Toxicology and Experimental Medicine Division, CSIR-Central Drug Research Institute, Sector-10, Janakipuram Extension, Sitapur Road, Lucknow 226031, U.P., India.
Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Sector-10, Janakipuram Extension, Sitapur Road, Lucknow 226031, U.P., India.
Life Sci. 2019 Jul 15;229:13-20. doi: 10.1016/j.lfs.2019.04.008. Epub 2019 Apr 3.
HIV-1 Nef downregulates surface MHC-I to protect the infected cells from CTLs-mediated killing. Although MHC-I downregulation has been extensively studied, the Nef-dependent assembly of the multi-protein complex and subsequent pathways activation has not yet been well explored. The present study is aimed for the identification of Nef-mediated sequential recruitment of cellular proteins that constitute the functional multi-protein complex, required for the downregulation of MHC-I.
Different Cellular protein complexes were identified by co-immunoprecipitation in Nef or NefE4A mutant-expressing Jurkat T, and THP-1 cells followed by exposure to Nef-specific peptides 24 h post infection. The MHC-I downregulation was analyzed by confocal microscopy and flow cytometry.
We found the association of Nef with PACS-2, GCC185, PI3K, AP-1, SFK, and MHC-I proteins that probably constitute a functional multi-protein complex. Furthermore, the immunoprecipitations with PACS-2 and GCC185 in the presence or absence of Nef, Nef E4A mutant and Nef with CP-inhibitor divide the functional complex of Nef into Nef-dependent (AP-1 and PI3K) and GCC185-dependent complex (MHC-I and SFK). The molecular mechanisms for activation of cellular pathways have been deciphered on the basis of these interactions that are brought in close proximity through Nef-GCC185 interaction. Knockdown of GCC185 using siRNA in Jurkat T cells showed a direct relationship between the assembly of functional multi-protein complex and MHC-I accumulation at GCC185.
Overall, our study elucidates that GCC185 is a focal point for the assembly of the Nef-mediated multi-protein complex at TGN.
HIV-1 的 Nef 下调表面 MHC-I 以保护受感染的细胞免受 CTL 介导的杀伤。尽管 MHC-I 的下调已经得到了广泛的研究，但 Nef 依赖性多蛋白复合物的组装以及随后的途径激活尚未得到很好的探索。本研究旨在鉴定 Nef 介导的细胞蛋白的顺序募集，这些蛋白构成功能性多蛋白复合物，是 MHC-I 下调所必需的。
在感染后 24 小时用 Nef 或 NefE4A 突变体表达的 Jurkat T 和 THP-1 细胞中通过共免疫沉淀鉴定不同的细胞蛋白复合物，然后用 Nef 特异性肽进行暴露。通过共聚焦显微镜和流式细胞术分析 MHC-I 的下调。
我们发现 Nef 与 PACS-2、GCC185、PI3K、AP-1、SFK 和 MHC-I 蛋白的关联，这些蛋白可能构成一个功能性的多蛋白复合物。此外，用 PACS-2 和 GCC185 进行免疫沉淀，无论是否存在 Nef、Nef E4A 突变体和 Nef 与 CP 抑制剂，都将 Nef 的功能复合物分为 Nef 依赖性（AP-1 和 PI3K）和 GCC185 依赖性复合物（MHC-I 和 SFK）。这些相互作用通过 Nef-GCC185 相互作用使细胞途径的激活的分子机制得以阐明。用 Jurkat T 细胞中的 siRNA 敲低 GCC185 显示，功能性多蛋白复合物的组装与 MHC-I 在 GCC185 处的积累之间存在直接关系。
总的来说，我们的研究阐明了 GCC185 是 TGN 中 Nef 介导的多蛋白复合物组装的焦点。
