STRUCTURE-INFORMED STUDIES OF THE EXTRACELLULAR REGION OF THE HUMAN NOTCH RECEPTOR IN HEALTH & DISEASE

Notch通路是一个重要的信号转导系统，调节细胞生物学中的关键决策。这些对于胚胎发育和成人组织的维持是至关重要的。在其最简单的形式中，细胞表面表达的配体通过与相邻细胞外部呈现的Notch受体蛋白结合来激活该途径。这会导致切割事件，从而使受体的细胞内部分释放并运往细胞核，在那里它与其他蛋白质形成复合体，激活负责调节细胞行为的特定基因。Notch活性的异常丧失或增加与许多人类发育障碍、成人发病疾病和癌症有关，使其成为治疗干预的关键靶点。尽管对激活的下游后果进行了广泛的研究，但导致Notch切割和信号产生的早期事件的许多方面仍然不清楚。这是因为我们对Notch的整体形状以及它与其配体相互作用的方式缺乏了解。由于Notch是一种弹性未知的大蛋白质，具有纤维状而不是球状组织，因此在技术上研究Notch的完整、全长状态是不可行的。相反，由于其模块化结构，可以根据功能重要性将其划分为不同的区域。在以前的工作中，我们已经建立了EGF4-13多肽的结构模型，它包含一个以前未被识别的EGF9和EGF10之间的灵活性区域。我们现在希望将我们的结构和功能研究扩展到EGF20-27和将其连接到配体结合区的结构域(EGF14-20)。我们热衷于了解EGF20-27内含有Abruptex(Ax)区域的结构域是否可以与配体结合区(LBR)在分子内或分子间相互作用，这将使我们能够了解从EGF4到EGF27的Notch胞外区域的整体形状，识别刚性区域和柔性区域，了解Ax/LBR相互作用对细胞表面Notch活性的结构、功能和调节的意义。对Notch信号是如何产生的基本了解应该使我们能够开发试剂来操纵Notch的活性，并促进细胞生物学的许多方面的进展，包括干细胞生物学。此外，由于该途径的功能障碍会导致许多遗传性和获得性疾病，如癌症，这些试剂很可能具有治疗潜力。

The Notch pathway is an essential signal transduction system which regulates crucial decisions in cell biology. These are vital for embryonic development and for the maintenance of adult tissue. In its simplest form, a cell-surface expressed ligand activates the pathway by binding to the Notch receptor protein presented on the outside of a neighbouring cell. This leads to cleavage events such that the intracellular portion of the receptor is released and travels to the nucleus where it forms a complex with other proteins to activate specific genes responsible for regulating cell behaviour. Abnormal loss or gain of Notch activity is associated with many human developmental disorders, adult onset diseases and cancers, making it a key target for therapeutic intervention. Despite extensive study of the downstream consequences of activation, many aspects of the early events leading to Notch cleavage and generation of the signal remain unclear. This is because we lack understanding of the overall shape of Notch and the way in which it can interact with its ligands. Because it is such a large protein of unknown flexibility, with a fibrous rather than globular organisation, it is not technically feasible to study Notch in its intact, full-length state. Instead, due to its modular construction it can be divided into different regions based on functional importance. In previous work, we have established a structural model for the EGF4-13 polypeptide which contains a previously unrecognized region of flexibility between EGF9 and EGF10. We now wish to extend our structural and functional studies to EGF20-27 and the domains (EGF14-20) that link this to the ligand-binding region. We are keen to understand whether domains within EGF20-27 which incorporate a region known as Abruptex (Ax) can interact intra or intermolecularly with the ligand-binding region (LBR).This will enable us to understand the overall shape of the extracellular domain of Notch from EGF4 to EGF27, to identify regions that are rigid and those that are flexible, to understand the significance of the Ax/LBR interaction for structure, function and regulation of Notch activity at the cell surface. Fundamental understanding of how the Notch signal is generated should allow us to develop reagents to manipulate Notch activity and facilitate advances in many aspects of cell biology, including stem cell biology. In addition, since dysfunction of the pathway results in many inherited and acquired forms of disease, such as cancer, these reagents are likely to have therapeutic potential.