Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 2Y2, Canada.
Life Sciences Institute, Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Structure. 2021 Dec 2;29(12):1371-1381.e6. doi: 10.1016/j.str.2021.07.002. Epub 2021 Aug 3.
There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism, and immunity. The class IB PI3K, PI3Kγ, is a heterodimeric complex composed of a catalytic p110γ subunit bound to a p101 or p84 regulatory subunit. PI3Kγ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and G protein-coupled receptors (GPCRs) to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3Kγ binding single-chain camelid nanobodies using hydrogen-deuterium exchange (HDX) mass spectrometry (MS) for structural and biochemical studies. We identify nanobodies that stimulated lipid kinase activity, block Ras activation, and specifically inhibited p101-mediated GPCR activation. Overall, our work reveals insight into PI3Kγ regulation and identifies sites that may be exploited for therapeutic development.
人们对开发抗体作为信号通路调节剂表现出浓厚的兴趣。在高等真核生物中，磷酸肌醇 3-激酶（PI3K）通路是最重要的信号通路之一，它在生长、代谢和免疫中发挥着基本作用。PI3K 家族的 IB 型 PI3Kγ是一种异二聚体复合物，由与 p101 或 p84 调节亚基结合的催化 p110γ亚基组成。PI3Kγ是多种免疫信号转导过程的关键组成部分，并且依赖于 Ras 和 G 蛋白偶联受体（GPCR）的激活来介导其细胞功能。在这里，我们使用氢氘交换（HDX）质谱（MS）技术快速有效地描述了多种 PI3Kγ结合单链骆驼纳米抗体的特征，用于结构和生化研究。我们鉴定出了能够刺激脂质激酶活性、阻断 Ras 激活以及特异性抑制 p101 介导的 GPCR 激活的纳米抗体。总的来说，我们的工作揭示了对 PI3Kγ 调节的深入了解，并确定了可能用于治疗开发的位点。
