Su Guoping, Min Wang, Taylor Ethan Will
Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602-2352, USA.
Mutat Res. 2005 Nov 11;579(1-2):133-48. doi: 10.1016/j.mrfmmm.2005.02.019. Epub 2005 Jul 28.
Pro-fs is a human immunodeficiency virus type 1 (HIV-l)-encoded putative selenoprotein, predicted by a theoretical analysis of the viral genome; it is potentially expressed by a -1 frameshift from the protease coding region. Pro-fs has significant sequence similarity to the DNA binding loop of nuclear factor kappa B (NF-kappaB), which is known to bind thioredoxin (Trx). We hypothesize that the putative HIV-1 pro-fs gene product functions by mimicry of NF-kappaB via binding to Trx. The hypothesis was tested in vitro by co-immunoprecipitation and GST-pull down assays, using a purified mutant pro-fs protein, in which the two potential selenocysteine residues were mutated to cysteines, in order to permit expression in bacteria. Both experiments showed that pro-fs binds to human wild type Trx (Trx-wt) with high affinity. Mutation of the two conserved cysteine residues in the Trx active site redox center to serine (Ser) (Trx-CS) weakened but failed to abolish the interaction. In pro-fs-transfected 293T cells, using confocal microscopy and fluorescence resonance energy transfer (FRET), we have observed that pro-fs localizes in cell nuclei and forms oligomers. Upon stimulation by phorbol 12-myristate 13-acetate (PMA), Trx translocates into cell nuclei. Significant FRET efficiency was detected in the nuclei of PMA-stimulated 293T cells co-expressing fluorescence-tagged pro-fs and Trx-wt or Trx-CS. These results indicate that in living cells the double cysteine mutant of pro-fs binds to both Trx and Trx-CS with high affinity, suggesting that Trx-pro-fs binding is a structurally-specific interaction, involving more of the Trx molecule than just its active site cysteine residues. These results establish the capacity for functional mimicry of the Trx binding ability of the NF-kappaB/Rel family of transcription factors by the putative HIV-1 pro-fs protein.
Pro-fs是一种由1型人类免疫缺陷病毒（HIV-1）编码的假定硒蛋白，通过对病毒基因组的理论分析预测得出；它可能由蛋白酶编码区发生 -1 移码表达产生。Pro-fs与核因子κB（NF-κB）的DNA结合环具有显著的序列相似性，已知NF-κB可结合硫氧还蛋白（Trx）。我们推测假定的HIV-1 pro-fs基因产物通过与Trx结合来模拟NF-κB发挥功能。为了在细菌中表达，使用纯化的突变型pro-fs蛋白（其中两个潜在的硒代半胱氨酸残基突变为半胱氨酸），通过共免疫沉淀和GST下拉试验在体外对该假说进行了验证。两个实验均表明pro-fs与人类野生型Trx（Trx-wt）具有高亲和力结合。将Trx活性位点氧化还原中心的两个保守半胱氨酸残基突变为丝氨酸（Ser）（Trx-CS）会削弱但并未消除这种相互作用。在转染了pro-fs的293T细胞中，使用共聚焦显微镜和荧光共振能量转移（FRET），我们观察到pro-fs定位于细胞核并形成寡聚体。在佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）刺激下，Trx会转位到细胞核中。在共表达荧光标记的pro-fs和Trx-wt或Trx-CS的PMA刺激的293T细胞的细胞核中检测到了显著的FRET效率。这些结果表明，在活细胞中，pro-fs的双半胱氨酸突变体与Trx和Trx-CS均具有高亲和力结合，这表明Trx-pro-fs结合是一种结构特异性相互作用，涉及的Trx分子部分不仅仅是其活性位点的半胱氨酸残基。这些结果证实了假定的HIV-1 pro-fs蛋白具有模拟转录因子NF-κB/Rel家族Trx结合能力的功能。
