Regulatory Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, United States of America.
Mass Spectrometry Core for Proteomics and Metabolomics, The Salk Institute for Biological Studies, La Jolla, CA, United States of America.
PLoS Pathog. 2018 May 23;14(5):e1007071. doi: 10.1371/journal.ppat.1007071. eCollection 2018 May.
HIV-1 Tat is a key regulator of viral transcription, however little is known about the mechanisms that control its turnover in T cells. Here we use a novel proteomics technique, called DiffPOP, to identify the molecular target of JIB-04, a small molecule compound that potently and selectively blocks HIV-1 Tat expression, transactivation, and virus replication in T cell lines. Mass-spectrometry analysis of whole-cell extracts from 2D10 Jurkat T cells revealed that JIB-04 targets Serine Hydroxymethyltransferase 2 (SHMT2), a regulator of glycine biosynthesis and an adaptor for the BRCC36 K63Ub-specific deubiquitinase in the BRISC complex. Importantly, knockdown of SHMT1,2 or BRCC36, or exposure of cells to JIB-04, strongly increased Tat K63Ub-dependent destruction via autophagy. Moreover, point mutation of multiple lysines in Tat, or knockdown of BRCC36 or SHMT1,2, was sufficient to prevent destruction of Tat by JIB-04. We conclude that HIV-1 Tat levels are regulated through K63Ub-selective autophagy mediated through SHMT1,2 and the BRCC36 deubiquitinase.
HIV-1 Tat 是病毒转录的关键调节因子，但对于控制其在 T 细胞中周转的机制知之甚少。在这里，我们使用一种称为 DiffPOP 的新型蛋白质组学技术来鉴定小分子化合物 JIB-04 的分子靶标，该化合物可有效且选择性地阻断 T 细胞系中的 HIV-1 Tat 表达、转录激活和病毒复制。对 2D10 Jurkat T 细胞的全细胞提取物进行质谱分析表明，JIB-04 靶向丝氨酸羟甲基转移酶 2（SHMT2），它是甘氨酸生物合成的调节剂，也是 BRISC 复合物中 BRCC36 K63Ub 特异性去泛素化酶的衔接蛋白。重要的是，敲低 SHMT1、2 或 BRCC36，或用 JIB-04 处理细胞，均可通过自噬强烈增加 Tat K63Ub 依赖性降解。此外，Tat 中的多个赖氨酸的点突变，或敲低 BRCC36 或 SHMT1、2，足以防止 Tat 被 JIB-04 破坏。我们得出结论，HIV-1 Tat 水平通过 SHMT1、2 和 BRCC36 去泛素化酶介导的 K63Ub 选择性自噬来调节。
