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首页> 外文期刊>Biochemistry >Dithiothreitol Causes HIV-1 Integrase Dimer Dissociation While Agents Interacting With the Integrase Dimer Interface Promote Dimer Formation
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Dithiothreitol Causes HIV-1 Integrase Dimer Dissociation While Agents Interacting With the Integrase Dimer Interface Promote Dimer Formation

机译：二硫苏糖醇引起HIV-1整合酶二聚体解离，而与整合酶二聚体界面相互作用的试剂促进二聚体形成。

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We have developed a homogeneous time-resolved fluorescence resonance energy transfer (FRET)-nbased assay that detects the formation of HIV-1 integrase (IN) dimers. The assay utilizes IN monomers thatnexpress two different epitope tags that are recognized by their respective antibodies, coupled to distinctnfluorophores. Surprisingly, we found that dithiothreitol (DTT), a reducing agent essential for in vitronenzymatic activity of IN, weakened the interaction between IN monomers. This effect of DTT on IN isndependent on its thiol groups, since the related chemical threitol, which contains hydroxyls in place of thiols,nhad no effect on INdimer formation. By studyingmutants of IN, we determined that cysteines in INappear tonbe dispensable for the dimer dissociation effect of DTT. Peptides derived from the IN binding domain (IBD)nof lens epithelium derived growth factor/transcriptional coactivator p75 (LEDGF), a cellular cofactor thatninteracts with the INdimer interface, were tested in this INdimerization assay. These peptides, which competenwith LEDGF for binding to IN, displayed an intriguing equilibrium binding dose-response curvencharacterized by a plateau rising to a peak, then descending to a second plateau. Mathematical modelingnof this binding system revealed that these LEDGF-derived peptides promote IN dimerization and blocknsubunit exchange between IN dimers. This dose-response behavior was also observed with a small moleculenthat interacts with the IN dimer interface and inhibits LEDGF binding to IN. In conclusion, this novel INndimerization assay revealed that peptide and small molecule inhibitors of the IN-LEDGF interaction alsonstabilize IN dimers and promote their formation.
机译：我们已经开发出一种基于均相时间分辨荧光共振能量转移（FRET）-n的检测方法，可检测HIV-1整合酶（IN）二聚体的形成。该测定利用使两个不同的表位标签紧贴的IN单体，所述两个不同的表位标签被其各自的抗体识别，并偶联至不同的荧光团。令人惊讶地，我们发现二硫苏糖醇（DTT），一种对IN的体外酶活性必不可少的还原剂，削弱了IN单体之间的相互作用。 DTT对IN的这种作用不依赖于其巯基，因为相关的化学苏糖醇含有羟基取代了巯基，对INdimer的形成没有影响。通过研究IN的突变体，我们确定IN上的半胱氨酸对于DTT的二聚体解离作用是可有可无的。在这种二聚化测定中测试了源自晶状体上皮的生长因子/转录共激活因子p75（LEDGF）的晶状体上皮的IN结合域（IBD）衍生的肽。这些与LEDGF竞争与IN结合的肽，表现出有趣的平衡结合剂量-反应曲线，其特征在于平台上升到峰值，然后下降到第二平台。该结合系统的数学建模表明，这些LEDGF衍生的肽促进IN二聚化和IN二聚体之间的嵌段亚基交换。还使用与IN二聚体界面相互作用并抑制LEDGF与IN结合的小分子观察到这种剂量反应行为。总而言之，这种新颖的INndimerization方法揭示了IN-LEDGF相互作用的肽和小分子抑制剂还可稳定IN二聚体并促进其形成。

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