Investigating the functional significance of lipid-binding properties of human Notch ligands in health and disease

Notch信号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定。这些对胚胎发育和维持成年组织至关重要。在其最简单的形式中，细胞表面表达的配体通过与邻近细胞外部存在的Notch受体蛋白结合来激活该途径。这导致裂解事件，使得受体的细胞内部分被释放并行进到细胞核，在那里它与其他蛋白质形成复合物以激活负责调节细胞行为的特定基因。Notch活性的异常丧失或获得与许多人类发育障碍、成人发病疾病和癌症相关，使其成为治疗干预的关键靶标。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配体相互作用的方式缺乏了解。我们最近建立了Notch配体的一个新特性：与细胞膜中发现的各种脂质结合的能力。脂质与配体的结合似乎与Notch结合偶联，表明依赖于可用的脂质和配体的类型的3组分复合物形式。这将进一步研究使用工程形式的配体，它应该形成更稳定的脂质/蛋白质复合物。此外，将使用高通量脂质结合测定来探测与配体相互作用的实际脂质的身份。最后，将通过测试各种突变配体激活Notch报告细胞系的能力，以及从肝细胞中Notch信号传导的研究中获得的细胞类型特异性见解，来研究脂质结合特性对遗传性和获得性疾病的重要性。对Notch信号如何产生的基本理解应该使我们能够开发试剂来操纵Notch活性，并促进细胞生物学（包括干细胞生物学）许多方面的进展。此外，由于该途径的功能障碍导致许多遗传性和获得性疾病，如癌症，因此这些试剂可能具有治疗潜力。

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. Aberrant 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. We have recently established a novel property of Notch ligands: the ability to bind to various lipids found in the cell membrane. Binding of lipid to ligand appears to be coupled to Notch binding, suggesting a 3-component complex forms dependent on the type of lipid and ligand available. This will be investigated further using an engineered form of ligand which should form more stable lipid/protein complexes. Furthermore the identity of the actual lipids which interact with ligand will be probed using high-throughput lipid binding assays. Lastly the importance of the lipid binding property for genetic and acquired disease will be investigated by testing the ability of various mutant ligands to activate a Notch reporter cell line , and cell type specific insights gained from the study of Notch signaling in hepatic cells. 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.