Bellingham Shayne A, Coleman Louise A, Masters Colin L, Camakaris James, Hill Andrew F
Department of Biochemistry & Molecular Biology, The University of Melbourne, Victoria, Australia.
J Biol Chem. 2009 Jan 9;284(2):1291-301. doi: 10.1074/jbc.M804755200. Epub 2008 Nov 6.
Prion diseases are associated with the conformational conversion of the host-encoded cellular prion protein into an abnormal pathogenic isoform. Reduction in prion protein levels has potential as a therapeutic approach in treating these diseases. Key targets for this goal are factors that affect the regulation of the prion protein gene. Recent in vivo and in vitro studies have suggested a role for prion protein in copper homeostasis. Copper can also induce prion gene expression in rat neurons. However, the mechanism involved in this regulation remains to be determined. We hypothesized that transcription factors SP1 and metal transcription factor-1 (MTF-1) may be involved in copper-mediated regulation of human prion gene. To test the hypothesis, we utilized human fibroblasts that are deleted or overexpressing the Menkes protein (MNK), a major mammalian copper efflux protein. Menkes deletion fibroblasts have high intracellular copper, whereas Menkes overexpressed fibroblasts have severely depleted intracellular copper. We have utilized this system previously to demonstrate copper-dependent regulation of the Alzheimer amyloid precursor protein. Here we demonstrate that copper depletion in MNK overexpressed fibroblasts decreases cellular prion protein and PRNP gene levels. Conversely, expression of transcription factors SP1 and/or MTF-1 significantly increases prion protein levels and up-regulates prion gene expression in copper-replete MNK deletion cells. Furthermore, siRNA "knockdown" of SP1 or MTF-1 in MNK deletion cells decreases prion protein levels and down-regulates prion gene expression. These data support a novel mechanism whereby SP1 and MTF-1 act as copper-sensing transcriptional activators to regulate human prion gene expression and further support a role for the prion protein to function in copper homeostasis. Expression of the prion protein is a vital component for the propagation of prion diseases; thus SP1 and MTF-1 represent new targets in the development of key therapeutics toward modulating the expression of the cellular prion protein and ultimately the prevention of prion disease.
朊病毒疾病与宿主编码的细胞朊病毒蛋白构象转变为异常致病异构体有关。降低朊病毒蛋白水平有可能成为治疗这些疾病的一种治疗方法。实现这一目标的关键靶点是影响朊病毒蛋白基因调控的因素。最近的体内和体外研究表明朊病毒蛋白在铜稳态中发挥作用。铜也可诱导大鼠神经元中的朊病毒基因表达。然而，这种调控所涉及的机制仍有待确定。我们假设转录因子SP1和金属转录因子-1（MTF-1）可能参与铜介导的人类朊病毒基因调控。为了验证这一假设，我们利用了缺失或过表达门克斯蛋白（MNK，一种主要的哺乳动物铜外排蛋白）的人成纤维细胞。缺失门克斯蛋白的成纤维细胞细胞内铜含量高，而过表达门克斯蛋白的成纤维细胞细胞内铜严重缺乏。我们之前利用这个系统证明了阿尔茨海默病淀粉样前体蛋白的铜依赖性调控。在此我们证明，过表达MNK的成纤维细胞中铜缺乏会降低细胞朊病毒蛋白和PRNP基因水平。相反，在铜充足的缺失MNK的细胞中，转录因子SP1和/或MTF-1的表达显著增加朊病毒蛋白水平并上调朊病毒基因表达。此外，在缺失MNK的细胞中用小干扰RNA（siRNA）“敲低”SP1或MTF-1会降低朊病毒蛋白水平并下调朊病毒基因表达。这些数据支持一种新机制，即SP1和MTF-1作为铜感应转录激活因子来调控人类朊病毒基因表达，并进一步支持朊病毒蛋白在铜稳态中发挥作用。朊病毒蛋白的表达是朊病毒疾病传播的重要组成部分；因此，SP1和MTF-1代表了开发关键治疗药物以调节细胞朊病毒蛋白表达并最终预防朊病毒疾病的新靶点。
