HDX-MS-optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases.
HDX-MS-optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases.
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HDX-MS 优化方法用于表征纳米抗体作为 IB 类磷酸肌醇 3-激酶生化和结构研究的工具。
DOI：
10.1016/j.str.2021.07.002
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Burke,JohnE
中科院分区：
文献类型：
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作者：
Rathinaswamy,ManojK;Fleming,KaelinD;Dalwadi,Udit;Pardon,Els;Harris,NoahJ;Yip,CalvinK;Steyaert,Jan;Burke,JohnE
开发抗体作为信号传导途径的调节剂具有相当大的兴趣。磷脂酰肌醇3-激酶（PI 3 K）信号通路是高等真核生物中最重要的信号通路之一，在生长、代谢和免疫中起着重要作用。IB类PI 3 K，PI 3 K γ，是一种异二聚体复合物，由催化性p110γ亚基与p101或p84调节亚基结合组成。PI 3 K γ是多种免疫信号传导过程中的关键组分，并且依赖于Ras和G蛋白偶联受体（GPCR）的激活来介导其细胞作用。在这里，我们描述了使用氢-氘交换（HDX）质谱（MS）进行结构和生化研究的多个PI 3 K γ结合单链骆驼科纳米抗体的快速和有效的表征。我们鉴定了刺激脂质激酶活性、阻断Ras活化和特异性抑制p101介导的GPCR活化的纳米抗体。总的来说，我们的工作揭示了PI 3 K γ调节的洞察力，并确定了可能用于治疗开发的位点。
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.