Byeong Jae Lee

Selenoprotein W is a glutathione‐dependent antioxidant in vivo

The function of selenoprotein W (Se‐W) was investigated by cloning the corresponding cDNA from mouse brain and expressing it in CHO cells and H1299 human lung cancer cells. Overexpression of Se‐W markedly reduced the sensitivity of both cell lines to H2O2 cytotoxicity. The intracellular peroxide concentration of the transfected cells was lower than that of the parental cells in the absence or presence of extracellular H2O2. The resistance to oxidative stress conferred by Se‐W was dependent on glutathione. Expression of Se‐W mutants in which selenocysteine‐13 or cysteine‐37 was replaced by serine did not confer resistance to H2O2, implicating these residues in the antioxidant activity of Se‐W in vivo.

Selective Inhibition of Selenocysteine tRNA Maturation and Selenoprotein Synthesis in Transgenic Mice Expressing Isopentenyladenosine-Deficient Selenocysteine tRNA

ABSTRACT Selenocysteine (Sec) tRNA (tRNA[Ser]Sec) serves as both the site of Sec biosynthesis and the adapter molecule for donation of this amino acid to protein. The consequences on selenoprotein biosynthesis of overexpressing either the wild type or a mutant tRNA[Ser]Sec lacking the modified base, isopentenyladenosine, in its anticodon loop were examined by introducing multiple copies of the corresponding tRNA[Ser]Sec genes into the mouse genome. Overexpression of wild-type tRNA[Ser]Sec did not affect selenoprotein synthesis. In contrast, the levels of numerous selenoproteins decreased in mice expressing isopentenyladenosine-deficient (i6A−) tRNA[Ser]Sec in a protein- and tissue-specific manner. Cytosolic glutathione peroxidase and mitochondrial thioredoxin reductase 3 were the most and least affected selenoproteins, while selenoprotein expression was most and least affected in the liver and testes, respectively. The defect in selenoprotein expression occurred at translation, since selenoprotein mRNA levels were largely unaffected. Analysis of the tRNA[Ser]Sec population showed that expression of i6A− tRNA[Ser]Sec altered the distribution of the two major isoforms, whereby the maturation of tRNA[Ser]Sec by methylation of the nucleoside in the wobble position was repressed. The data suggest that the levels of i6A− tRNA[Ser]Sec and wild-type tRNA[Ser]Sec are regulated independently and that the amount of wild-type tRNA[Ser]Sec is determined, at least in part, by a feedback mechanism governed by the level of the tRNA[Ser]Sec population. This study marks the first example of transgenic mice engineered to contain functional tRNA transgenes and suggests that i6A−tRNA[Ser]Sec transgenic mice will be useful in assessing the biological roles of selenoproteins.

Selective Removal of the Selenocysteine tRNA [Ser]Sec Gene ( Trsp ) in Mouse Mammary Epithelium

ABSTRACT Mice homozygous for an allele encoding the selenocysteine (Sec) tRNA[Ser]Sec gene (Trsp) flanked by loxP sites were generated. Cre recombinase-dependent removal of Trsp in these mice was lethal to embryos. To investigate the role of Trsp in mouse mammary epithelium, we deleted this gene by using transgenic mice carrying the Cre recombinase gene under control of the mouse mammary tumor virus (MMTV) long terminal repeat or the whey acidic protein promoter. While both promoters target Cre gene expression to mammary epithelium, MMTV-Cre is also expressed in spleen and skin. Sec tRNA[Ser]Sec amounts were reduced by more than 70% in mammary tissue with either transgene, while in skin and spleen, levels were reduced only with MMTV-Cre. The selenoprotein population was selectively affected with MMTV-Cre in breast and skin but not in the control tissue, kidney. Moreover, within affected tissues, expression of specific selenoproteins was regulated differently and often in a contrasting manner, with levels of Sep15 and the glutathione peroxidases GPx1 and GPx4 being substantially reduced. Expression of the tumor suppressor genes BRCA1 and p53 was also altered in a contrasting manner in MMTV-Cre mice, suggesting greater susceptibility to cancer and/or increased cell apoptosis. Thus, the conditional Trsp knockout mouse allows tissue-specific manipulation of Sec tRNA and selenoprotein expression, suggesting that this approach will provide a useful tool for studying the role of selenoproteins in health.

Chromatographic analysis of the aminoacyl-trnas which are required for translation of codons at and around the ribosomal frameshift sites of HIV, HTLV-1, and BLV

Abstract An examination of the frameshift signals or proposed signals within published sequences of retroviruses and other genetic elements from higher animals shows that each site utilizes a tRNA which normally contains Wybutoxine (Wye) base or Queuine (Q) base in the anticodon loop. We find experimentally that most of the Phe-tRNA present in HIV-1 infected cells lacks the highly modified Wye base in its anticodon loop and most of the Asn-tRNA in HTLV-1 and BLV infected cells lacks the highly modified Q base in its anticodon loop. Interestingly, Phe-tRNA translates a UUU codon within the ribosomal frameshift signal in HIV and Asn-tRNA translates a AAC codon within the proposed frameshift signals in HTLV-1 and BLV. Thus, the lack of a highly modified base in the anticodon loop of tRNAs in retroviral infected cells is correlated with the participation of these undermodified tRNAs in the corresponding frameshift event. This suggests that the “shifty” tRNAs proposed by Jacks et al. (Cell 55, 447–458, 1988) to carry out frameshifting may be hypomodified isoacceptors.

Structural study of novel antimicrobial peptides, nigrocins, isolated from Rana nigromaculata

Novel cationic antimicrobial peptides, named nigrocin 1 and 2, were isolated from the skin of Rana nigromaculata and their amino acid sequences were determined. These peptides manifested a broad spectrum of antimicrobial activity against various microorganisms with different specificity. By primary structural analysis, it was revealed that nigrocin 1 has high sequence homology with brevinin 2 but nigrocin 2 has low sequence homology with any other known antimicrobial peptides. To investigate the structure–activity relationship of nigrocin 2, which has a unique primary structure, circular dichroism (CD) and homonuclear nuclear magnetic resonance spectroscopy (NMR) studies were performed. CD investigation revealed that nigrocin 2 adopts mainly an α‐helical structure in trifluoroethanol (TFE)/H2O solution, sodium dodecyl sulfate (SDS) micelles, and dodecylphosphocholine micelles. The solution structures of nigrocin 2 in TFE/H2O (1:1, v/v) solution and in SDS micelles were determined by homonuclear NMR. Nigrocin 2 consists of a typical amphipathic α‐helix spanning residues 3–18 in both 50% TFE solution and SDS micelles. From the structural comparison of nigrocin 2 with other known antimicrobial peptides, nigrocin 2 could be classified into the family of antimicrobial peptides containing a single linear amphipathic α‐helix that potentially disrupts membrane integrity, which would result in cell death.

Selenoprotein Gene Nomenclature

The human genome contains 25 genes coding for selenocysteine-containing proteins (selenoproteins). These proteins are involved in a variety of functions, most notably redox homeostasis. Selenoprotein enzymes with known functions are designated according to these functions: TXNRD1, TXNRD2, and TXNRD3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4, and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine sulfoxide reductase B1), and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15-kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV), and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing, and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

Selective Restoration of the Selenoprotein Population in a Mouse Hepatocyte Selenoproteinless Background with Different Mutant Selenocysteine tRNAs Lacking Um34

Novel mouse models were developed in which the hepatic selenoprotein population was targeted for removal by disrupting the selenocysteine (Sec) tRNA[Ser]Sec gene (trsp), and selenoprotein expression was then restored by introducing wild type or mutant trsp transgenes. The selenoprotein population was partially replaced in liver with mutant transgenes encoding mutations at either position 34 (34T→A) or 37 (37A→G) in tRNA[Ser]Sec. The A34 transgene product lacked the highly modified 5-methoxycarbonylmethyl-2′-O-methyluridine, and its mutant base A was converted to I34. The G37 transgene product lacked the highly modified N6-isopentenyladenosine. Both mutant tRNAs lacked the 2′-methylribose at position 34 (Um34), and both supported expression of housekeeping selenoproteins (e.g. thioredoxin reductase 1) in liver but not stress-related proteins (e.g. glutathione peroxidase 1). Thus, Um34 is responsible for synthesis of a select group of selenoproteins rather than the entire selenoprotein population. The ICA anticodon in the A34 mutant tRNA decoded Cys codons, UGU and UGC, as well as the Sec codon, UGA. However, metabolic labeling of A34 transgenic mice with 75Se revealed that selenoproteins incorporated the label from the A34 mutant tRNA, whereas other proteins did not. These results suggest that the A34 mutant tRNA did not randomly insert Sec in place of Cys, but specifically targeted selected selenoproteins. High copy numbers of A34 transgene, but not G37 transgene, were not tolerated in the absence of wild type trsp, further suggesting insertion of Sec in place of Cys in selenoproteins.

Correlation between the activities of α-helical antimicrobial peptides and hydrophobicities represented as RP HPLC retention times

PTP7 is a 13-amino acid residue peptide designed from gaegurin 6, an antimicrobial peptide isolated from skin secretions of Rana rugosa. In order to examine the effect of hydrophobicity on antimicrobial activity, a series of PTP7 derivatives were constructed and analyzed the activity against bacteria and artificial membrane. We found that the mean hydrophobicity by simple summation of hydrophobicity of each constituent amino acid did not necessarily describe the hydrophobic property of antimicrobial peptides. The mean hydrophobicity did not show close correlation with the observed hydrophobicity by measuring reverse phase high performance liquid chromatography (RP HPLC) retention time. The observed hydrophobicity represented as RP HPLC retention time correlated well with the activity against artificial membrane and Gram positive bacterial species, such as Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus, rather than mean hydrophobicity. However, antimicrobial activity against Gram negative bacteria, such as Escherichia coli, did not show correlation with RP HPLC retention time. These data indicate that the RP HPLC retention time should be exploited rather than the mean hydrophobicity in the analysis of the relationship between hydrophobicity and antimicrobial activity.

Association between apolipoprotein E polymorphism and Alzheimer's disease in Koreans

We analyzed the aplolipoprotein E (APOE) genotypes of 110 probable AD patients and 226 cognitively normal controls in Koreans. The APOE epsilon4 allele was more prevalent in both early- and late-onset AD patients (P < 0.01) than in controls. The odds for the APOE epsilon4-heterozygous subjects were 2.7 (95% CI = 1.6-4.5), and those for the APOE epsilon4-homozygous subjects were 17.4 (95% CI = 2.0-147.3). But the odds were not uniform across age groups, and were higher in women than in men. Although the APOE epsilon2 allele frequency did not differ by diagnosis, the patients carrying an APOE epsilon2 allele showed delayed age-at-onset (P = 0.02). In conclusion, the APOE e4 allele increased the risk for AD in dose-dependent manner, and the APOE epsilon4-conferred AD risk was age- and sex-dependent in Koreans.

Homeodomain-interacting protein kinase 2 (HIPK2) targets β-catenin for phosphorylation and proteasomal degradation

The regulation of intracellular beta-catenin levels is central in the Wnt/beta-catenin signaling cascade and the activation of the Wnt target genes. Here, we show that homeodomain-interacting protein kinase 2 (HIPK2) acts as a negative regulator of the Wnt/beta-catenin pathway. Knock-down of endogenous HIPK2 increases the stability of beta-catenin and results in the accumulation of beta-catenin in the nucleus, consequently enhancing the expression of Wnt target genes and cell proliferation both in vivo and in cultured cells. HIPK2 inhibits TCF/LEF-mediated target gene activation via degradation of beta-catenin. HIPK2 phosphorylates beta-catenin at its Ser33 and Ser37 residues without the aid of a priming kinase. Substitutions of Ser33 and Ser37 for alanines abolished the degradation of beta-catenin associated with HIPK2. In ex vivo mouse model, HIPK2 knock-down resulted in accumulation of beta-catenin, thereby potentiated beta-catenin-mediated cell proliferation and tumor formation. Furthermore, the axis duplication induced by the ectopic expression of beta-catenin was blocked by co-injection of HIPK2 mRNAs into Xenopus embryos. Taken together, HIPK2 appears to function as a novel negative regulator of beta-catenin through its phosphorylation and proteasomal degradation.