e-Ming Xu

Thioredoxin Reductase 1 Deficiency Reverses Tumor Phenotype and Tumorigenicity of Lung Carcinoma Cells

Dietary selenium has potent cancer prevention activity. Both low molecular weight selenocompounds and selenoproteins are implicated in this effect. Thioredoxin reductase 1 (TR1) is one of the major antioxidant and redox regulators in mammals that supports p53 function and other tumor suppressor activities. However, this selenium-containing oxidoreductase is also overexpressed in many malignant cells and has been proposed as a target for cancer therapy. To further assess the role of TR1 in the malignancy process, we used RNA interference technology to decrease its expression in mouse lung carcinoma (LLC1) cells. Stable transfection of LLC1 cells with a small interfering RNA construct that specifically targets TR1 removal manifested a reversal in the morphology and anchorage-independent growth properties of these cancer cells that made them similar to those of normal cells. The expression of at least two cancer-related protein mRNAs, Hgf and Opn1, were reduced dramatically in the TR1 knockdown cells. Mice injected with the TR1 knockdown showed a dramatic reduction in tumor progression and metastasis compared with those mice injected with the corresponding control vector. In addition, tumors that arose from injected TR1 knockdown cells lost the targeting construct, suggesting that TR1 is essential for tumor growth in mice. These observations provide direct evidence that the reduction of TR1 levels in malignant cells is antitumorigenic and suggest that the enzyme is a prime target for cancer therapy.

Selenocysteine incorporation machinery and the role of selenoproteins in development and health.

Publisher Summary One of the major areas of emphasis has understood the role of selenium in health. Selenium is an essential micronutrient in the diet of mammals, and this element has numerous health benefits. It has roles in cancer and heart disease prevention, inhibiting viral expression, and delaying the progression of AIDS in HIV positive patients. Selenium has been reported to have roles in immune function, male reproduction, mammalian development, and slowing the aging process. This chapter discusses the means by which amino acid selenocysteine (Sec) is biosynthesized and incorporated into protein; the generation of mouse models for elucidating the role of selenoproteins in development and health; the identity and functions of selenoproteins; and the distribution and evolution of the amino acid Sec insertion machinery among eukaryotes. Sec is biosynthesized, unlike the common biosynthetic pathways of the other 20 protein amino acids, on its transfer RNA (tRNA). The machinery for inserting Sec into protein is novel and unique to this amino acid. It is apparent that tremendous effort has been expended in evolution for inserting selenium into protein in the form of Sec as discussed in the chapter.

Supramolecular complexes mediate selenocysteine incorporation in vivo.

ABSTRACT Selenocysteine incorporation in eukaryotes occurs cotranslationally at UGA codons via the interactions of RNA-protein complexes, one comprised of selenocysteyl (Sec)-tRNA[Ser]Sec and its specific elongation factor, EFsec, and another consisting of the SECIS element and SECIS binding protein, SBP2. Other factors implicated in this pathway include two selenophosphate synthetases, SPS1 and SPS2, ribosomal protein L30, and two factors identified as binding tRNA[Ser]Sec, termed soluble liver antigen/liver protein (SLA/LP) and SECp43. We report that SLA/LP and SPS1 interact in vitro and in vivo and that SECp43 cotransfection increases this interaction and redistributes all three proteins to a predominantly nuclear localization. We further show that SECp43 interacts with the selenocysteyl-tRNA[Ser]Sec-EFsec complex in vitro, and SECp43 coexpression promotes interaction between EFsec and SBP2 in vivo. Additionally, SECp43 increases selenocysteine incorporation and selenoprotein mRNA levels, the latter presumably due to circumvention of nonsense-mediated decay. Thus, SECp43 emerges as a key player in orchestrating the interactions and localization of the other factors involved in selenoprotein biosynthesis. Finally, our studies delineating the multiple, coordinated protein-nucleic acid interactions between SECp43 and the previously described selenoprotein cotranslational factors resulted in a model of selenocysteine biosynthesis and incorporation dependent upon both cytoplasmic and nuclear supramolecular complexes.

Targeting Thioredoxin Reductase 1 Reduction in Cancer Cells Inhibits Self-Sufficient Growth and DNA Replication

Thioredoxin reductase 1 (TR1) is a major redox regulator in mammalian cells. As an important antioxidant selenoprotein, TR1 is thought to participate in cancer prevention, but is also known to be over-expressed in many cancer cells. Numerous cancer drugs inhibit TR1, and this protein has been proposed as a target for cancer therapy. We previously reported that reduction of TR1 levels in cancer cells reversed many malignant characteristics suggesting that deficiency in TR1 function is antitumorigenic. The molecular basis for TR1s role in cancer development, however, is not understood. Herein, we found that, among selenoproteins, TR1 is uniquely overexpressed in cancer cells and its knockdown in a mouse cancer cell line driven by oncogenic k-ras resulted in morphological changes characteristic of parental (normal) cells, without significant effect on cell growth under normal growth conditions. When grown in serum-deficient medium, TR1 deficient cancer cells lose self-sufficiency of growth, manifest a defective progression in their S phase and a decreased expression of DNA polymerase α, an enzyme important in DNA replication. These observations provide evidence that TR1 is critical for self-sufficiency in growth signals of malignant cells, that TR1 acts largely as a pro-cancer protein and it is indeed a primary target in cancer therapy.

Selenoprotein H Is a Nucleolar Thioredoxin-like Protein with a Unique Expression Pattern

The human selenoproteome consists of 25 known selenoproteins, but functions of many of these proteins are not known. Selenoprotein H (SelH) is a recently discovered 14-kDa mammalian protein with no sequence homology to functionally characterized proteins. By sensitive sequence and structure analyses, we identified SelH as a thioredoxin fold-like protein in which a conserved CXXU motif (cysteine separated by two other residues from selenocysteine) corresponds to the CXXC motif in thioredoxins. These data suggest a redox function of SelH. Indeed, a recombinant SelH shows significant glutathione peroxidase activity. In addition, SelH has a conserved RKRK motif in the N-terminal sequence. We cloned wild-type and cysteine mutant forms of SelH either upstream or downstream of green fluorescent protein (GFP) and localized this fusion protein to the nucleus in transfected mammalian cells, whereas mutations in the RKRK motif resulted in the cytosolic protein. Interestingly, the full-length SelH-GFP fusion protein localized specifically to nucleoli, whereas the N-terminal sequence of SelH fused to GFP had a diffuse nucleoplasm location. Northern blot analyses revealed low expression levels of SelH mRNA in various mouse tissues, but it was elevated in the early stages of embryonic development. In addition, SelH mRNA was overexpressed in human prostate cancer LNCaP and mouse lung cancer LCC1 cells. Down-regulation of SelH by RNA interference made LCC1 cells more sensitive to hydrogen peroxide but not to other peroxides tested. Overall, these data establish SelH as a novel nucleolar oxidoreductase and suggest that some functions in this compartment are regulated by redox and dependent on the trace element selenium.

Evidence for Direct Roles of Two Additional Factors, SECp43 and Soluble Liver Antigen, in the Selenoprotein Synthesis Machinery

Selenocysteine (Sec) is inserted into selenoproteins co-translationally with the help of various cis- and trans-acting factors. The specific mechanisms of Sec biosynthesis and insertion into protein in eukaryotic cells, however, are not known. Two proteins, SECp43 and the soluble liver antigen (SLA), were previously reported to interact with tRNA[Ser]Sec, but their functions remained elusive. Herein, we report that knockdown of SECp43 in NIH3T3 or TCMK-1 cells using RNA interference technology resulted in a reduction in the level of methylation at the 2′-hydroxylribosyl moiety in the wobble position (Um34) of Sec tRNA[Ser]Sec, and consequently reduced glutathione peroxidase 1 expression. Double knockdown of SECp43 and SLA resulted in decreased selenoprotein expression. SECp43 formed a complex with Sec tRNA[Ser]Sec and SLA, and the targeted removal of one of these proteins affected the binding of the other to Sec tRNA[Ser]Sec. SECp43 was located primarily in the nucleus, whereas SLA was found in the cytoplasm. Co-transfection of both proteins resulted in the nuclear translocation of SLA suggesting that SECp43 may also promote shuttling of SLA and Sec tRNA[Ser]Sec between different cellular compartments. Taken together, these data establish the role of SECp43 and SLA in selenoprotein biosynthesis through interaction with tRNA[Ser]Sec in a multiprotein complex. The data also reveal a role of SECp43 in regulation of selenoprotein expression by affecting the synthesis of Um34 on tRNA[Ser]Sec and the intracellular location of SLA.

Biosynthesis of Selenocysteine, the 21st Amino Acid in the Genetic Code, and a Novel Pathway for Cysteine Biosynthesis

The biosynthetic pathway for selenocysteine (Sec), the 21st amino acid in the genetic code whose codeword is UGA, was recently determined in eukaryotes and archaea. Sec tRNA, designated tRNA([Ser]Sec), is initially aminoacylated with serine by seryl-tRNA synthetase and the resulting seryl moiety is converted to phosphoserine by O-phosphoseryl-tRNA kinase to form O-phosphoseryl-tRNA([Ser]Sec). Sec synthase (SecS) then uses O-phosphoseryl-tRNA([Ser]Sec) and the active donor of selenium, selenophosphate, to form Sec-tRNA([Ser]Sec). Selenophosphate is synthesized from selenide and ATP by selenophosphate synthetase 2 (SPS2). Sec was the last protein amino acid in eukaryotes whose biosynthesis had not been established and the only known amino acid in eukaryotes whose biosynthesis occurs on its tRNA. Interestingly, sulfide can replace selenide to form thiophosphate in the SPS2-catalyzed reaction that can then react with O-phosphoseryl-tRNA([Ser]Sec) in the presence of SecS to form cysteine-(Cys-)tRNA([Ser]Sec). This novel pathway of Cys biosynthesis results in Cys being decoded by UGA and replacing Sec in normally selenium-containing proteins (selenoproteins). The selenoprotein, thioredoxin reductase 1 (TR1), was isolated from cells in culture and from mouse liver for analysis of Cys/Sec replacement by MS. The level of Cys/Sec replacement in TR1 was proportional to the level of selenium in the diet of the mice. Elucidation of the biosynthesis of Sec and Sec/Cys replacement provides novel ways of regulating selenoprotein functions and ultimately better understanding of the biological roles of dietary selenium.

Deficiency in the 15-kDa Selenoprotein Inhibits Tumorigenicity and Metastasis of Colon Cancer Cells

Selenium has cancer-preventive activity that is mediated, in part, through selenoproteins. The role of the 15-kDa selenoprotein (Sep15) in colon cancer was assessed by preparing and using mouse colon CT26 cells stably transfected with short hairpin RNA constructs targeting Sep15. Metabolic 75Se labeling and Northern and Western blot analyses revealed that >90% of Sep15 was downregulated. Growth of the resulting Sep15-deficient CT26 cells was reduced (P < 0.01), and cells formed significantly (P < 0.001) fewer colonies in soft agar compared with control CT26 cells. Whereas most (14 of 15) BALB/c mice injected with control cells developed tumors, few (3 of 30) mice injected with Sep15-deficient cells developed tumors (P < 0.0001). The ability to form pulmonary metastases had similar results. Mice injected with the plasmid-transfected control cells had >250 lung metastases per mouse; however, mice injected with cells with downregulation of Sep15 only had 7.8 ± 5.4 metastases. To investigate molecular targets affected by Sep15 status, gene expression patterns between control and knockdown CT26 cells were compared. Ingenuity Pathways Analysis was used to analyze the 1,045 genes that were significantly (P < 0.001) affected by Sep15 deficiency. The highest-scored biological functions were cancer and cellular growth and proliferation. Consistent with these observations, subsequent analyses revealed a G2-M cell cycle arrest in cells with targeted downregulation of Sep15. In contrast to CT26 cells, Sep15-targeted downregulation in Lewis lung carcinoma (LLC1) cells did not affect anchorage-dependent or anchorage-independent cell growth. These data suggest tissue specificity in the cancer-protective effects of Sep15 downregulation, which are mediated, at least in part, by influencing the cell cycle. Cancer Prev Res; 3(5); 630–9. ©2010 AACR.

Structure and Catalytic Mechanism of Eukaryotic Selenocysteine Synthase

In eukaryotes and Archaea, selenocysteine synthase (SecS) converts O-phospho-l-seryl-tRNA[Ser]Sec into selenocysteyl-tRNA[Ser]Sec using selenophosphate as the selenium donor compound. The molecular mechanisms underlying SecS activity are presently unknown. We have delineated a 450-residue core of mouse SecS, which retained full selenocysteyl-tRNA[Ser]Sec synthesis activity, and determined its crystal structure at 1.65Å resolution. SecS exhibits three domains that place it in the fold type I family of pyridoxal phosphate (PLP)-dependent enzymes. Two SecS monomers interact intimately and together build up two identical active sites around PLP in a Schiff-base linkage with lysine 284. Two SecS dimers further associate to form a homotetramer. The N terminus, which mediates tetramer formation, and a large insertion that remodels the active site set SecS aside from other members of the family. The active site insertion contributes to PLP binding and positions a glutamate next to the PLP, where it could repel substrates with a free α-carboxyl group, suggesting why SecS does not act on free O-phospho-l-serine. Upon soaking crystals in phosphate buffer, a previously disordered loop within the active site insertion contracted to form a phosphate binding site. Residues that are strictly conserved in SecS orthologs but variant in related enzymes coordinate the phosphate and upon mutation corrupt SecS activity. Modeling suggested that the phosphate loop accommodates the γ-phosphate moiety of O-phospho-l-seryl-tRNA[Ser]Sec and, after phosphate elimination, binds selenophosphate to initiate attack on the proposed aminoacrylyl-tRNA[Ser]Sec intermediate. Based on these results and on the activity profiles of mechanism-based inhibitors, we offer a detailed reaction mechanism for the enzyme.

Simultaneous knockdown of the expression of two genes using multiple shRNAs and subsequent knock-in of their expression

Small hairpin RNA (shRNA) is a powerful tool for inhibiting gene expression. One limitation has been that this technique has been used primarily to target a single gene. This protocol expands upon previous methods by describing a knockdown vector that facilitates cloning of multiple shRNAs; this allows targeted knockdown of more than one gene or of a single gene that may otherwise be difficult to knockdown using a single shRNA. The targeted gene(s) can be readily re-expressed by transfecting knockdown cells with a knock-in vector, containing an shRNA-refractive cDNA that will express the protein-of-interest even in the presence of shRNAs. The constructed knockdown and knock-in vectors can be easily used concurrently to assess possible interrelationships between genes, the effects of gene loss on cell function and/or their restoration by replacing targeted genes one at a time. The entire knockdown or knock-in procedure can be completed in ∼3–4 months.