Expression of human papilloma virus type 16 E5 protein in amelanotic melanoma cells regulates endo-cellular pH and restores tyrosinase activity
Expression of human papilloma virus type 16 E5 protein in amelanotic melanoma cells regulates endo-cellular pH and restores tyrosinase activity
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DOI：
10.1186/1756-9966-28-4
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发表时间：
2009-01-08
影响因子：
11.3
通讯作者：
De Marco, Federico
中科院分区：
文献类型：
--
作者：
Di Domenico, Fabio;Foppoli, Cesira;De Marco, Federico
背景：黑色素合成是黑色素细胞的选择性特性，受酪氨酸酶活性的调节。在酪氨酸酶阳性的无色素黑素瘤中，由于酸性内黑素体ph值，这种限速酶失去活性。人乳头瘤病毒16型的E5致癌基因是一种小的跨膜蛋白，具有弱的转化活性，在病毒感染的早期阶段起作用。E5被证明与跨膜液泡atp酶质子泵的16 kDa亚基C相互作用，最终导致其功能抑制。然而，这种相互作用对细胞的影响仍在争论中。通过这项工作，我们打算探索HPV16 E5癌蛋白是否确实与液泡atp酶质子泵相互作用，一旦在完整的人类细胞中表达，以及这种相互作用是否对细胞代谢和表型具有功能影响。方法：用逆转录病毒构建体诱导两株酪氨酸酶阳性无色素黑素瘤（FRM和M14细胞系）表达HPV16-E5癌蛋白。用吖啶橙和荧光显微镜测量细胞内pH的调节。RT-PCR、Western Blot和酶法检测酪氨酸酶的表达和活性。同时监测表达E5的细胞的非锚定生长和代谢活性。结果：我们提供的证据表明，在表达E5的细胞中，E5和v - atp酶之间的相互作用决定了细胞内ph的增加。细胞碱化反过来导致酪氨酸酶的翻译后激活，黑色素合成和表型调节。这些作用与酪氨酸类似物抗肿瘤药物的激活增加有关。结论：HPV16-E5癌蛋白一旦在完整的人类细胞中表达，确实与液泡v - atp酶质子泵相互作用，这种相互作用诱导了许多功能效应。在无色素黑素瘤中，这些作用可以调节细胞表型，并诱导对酪氨酸相关抗母细胞药物的更高敏感性。
Background: Melanin synthesis, the elective trait of melanocytes, is regulated by tyrosinase activity. In tyrosinase-positive amelanotic melanomas this rate limiting enzyme is inactive because of acidic endo-melanosomal pH. The E5 oncogene of the Human Papillomavirus Type 16 is a small transmembrane protein with a weak transforming activity and a role during the early steps of viral infections. E5 has been shown to interact with 16 kDa subunit C of the transmembrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions. However, the cellular effects of such an interaction are still under debate. With this work we intended to explore whether the HPV16 E5 oncoprotein does indeed interact with the vacuolar ATPase proton pump once expressed in intact human cells and whether this interaction has functional consequences on cell metabolism and phenotype.Methods: The expression of the HPV16-E5 oncoproteins was induced in two Tyrosinase-positive amelanotic melanomas (the cell lines FRM and M14) by a retroviral expression construct. Modulation of the intracellular pH was measured with Acridine orange and fluorescence microscopy. Expression of tyrosinase and its activity was followed by RT-PCR, Western Blot and enzyme assay. The anchorage-independence growth and the metabolic activity of E5 expressing cells were also monitored.Results: We provide evidence that in the E5 expressing cells interaction between E5 and V-ATPase determines an increase of endo-cellular pH. The cellular alkalinisation in turn leads to the post-translational activation of tyrosinase, melanin synthesis and phenotype modulation. These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs.Conclusion: Once expressed within intact human cells the HPV16-E5 oncoprotein does actually interact with the vacuolar V-ATPase proton pump and this interaction induces a number of functional effects. In amelanotic melanomas these effects can modulate the cell phenotype and can induce a higher sensitivity to tyrosine related anti-blastic drugs.