Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, Dartmouth, Massachusetts, USA; The Biomedical Engineering and Biotechnology Program, University of Massachusetts Dartmouth, Dartmouth, Massachusetts, USA.
Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth, Dartmouth, Massachusetts, USA; The Biomedical Engineering and Biotechnology Program, University of Massachusetts Dartmouth, Dartmouth, Massachusetts, USA.
J Biol Chem. 2024 Aug;300(8):107529. doi: 10.1016/j.jbc.2024.107529. Epub 2024 Jul 1.
The multifunctional, HIV-1 accessory protein Nef enables infected cells to evade host immunity and thus plays a key role in viral pathogenesis. One prominent function of Nef is the downregulation of major histocompatibility complex class I (MHC-I), which disrupts antigen presentation and thereby allows the infected cells to evade immune surveillance by the cytotoxic T cells. Therapeutic inhibition of this Nef function is a promising direction of antiretroviral drug discovery as it may revitalize cytotoxic T cells to identify, and potentially clear, hidden HIV-1 infections. Guided by the crystal structure of the protein complex formed between Nef, MHC-I, and the hijacked clathrin adaptor protein complex 1, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef's activity on MHC-I. The optimized assay has a good signal-to-noise ratio, substantial tolerance of dimethylsulfoxide, and excellent ability to detect competitive inhibition, indicating that it is suitable for high-throughput screening.
多功能 HIV-1 辅助蛋白 Nef 使受感染的细胞能够逃避宿主的免疫，因此在病毒发病机制中起着关键作用。Nef 的一个突出功能是下调主要组织相容性复合体 I（MHC-I），这破坏了抗原呈递，从而使受感染的细胞能够逃避细胞毒性 T 细胞的免疫监视。抑制这种 Nef 功能是抗逆转录病毒药物发现的一个有前途的方向，因为它可能使细胞毒性 T 细胞恢复活力，以识别和潜在清除潜伏的 HIV-1 感染。受 Nef、MHC-I 和劫持的网格蛋白衔接蛋白复合物 1 之间形成的蛋白质复合物的晶体结构的指导，我们开发了一种基于荧光偏振的测定法，用于筛选针对 Nef 对 MHC-I 活性的抑制剂。优化后的测定法具有良好的信噪比、对二甲亚砜有很大的耐受性以及出色的检测竞争抑制的能力，表明它适合高通量筛选。
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