling; Molecular; Molecular Conformation; Mutation; Negative Staining; Oncogenic; Orphan; Outcome; Patients; Phosphotransferases; Plant Roots; Rationalization; Receptor Protein-Tyrosine Kinases; Recombinants; Regulation; Resistance development; Resolution; Role; Sampling; Side; Signal Pathway; Signal Transduction; Somatic Mutation; Structure; Structure-Activity Relationship; System; Tail; Technology; Therapeutic; Time; base; biophysical analysis; biophysical techniques; extracellular; falls; flexibility; innovation; insight; malignant breast neoplasm; member; microscopic imaging; multidisciplinary; mutant; nanodisk; novel therapeutics; particle; prevent; receptor; reconstitution; response; small molecule; standard care; success; targeted treatment; therapy resistant; tool

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.

项目总结/摘要 该提案的目标是解决我们对HER 2受体酪氨酸的理解中存在的根本性差距 通过直接的结构和生物物理学研究， 全长受体通过扩增或致癌突变的异常HER 2信号传导是HER 2基因突变的根源。 许多癌症，并且仍然是治疗的关键目标。HER 2与EGFR、HER 3和HER 4一起包含 RTKs的人表皮生长因子受体（HER）家族，对于细胞内稳态是不可或缺的。 这些受体通过同源和异源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寡聚化。HER 2是一种孤儿受体， 已知的配体和信号通过与HER家族的其他成员异二聚化。我们不理解 HER 2如何在缺乏配体结合的辅助受体的情况下调节其催化活性，但许多HER 2 致癌突变损害这些机制并以非共受体依赖性方式赋予活性。 这些突变中有几个落在激酶结构域之外，但在缺乏对激酶结构的理解的情况下， 生长因子如何结合在受体的细胞外侧增加了受体的催化活性， 受体的胞内激酶结构域，我们无法预测这些突变如何提高HER 2信号传导， 最重要的是改变HER 2对已知疗法的脆弱性。我们假设孤儿受体 HER 2具有内在的结构机制，在缺乏配体或共-配体的情况下调节催化活性。 激酶结构域外的受体和致癌突变通过以下途径克服这些调节机制： 寡聚化或构象状态的改变以产生异常活化。 解决我们的假设依赖于受体作为一个整体的生物物理分析。目标1： 通过冷冻电子显微镜（cryo-EM）确定近全长HER 2的高分辨率结构。的 缺乏全长RTK的任何高分辨率结构归因于在表达、纯化和表达中的挑战。 稳定同质受体样品。我们最近通过以下方式克服了HER 2的这些挑战： 工程化以稳定形式稳健表达和纯化的近全长HER 2构建体。我们 初步的负染色电子显微镜成像显示了高的样品均匀性， cryo-EM的结构研究。在目标2中，我们将利用我们分离HER 2的能力， 通过EM表征HER 2致癌突变对寡聚化状态和结构的影响。我们将 然后将体外观察结果与下游信号联系起来。完成这一多学科 该项目将代表一个重大的科学贡献，不仅是因为需要技术进步， 研究单程跨膜受体，但也要了解HER 2如何调节自身， 配体或共受体。这些知识可以应用于开发新的药物，选择性地针对突变体 HER 2形式，并抵消抗HER 2疗法常见的耐药性。