Role of Selenium in Cancer, HIV Infection and Human Health

硒是公认的癌症和心脏病的化学预防剂，作为抗病毒药物，它可能在延缓HIV阳性患者的衰老过程和延缓艾滋病的进展中发挥作用。这种元素的有益作用至少部分是由于它作为氨基酸硒半胱氨酸（Sec）存在于硒蛋白中。我们的项目侧重于硒与蛋白质结合的方式，特定硒蛋白在人体健康中的作用以及硒在艾滋病毒感染中的作用。由于Sec tRNA是硒蛋白生物合成的关键分子，我们用转基因小鼠分别编码2 ~ 20个野生型和2 ~ 40个异戊烯腺苷（i6A）突变型Sec tRNA基因，研究其表达对硒代谢的影响。野生型Sec tRNA的过表达不影响硒蛋白的合成，表明Sec tRNA不限制蛋白质的翻译。然而，i6A突变体Sec tRNA的表达选择性地抑制内源性Sec tRNA的成熟和特定硒蛋白的翻译。硒蛋白缺乏对i6A突变型Sec tRNA小鼠整体影响的DNA微阵列分析显示，参与氧化还原反应的许多其他酶都大幅减少。这些数据表明，硒蛋白表达与参与氧化还原反应的其他酶之间存在胞内联系。此外，我们（与MJB一起，见合作者）参与鉴定了与硒半胱氨酸- trna结合以将Sec插入蛋白质的特定延伸因子。在我们对特定硒蛋白在人类健康中的作用的研究中，我们发现一个15 kDa的硒蛋白（Sep15）位于染色体1p31上，在人类癌症中通常发生突变或缺失。该基因全长51 kb，由5个外显子和4个内含子组成。它以两个等位基因的形式存在于人类群体中，它们的差异在于两个单核苷酸多态性位点。其中一个多态性似乎需要更高水平的硒才能表达Sep15。Sep15基因在前列腺、肝脏、睾丸、肾脏和大脑中表达水平最高，在相应的恶性组织或细胞中表达水平显著降低。这些观察结果都表明Sep15在癌症发展中起作用。有趣的是，Sep15存在于哺乳动物细胞的内质网中，与udp -葡萄糖：糖蛋白葡萄糖基转移酶形成复合物，提示硒在蛋白质折叠的质量控制中起作用。在一项关于硒对人体健康影响的相关研究中，我们研究了艾滋病毒感染和随后的艾滋病发展导致患者缺乏硒和谷胱甘肽过氧化物酶（一种硒蛋白）的途径。接种猴免疫缺陷病毒的恒河猴在疾病的发展过程中会出现典型的硒和谷胱甘肽过氧化物酶缺乏，这在HIV阳性患者中也有发现。这一观察结果为我们研究与艾滋病相关的硒缺乏提供了一个模型。我们之前观察到，HIV感染的细胞不像未感染的对照细胞那样有效地标记硒蛋白群体。我们已经完全排除了HIV TAT蛋白在这一过程中的作用。然而，我们观察到，在慢性感染的HIV细胞中诱导HIV表达导致Sec tRNA亚型的分布与通常在缺硒组织中看到的相同，而在相应的未诱导细胞中Sec tRNA亚型的分布与在富硒组织中观察到的相似。因此，Sec tRNA群体似乎在该疾病的硒缺乏中起直接作用。为了更好地了解tRNA在HIV和其他哺乳动物逆转录病毒核糖体移框中的作用，我们检测了在其反密码子3侧缺乏高度修饰的wybutoxosine碱基的苯丙氨酸tRNA刺激移框事件的能力。这种低修饰的tRNA显著增强了移框，这表明通过在细胞内维持完全修饰的tRNA来抑制某些逆转录病毒表达的另一种途径。

Selenium is recognized as a chemopreventive agent against cancer and heart disease, as an antiviral agent and it likely plays a role in delaying the aging process and in delaying the progression of AIDS in HIV positive patients. The beneficial effects of this element are due, at least in part, to its presence in selenoproteins as the amino acid, selenocysteine (Sec). Our program focuses on the means by which Sec is incorporated into protein, the role of specific selenoproteins in human health and the role of selenium in HIV infection. As Sec tRNA is the key molecule in selenoprotein biosynthesis, we have produced transgenic mice encoding from 2 to 20 wild type or 2 to 40 isopentenyladenosine (i6A) mutant Sec tRNA transgenes to examine the effect of their expression on selenium metabolism. Over-expression of wild type Sec tRNA does not affect selenoprotein synthesis demonstrating that Sec tRNA is not limiting in protein translation. Expression of i6A mutant Sec tRNA, however, selectively inhibits endogenous Sec tRNA maturation and translation of specific selenoproteins. DNA microarray analysis of the global effects of selenoprotein deficiency in these i6A mutant Sec tRNA mice show that many other enzymes involved in redox reactions are substantially reduced. These data suggest an intracellular connection between selenoprotein expression and other enzymes involved in redox reactions. In addition, we participated (with MJB, see collaborators) in identifying the specific elongation factor that is responsible for binding with selenocysteyl-tRNA for insertion of Sec into protein. In our studies on examining the role of specific selenoproteins in human health, we found that a 15 kDa selenoprotein (Sep15) is localized on chromosome 1p31 at a site commonly mutated or deleted in human cancers. The gene spans 51 kb consisting of 5 exons and 4 introns. It exists as two alleles in the human population that differ by two single nucleotide polymorphic sites. One of the polymorphisms appears to require higher levels of selenium for Sep15 expression. Sep15 gene expression occurs in highest levels in prostate, liver, testes, kidney and brain and the level of expression was reduced dramatically in the corresponding malignant tissues or cells. Each of these observations suggests that Sep15 has a role in cancer development. Interestingly, Sep15 exists in the endoplasmic reticulum of mammalian cells in a complex with UDP-glucose:glycoprotein glucosyltransferase suggesting a role of selenium in quality control of protein folding. In a related study on the effects of selenium on human health, we have examined the means by which HIV infection and the subsequent development of AIDS results in selenium and glutathione peroxidase (a selenoprotein) deficiency in afflicted patients. Rhesus monkeys that are inoculated with simian immunodeficiency virus become characteristically selenium and glutathione peroxidase deficient in the progression of the resulting disease as is found in HIV positive patients. This observation provides us with a model for studying selenium deficiency associated with AIDS. We previously observed that HIV infected cells do not label the selenoprotein population as efficiently as uninfected control cells. We have completely ruled out a role of the HIV TAT protein in this process. However, we observed that induction of HIV expression in chronically infected HIV cells results in the distribution of the Sec tRNA isoforms that is normally seen in selenium deficient tissues, while the Sec tRNA distribution in the corresponding uninduced cells is like that observed in selenium sufficient tissues. Thus, the Sec tRNA population appears to have a direct role in selenium deficiency in this disease. To better understand the role of tRNA in ribosomal frameshifting in HIV and other mammalian retroviruses, we examined the ability of phenylalanine tRNA lacking the highly modified wybutoxosine base on the 3 side of its anticodon to stimulate the frameshift event. This hypomodified tRNA dramatically enhances frameshifting suggesting an alternative avenue for inhibiting expression of some retroviruses by maintaining fully modified tRNAs intracellularly.