PDF(Pubmed)
Abstract：
The transmembrane protein Agp2, initially shown as a transporter of L-carnitine, mediates the high-affinity transport of polyamines and the anticancer drug bleomycin-A5. Cells lacking Agp2 are hyper-resistant to polyamine and bleomycin-A5. In these earlier studies, we showed that the protein synthesis inhibitor cycloheximide blocked the uptake of bleomycin-A5 into the cells suggesting that the drug uptake system may require de novo synthesis. However, our recent findings demonstrated that cycloheximide, instead, induced rapid degradation of Agp2, and in the absence of Agp2 cells are resistant to cycloheximide. These observations raised the possibility that the degradation of Agp2 may allow the cell to alter its drug resistance network to combat the toxic effects of cycloheximide. In this study, we show that membrane extracts from agp2Δ mutants accentuated several proteins that were differentially expressed in comparison to the parent. Mass spectrometry analysis of the membrane extracts uncovered the pleiotropic drug efflux pump, Pdr5, involved in the efflux of cycloheximide, as a key protein upregulated in the agp2Δ mutant. Moreover, a global gene expression analysis revealed that 322 genes were differentially affected in the agp2Δ mutant versus the parent, including the prominent PDR5 gene and genes required for mitochondrial function. We further show that Agp2 is associated with the upstream region of the PDR5 gene, leading to the hypothesis that cycloheximide resistance displayed by the agp2Δ mutant is due to the derepression of the PDR5 gene.
摘要：
跨膜蛋白Agp2，最初显示为L-肉碱的转运蛋白，介导多胺和抗癌药物博来霉素-A5的高亲和力转运。缺乏Agp2的细胞对多胺和博来霉素-A5具有高抗性。在这些早期的研究中，我们发现蛋白质合成抑制剂环己酰亚胺阻断了博来霉素A5向细胞的摄取，这表明药物摄取系统可能需要从头合成。然而,我们最近的研究结果表明，环己酰亚胺，相反，Agp2诱导的快速降解，并且在不存在Agp2的情况下，细胞对环己酰亚胺具有抗性。这些观察结果提出了Agp2的降解可能允许细胞改变其耐药性网络以对抗环己酰亚胺的毒性作用的可能性。在这项研究中,我们表明，与亲本相比，agp2Δ突变体的膜提取物增强了几种差异表达的蛋白质。膜提取物的质谱分析揭示了多效性药物外排泵，参与环己酰亚胺外排的Pdr5，作为在agp2Δ突变体中上调的关键蛋白。此外，全球基因表达分析显示，在agp2Δ突变体与亲本中，有322个基因受到差异影响，包括突出的PDR5基因和线粒体功能所需的基因。我们进一步显示，Agp2与PDR5基因的上游区域相关，导致这样的假设，即agp2Δ突变体显示的环己酰亚胺抗性是由于PDR5基因的去抑制。
