We have characterized a new isozyme of selenium-dependent glutathione peroxidase, GSHPx-GI. Its mRNA is mainly detected in gastrointestinal (GI) tract tissues, and human but not rodent liver. Since GSHPx-GI mRNA is the major species of GSHPx detected in the intestinal epithelial fraction, GSHPx-GI could play a major role in detoxifying dietary lipid hydroperoxides. Because antioxidant activity, including that of GSHPx, may play an important role in the chemoprevention of those cancers elicited by oxidant stress, and since the GI tract is at the forefront in dealing with dietary oxidant insult, it would be important to know the basic biology of GSHPx-GI gene expression. Thus, our goals are: To identify which cell types in intestinal mucosa express GSHPx-GI. Since four cell types are present in intestinal epithelium including absorptive enterocytes, mucus-producing goblet cells, enteroendocrine cells, and defensin/lysozyme-producing Paneth cells, we will use immunocytochemistry and in situ hybridization with nucleic acid probes to identify the cell types express GSHPx-GI. Cellular fractionation will also be performed to determine whether GSHPx-GI mRNA and protein are expressed in differentiated villi or undifferentiated crypt cells. Although the expression of GSHPx-GI is selenium-dependent, the mechanism of selenium involvement is not fully understood. We have identified a sequence containing a partial selenocysteine-insertion (SECIS) motif in the 3' untranslated (3'UT) region of GSHPx-GI mRNA. We will test the sequence requirement for 75Se-incorporation into GSHPx-GI and its mutants lacking the UGA selenocysteine codon. Constructs will be made to determine the minimal sequence requirement to suppress the UGA opal codon. We will also analyze whether the SECIS sequence will induce selenocysteine incorporation when UGA is mutated into a Cys codon similarly to a formate dehydrogenase gene. On the other hand, selenium deficiency has been reported to enhance the degradation of several mammalian selenium- dependent genes. We hypothesize a mechanism which involves the SECIS motif in determining the mRNA degradation of these genes. This will be tested in our constructs of GSHPx-GI in the plasmids studied in vitro. Accomplishing the proposed research, we will uncover the site that the majority of lipid hydroperoxides are being reduced. We will also understand the mechanism involved in decoding the UGA codon in mammalian cells, as well as the mechanism involved in the selenium-dependent mRNA stability.
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