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© 2003 The American Society for Nutritional Sciences J. Nutr. 133:2494S-2496S, July 2003

Supplement: Nutritional Genomics and Proteomics in Cancer Prevention

Models for Assessing the Role of Selenoproteins in Health1

Mohamed E. Moustafa, Easwari Kumaraswamy, Nianxin Zhong, Mahadev Rao, Bradley A. Carlson and Dolph L. Hatfield2

Section on the Molecular Biology of Selenium, Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892

2 To whom correspondence should be addressed. E-mail: hatfield{at}dc37a.nci.nih.gov.


   ABSTRACT
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 ABSTRACT
DISCUSSION
 LITERATURE CITED
 
Two model systems for examining the role of selenoproteins in health are discussed. One utilizes transgenic mice that carry mutant selenocysteine (Sec) tRNA transgenes that result in the reduction of selenoprotein expression in a protein- and tissue-specific manner. The other utilizes loxP-Cre technology to selectively remove the Sec tRNA gene in mammary epithelium that results in the reduction of only certain selenoproteins in this tissue. Both approaches provide important tools for examining the role of selenoproteins in health.

KEY WORDS: • conditional knockout mice • selenium • selenocysteine • selenocysteine tRNA • transgenic mice

Selenium is an essential micronutrient in the diet of many life forms including humans and other mammals [reviewed in ( 1)]. It is recognized as one of the most promising cancer chemopreventive agents ( 2). As an indication of the anticipated beneficial role of selenium in preventing cancer, the National Cancer Institute, based in part on an earlier human clinical trial that suggested that selenium dramatically reduces the incidence of prostate cancer ( 3), undertook a new human clinical trial that examines the role of selenium and/or vitamin E in the prevention of prostate cancer ( 4). This trial, which is known as the Selenium and Vitamin E Cancer Prevention Trial (SELECT), employs 32,000 men whose diets are being supplemented with selenium, vitamin E, selenium + vitamin E or a placebo over a 12-y period. What is so important about this study is that it not only assesses the role of selenium and/or vitamin E in preventing prostate cancer, but also provides insights into the roles of these two components in many other disorders such as lung and liver cancers and heart disease as well as in aging.

In addition to cancer, there is evidence to suggest that selenium has a role in the following: inhibiting viral expression ( 5), slowing the aging process ( 6), delaying the progression of AIDS in HIV-infected patients ( 7), preventing heart disease and other cardiovascular and muscle disorders ( 8), male reproduction ( 9), mammalian development ( 10) and immune function ( 6). Despite the many health benefits attributed to selenium, knowledge of the specific action of this element at the molecular level is lacking. One likely possibility is that selenium-containing proteins play a major role in providing better health. At least two selenoproteins, Sep15 ( 11, 12) and glutathione peroxidase (GPx)3 1 ( 13), were shown recently to have possible links to cancer prevention. However, there is a need for generation of model systems that can define the role of selenoproteins as a whole as well as individually in health.

One of our goals is to provide model systems that will permit assessment of selenoproteins in health. We have taken advantage of the fact that selenoprotein expression is totally dependent on the presence of selenocysteine (Sec) tRNA (designated Sec tRNA[Ser]Sec), which serves as the donor of selenium in the form of the amino acid Sec to protein [reviewed in ( 1)]. In fact, Sec tRNA[Ser]Sec is the only known tRNA that governs the expression of an entire class of proteins, namely, the selenoproteins. Recently, we generated a transgenic mouse line that carries a mutation at position 37 within the anticodon loop of Sec tRNA[Ser]Sec ( 14). Position 37 contains an adenosine that is modified to a much larger base, isopentenyladenosine (i6A). Mice that express Sec tRNA[Ser]Sec and lack i6A manifest a reduction in the level of numerous selenoproteins. In addition, using the loxP-Cre recombination system, we have generated a conditional knockout of the Sec tRNA gene ( 15) to target specific tissues and organs for removal of selenoprotein expression. As described herein, both of these mouse systems provide novel approaches for examination of the role of selenoproteins in health.


   DISCUSSION
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 ABSTRACT
 DISCUSSION
LITERATURE CITED
 
The recent symposium sponsored by the National Cancer Institute entitled Nutritional Genomics and Proteomics in Cancer Prevention ( 16) was not only a highly significant meeting on nutrition but also was very timely. The symposium emphasized the point that nutritional scientists and molecular biologists are more and more recognizing that health is governed in large part by the interplay between genetic background and nutritional status. One of our major goals has been to provide genetic models for understanding the role of selenium in health, and therefore, to elucidate the interplay between selenium and selenoproteins (whose expression can be altered genetically) in promoting better health.

Before examining specific approaches for generating model systems for elucidating the role of selenium through the action of selenoproteins in health, the mechanism of selenoprotein biosynthesis in mammals is discussed. Selenium is incorporated into protein as the amino acid Sec. Unlike the other 20 amino acids in mammalian proteins, Sec is biosynthesized on its tRNA, Sec tRNA[Ser]Sec. The genetic codeword for Sec is UGA, which was known to terminate protein synthesis at the time the code was first deciphered ( 17). It was not realized at that time that any codeword other than AUG, which was known to initiate protein synthesis and to serve as an internal codon for methionine, might have a dual function. However, UGA was found to also dictate Sec [reviewed in ( 1)], and its addition to the genetic code marked the first expansion of the code since it was deciphered in the mid-1960s ( 17).

What distinguishes UGA as a Sec codon and not a stop codon is the presence of a stem-loop structure in the 3'-untranslated region of eukaryotic selenoprotein mRNA [reviewed in ( 1)]. This stem-loop structure is designated as the selenocysteine insertion sequence (SECIS) element ( 18). The SECIS element is recognized by a protein factor designated as the SECIS binding protein ( 19). The resulting complex of these two components acts in concert with Sec tRNA[Ser]Sec bound to its specific elongation factor to donate Sec to the growing selenopeptide chain in protein synthesis (see Fig. 1).



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  		FIGURE 1  Incorporation of selenocysteine (Sec) into protein in eukaryotes. Sec tRNA[Ser]Sec is attached to its elongation factor (EFsec), and this complex is bound to SECIS binding protein 2 (SBP2) that recognizes the stem-loop structure [Sec insertion sequence (SECIS) element] in the 3'-untranslated region of selenoprotein mRNA. Sec tRNA[Ser]Sec is poised for insertion into the ribosomal acceptor site (dark-shaded oval on the right side of the large ribosomal subunit). The growing selenopeptide is shown at the ribosomal peptidyl site (dark-shaded center oval) as alternating light and dark balls. The left dark-shaded oval is the unacylated tRNA exit site. Selenoprotein mRNA is shown as a long dark line with its 5' and 3' ends, start and stop codons, SECIS element and UGA Sec codon ready to be decoded by Sec tRNA[Ser]Sec at the ribosomal acceptor site. After Sec tRNA[Ser]Sec is decoded and Sec is donated to the growing selenopeptide, EFsec is recycled and unacylated Sec tRNA[Ser]Sec leaves via the ribosomal exit site, but SBP2 remains attached to the SECIS element. [See ( 1) and text.]

 
There are about two dozen selenoproteins in mammals (Vadim N. Gladyshev, personal communication). Most certainly, selenoproteins play a major role in accounting for the many beneficial effects of selenium on health. To explore the role of selenoproteins in health, we generated (as noted earlier) a transgenic mouse that carries a mutant Sec tRNA[Ser]Sec that lacks the highly modified base, i6A, in its anticodon loop. Most likely, i6A functions to prevent misreading [see ( 14) and references therein]. The presence of the i6A mutant tRNA[Ser]Sec resulted in an inhibition in selenoprotein synthesis and in the maturation of tRNA[Ser]Sec ( 14). The reduction in selenoprotein synthesis occurs in a protein- and tissue-specific manner, whereby GPx1 and thioredoxin reductase 3 (TR3) are the most- and least-affected selenoproteins, respectively, and liver and testes are the most- and least-affected tissues, respectively ( 14). In fact, the synthesis of TR3 appears to be elevated in liver. The fact that the levels of numerous selenoproteins are reduced provides us with a model system to determine how this mouse will respond to a variety of environmental stresses including infection, carcinogens and cancer driver genes. Furthermore, because these mice are selenium deficient, placing them on a selenium-free diet should make them even more deficient than mice used in previous studies to examine the role of selenium deficiency on health.

The inhibition in Sec tRNA[Ser]Sec maturation caused by the i6A mutant does not affect the overall amount of the wild-type Sec tRNA[Ser]Sec population but only the distribution of the two major isoforms ( 14). The final step in the maturation of Sec tRNA[Ser]Sec is the methylation of one of the major isoforms, designated methylcarboxymethyl-5'-uridine (mcm5U), at the 2'-O-methylribosyl moiety in the wobble position of its anticodon to yield the other major isoform, which is designated methylcarboxymethyl-5'-uridine-2'-O-methylribose [mcm5Um; ( 14)]. The distribution of the two isoforms within the Sec tRNA[Ser]Sec population changed from a more-enriched amount of mcm5Um in liver of wild-type mice to a more-enriched amount of mcm5U in liver of i6A mutant mice. Interestingly, higher amounts of mcm5U appear to be associated with TR expression, and higher amounts of mcm5Um appear to be associated with GPx1 expression ( 14, 20).

In addition, we recently generated a mouse line that encodes the Sec tRNA[Ser]Sec gene flanked by loxP sites such that the removal of the gene is dependent on expression of the Cre recombinase ( 15). The Cre recombinase is carried in transgenic mice as a transgene that may be under the control of any of a number of different promoters that target a specific tissue ( 21, 22). Initially, we investigated (in collaboration with Vadim N. Gladyshev, University of Nebraska, Lincoln, NE and Lothar Hennighausen, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health) the effect of removal of the Sec tRNA[Ser]Sec gene in mouse mammary tissue by Cre recombinase that was under the influence of two different promoters: the promoter for the mouse mammary tumor virus (MMTV) long-terminal repeat and that for the whey acidic protein. Both promoters target mammary epithelium, but MMTV also is expressed in skin and spleen. The level of Sec tRNA[Ser]Sec was reduced > 70% in mammary tissue with either promoter but only in skin and spleen with MMTV-Cre. Selenoprotein expression was selectively reduced in breast with MMTV-Cre, whereby Sep15, GPx1 and GPx4 were substantially reduced. In skin, the TR level was increased more than twofold, whereas the GPx1 level was reduced ~25% ( 15). This hierarchy in selenoprotein expression has been discussed in detail elsewhere ( 1).

Transgenic mice that carry Sec tRNA[Ser]Sec–deficient i6A transgenes ( 14) and mice that encode conditional knockout of the Sec tRNA[Ser]Sec gene permit us to selectively manipulate Sec tRNA[Ser]Sec levels and selenoprotein expression and provide us with important tools for studying the role of selenoproteins in health.


   FOOTNOTES
 
1 Published in a supplement to The Journal of Nutrition. Presented at the "Nutritional Genomics and Proteomics in Cancer Prevention Conference" held September 5–6, 2002, in Bethesda, MD. This meeting was sponsored by the Center for Cancer Research, National Cancer Institute; Division of Cancer Prevention, National Cancer Institute; National Center for Complementary and Alternative Medicine, National Institutes of Health; Office of Dietary Supplements, National Institutes of Health; Office of Rare Diseases, National Institutes of Health; and the American Society for Nutritional Sciences. Guest editors for the supplement were Young S. Kim and John A. Milner, Nutritional Science Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD. Back

3 Abbreviations used: GPx, glutathione peroxidase; i6A, isopentenyladenosine; mcm5U, methylcarboxymethyl-5'-uridine; mcm5Um, methylcarboxymethyl-5'-uridine-2'-O-methylribose; MMTV, mouse mammary tumor virus; Sec, selenocysteine; SECIS, selenocysteine insertion sequence; TR3, thioredoxin reductase. Back


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 DISCUSSION
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1. Hatfield, D. L. & Gladyshev, V. N. (2002) How selenium has altered our understanding of the genetic code. Mol. Cell. Biol. 22: 3565–3576.[Free Full Text]

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3. Clark, L. C., Combs, G. F., JR., Turnbull, B. W., Slate, E. H., Chalker, D. K., Chow, J., Davis, L. S., Glover, R. A., Graham, G. F., Gross, E. G., Krongrad, A., Lesher, J. L., JR., Park, H. K., Sanders, B. B., JR., Smith, C. L. & Taylor, J. R. (1996) Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. J. Am. Med. Assoc. 276: 1957–1963.[Abstract]

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7. Baum, M. K., Campa, A., Miguez-Burbano, M. J., Burbano, X. & Shor-Posner, G. (2001) Role of selenium in HIV/AIDS. In: Selenium: Its Molecular Biology and Role in Human Health (Hatfield, D. L., ed.), ch. 20, pp. 247–255. Kluwer Academic Publishers, Norwell, MA.

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