l(2)01810 is a novel type of glutamate transporter that is responsible for megamitochondrial formation.

Shim, Myoung Sup,Kim, Jin Young,Lee, Kwang Hee,Jung, Hee Kyoung,Carlson, Bradley A,Xu, Xue-Ming,Hatfield, Dolph L,Lee, Byeong Jae Biochemical Society 2011 The Biochemical journal Vol.439 No.2

<P>l(2)01810 causes glutamine-dependent megamitochondrial formation when it is overexpressed in Drosophila cells. In the present study, we elucidated the function of l(2)01810 during megamitochondrial formation. The overexpression of l(2)01810 and the inhibition of glutamine synthesis showed that l(2)01810 is involved in the accumulation of glutamate. l(2)01810 was predicted to contain transmembrane domains and was found to be localized to the plasma membrane. By using (14)C-labelled glutamate, l(2)01810 was confirmed to uptake glutamate into Drosophila cells with high affinity (K(m)=69.4 μM). Also, l(2)01810 uptakes glutamate in a Na(+)-independent manner. Interestingly, however, this uptake was not inhibited by cystine, which is a competitive inhibitor of Na(+)-independent glutamate transporters, but by aspartate. A signal peptide consisting of 34 amino acid residues targeting to endoplasmic reticulum was predicted at the N-terminus of l(2)01810 and this signal peptide is essential for the protein's localization to the plasma membrane. In addition, l(2)01810 has a conserved functional domain of a vesicular-type glutamate transporter, and Arg(146) in this domain was found to play a key role in glutamate transport and megamitochondrial formation. These results indicate that l(2)01810 is a novel type of glutamate transporter and that glutamate uptake is a rate-limiting step for megamitochondrial formation.</P>

Cell Proliferation and Motility Are Inhibited by G1 Phase Arrest in 15-kDa Selenoprotein-Deficient Chang Liver Cells

방지영,허장호,나지운,Qiao Lu,Bradley A. Carlson,Ryuta Tobe,Petra A. Tsuji,Vadim N. Gladyshev,Dolph L. Hatfield,이병재 한국분자세포생물학회 2015 Molecules and cells Vol.38 No.5

The 15-kDa selenoprotein (Sep15) is a selenoprotein residing in the lumen of the endoplasmic reticulum (ER) and implicated in quality control of protein folding. Herein, we established an inducible RNAi cell line that targets Sep15 mRNA in Chang liver cells. RNAi-induced Sep15 deficiency led to inhibition of cell proliferation, whereas cell growth was resumed after removal of the knockdown inducer. Sep15-deficient cells were arrested at the G1 phase by upregulating p21 and p27, and these cells were also characterized by ER stress. In addition, Sep15 deficiency led to the relocation of focal adhesions to the periphery of the cell basement and to the decrease of the migratory and invasive ability. All these changes were reversible depending on Sep15 status. Rescuing the knockdown state by expressing a silent mutant Sep15 mRNA that is resistant to siRNA also reversed the phenotypic changes. Our results suggest that SEP15 plays important roles in the regulation of the G1 phase during the cell cycle as well as in cell motility in Chang liver cells, and that this selenoprotein offers a novel functional link between the cell cycle and cell motility.

Cell Proliferation and Motility Are Inhibited by G1 Phase Arrest in 15-kDa Selenoprotein-Deficient Chang Liver Cells

Bang, Jeyoung,Huh, Jang Hoe,Na, Ji-Woon,Lu, Qiao,Carlson, Bradley A.,Tobe, Ryuta,Tsuji, Petra A.,Gladyshev, Vadim N.,Hatfield, Dolph L.,Lee, Byeong Jae Korean Society for Molecular and Cellular Biology 2015 Molecules and cells Vol.38 No.5

The 15-kDa selenoprotein (Sep15) is a selenoprotein residing in the lumen of the endoplasmic reticulum (ER) and implicated in quality control of protein folding. Herein, we established an inducible RNAi cell line that targets Sep15 mRNA in Chang liver cells. RNAi-induced Sep15 deficiency led to inhibition of cell proliferation, whereas cell growth was resumed after removal of the knockdown inducer. Sep15-deficient cells were arrested at the G1 phase by upregulating p21 and p27, and these cells were also characterized by ER stress. In addition, Sep15 deficiency led to the relocation of focal adhesions to the periphery of the cell basement and to the decrease of the migratory and invasive ability. All these changes were reversible depending on Sep15 status. Rescuing the knockdown state by expressing a silent mutant Sep15 mRNA that is resistant to siRNA also reversed the phenotypic changes. Our results suggest that SEP15 plays important roles in the regulation of the G1 phase during the cell cycle as well as in cell motility in Chang liver cells, and that this selenoprotein offers a novel functional link between the cell cycle and cell motility.

<i>MsrB1</i>(Methionine-R-sulfoxide Reductase 1) Knock-out Mice : <i>ROLES OF MsrB1 IN REDOX REGULATION AND IDENTIFICATION OF A NOVEL SELENOPROTEIN FORM</i>

Fomenko, Dmitri E.,Novoselov, Sergey V.,Natarajan, Sathish Kumar,Lee, Byung Cheon,Koc, Ahmet,Carlson, Bradley A.,Lee, Tae-Hyung,Kim, Hwa-Young,Hatfield, Dolph L.,Gladyshev, Vadim N. American Society for Biochemistry and Molecular Bi 2009 The Journal of biological chemistry Vol.284 No.9

<P>Protein oxidation has been linked to accelerated aging and is a contributing factor to many diseases. Methionine residues are particularly susceptible to oxidation, but the resulting mixture of methionine R-sulfoxide (Met-RO) and methionine S-sulfoxide (Met-SO) can be repaired by thioredoxin-dependent enzymes MsrB and MsrA, respectively. Here, we describe a knock-out mouse deficient in selenoprotein MsrB1, the main mammalian MsrB located in the cytosol and nucleus. In these mice, in addition to the deletion of 14-kDa MsrB1, a 5-kDa selenoprotein form was specifically removed. Further studies revealed that the 5-kDa protein occurred in both mouse tissues and human HEK 293 cells; was down-regulated by MsrB1 small interfering RNA, selenium deficiency, and selenocysteine tRNA mutations; and was immunoprecipitated and recognized by MsrB1 antibodies. Specific labeling with (75)Se and mass spectrometry analyses revealed that the 5-kDa selenoprotein corresponded to the C-terminal sequence of MsrB1. The MsrB1 knock-out mice lacked both 5- and 14-kDa MsrB1 forms and showed reduced MsrB activity, with the strongest effect seen in liver and kidney. In addition, MsrA activity was decreased by MsrB1 deficiency. Liver and kidney of the MsrB1 knock-out mice also showed increased levels of malondialdehyde, protein carbonyls, protein methionine sulfoxide, and oxidized glutathione as well as reduced levels of free and protein thiols, whereas these parameters were little changed in other organs examined. Overall, this study established an important contribution of MsrB1 to the redox control in mouse liver and kidney and identified a novel form of this protein.</P>

Selenophosphate synthetase 1 and its role in redox homeostasis, defense and proliferation

Na, Jiwoon,Jung, Jisu,Bang, Jeyoung,Lu, Qiao,Carlson, Bradley A.,Guo, Xiong,Gladyshev, Vadim N.,Kim, Jinhong,Hatfield, Dolph L.,Lee, Byeong Jae Elsevier 2018 FREE RADICAL BIOLOGY AND MEDICINE Vol.127 No.-

<P><B>Abstract</B></P> <P>Selenophosphate synthetase (SEPHS) synthesizes selenophosphate, the active selenium donor, using ATP and selenide as substrates. SEPHS was initially identified and isolated from bacteria and has been characterized in many eukaryotes and archaea. Two SEPHS paralogues, SEPHS1 and SEPHS2, occur in various eukaryotes, while prokaryotes and archaea have only one form of SEPHS. Between the two isoforms in eukaryotes, only SEPHS2 shows catalytic activity during selenophosphate synthesis. Although SEPHS1 does not contain any significant selenophosphate synthesis activity, it has been reported to play an essential role in regulating cellular physiology. Prokaryotic SEPHS contains a cysteine or selenocysteine (Sec) at the catalytic domain. However, in eukaryotes, SEPHS1 contains other amino acids such as Thr, Arg, Gly, or Leu at the catalytic domain, and SEPHS2 contains only a Sec. Sequence comparisons, crystal structure analyses, and ATP hydrolysis assays suggest that selenophosphate synthesis occurs in two steps. In the first step, ATP is hydrolyzed to produce ADP and gamma-phosphate. In the second step, ADP is further hydrolyzed and selenophosphate is produced using gamma-phosphate and selenide. Both SEPHS1 and SEPHS2 have ATP hydrolyzing activities, but Cys or Sec is required in the catalytic domain for the second step of reaction. The gene encoding SEPHS1 is divided by introns, and five different splice variants are produced by alternative splicing in humans. SEPHS1 mRNA is abundant in rapidly proliferating cells such as embryonic and cancer cells and its expression is induced by various stresses including oxidative stress and salinity stress. The disruption of the SEPHS1 gene in mice or <I>Drosophila</I> leads to the inhibition of cell proliferation, embryonic lethality, and morphological changes in the embryos. Targeted removal of SEPHS1 mRNA in insect, mouse, and human cells also leads to common phenotypic changes similar to those observed by in vivo gene knockout: the inhibition of cell growth/proliferation, the accumulation of hydrogen peroxide in mammals and an unidentified reactive oxygen species (ROS) in <I>Drosophila</I>, and the activation of a defense system. Hydrogen peroxide accumulation in SEPHS1-deficient cells is mainly caused by the down-regulation of genes involved in ROS scavenging, and leads to the inhibition of cell proliferation and survival. However, the mechanisms underlying SEPHS1 regulation of redox homeostasis are still not understood.</P> <P><B>Highlights</B></P> <P> <UL> <LI> The main determinant for SEPHS activity is Cys or Sec in the catalytic domain. </LI> <LI> SEPHS1 makes homodimers or heterodimers with its splice variants. </LI> <LI> SEPHS1 interacts with many other proteins including SEPHS2. </LI> <LI> SEPHS1 plays a role in maintaining cellular redox homeostasis. </LI> <LI> Redox status by SEPHS1 is responsible for cell proliferation and defense. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Elevation of glutamine level by selenophosphate synthetase 1 knockdown induces megamitochondrial formation in Drosophila cells.

Shim, Myoung Sup,Kim, Jin Young,Jung, Hee Kyoung,Lee, Kwang Hee,Xu, Xue-Ming,Carlson, Bradley A,Kim, Ki Woo,Kim, Ick Young,Hatfield, Dolph L,Lee, Byeong Jae American Society for Biochemistry and Molecular Bi 2009 The Journal of biological chemistry Vol.284 No.47

<P>Although selenophosphate synthetase 1 (SPS1/SelD) is an essential gene in Drosophila, its function has not been determined. To elucidate its intracellular role, we targeted the removal of SPS1/SelD mRNA in Drosophila SL2 cells using RNA interference technology that led to the formation of vacuole-like globular structures. Surprisingly, these structures were identified as megamitochondria, and only depolarized mitochondria developed into megamitochondria. The mRNA levels of l(2)01810 and glutamine synthetase 1 (GS1) were increased by SPS1/SelD knockdown. Blocking the expression of GS1 and l(2)01810 completely inhibited the formation of megamitochondria induced by loss of SPS1/SelD activity and decreased the intracellular levels of glutamine to those of control cells suggesting that the elevated level of glutamine is responsible for megamitochondrial formation. Overexpression of GS1 and l(2)01810 had a synergistic effect on the induction of megamitochondrial formation and on the synthesis of glutamine suggesting that l(2)01810 is involved in glutamine synthesis presumably by activating GS1. Our results indicate that, in Drosophila, SPS1/SelD regulates the intracellular glutamine by inhibiting GS1 and l(2)01810 expression and that elevated levels of glutamine lead to a nutritional stress that provides a signal for megamitochondrial formation.</P>

Selenoprotein Gene Nomenclature

Gladyshev, Vadim N.,Arné,r, Elias S.,Berry, Marla J.,Brigelius-Flohé,é,, Regina,Bruford, Elspeth A.,Burk, Raymond F.,Carlson, Bradley A.,Castellano, Sergi,Chavatte, Laurent,Conrad, M American Society for Biochemistry and Molecular Bi 2016 The Journal of biological chemistry Vol.291 No.46

<P>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.</P>

The zebrafish genome contains two distinct selenocysteine tRNA[Ser]Sec genes

(Xue Ming Xu),(Xuan Zhou),(Bradley A. Carlson),(Lark Kyun Kim),(Tae Lin Huh),(Byeong Jae Lee),(Dolph L. Hatfield) 경북대학교 유전공학연구소 2000 遺傳工學硏究所報 Vol.14 No.-

Regulation of HIF-1alpha Activity by Overexpression of Thioredoxin is Independent of Thioredoxin Reductase Status

Salvador Naranjo-Suarez,Dolph L. Hatfield,Bradley A. Carlson,Ryuta Tobe,유민혁,Petra A. Tsuji,Vadim N. Gladyshev 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.2

Under hypoxic conditions, cells activate a transcriptional response mainly driven by hypoxia-inducible factors (HIFs). HIF-1 stabilization and activity are known to be regulated by thioredoxin 1 (Txn1), but how the thioredoxin system regulates the hypoxic response is unknown. By examining the effects of Txn1 overexpression on HIF-1 function in HeLa, HT-29, MCF-7 and EMT6 cell lines, we found that this oxidoreductase did not stabilize HIF-1, yet could increase its activity. These effects were dependent on the redox function of Txn1. However, Txn1 deficiency did not affect HIF-1 hypoxic-stabilization and activity, and overexpression of thioredoxin reductase 1 (TR1), the natural Txn1 reductase, had no influence on HIF-1 activity. Moreover, overexpression of Txn1 in TR1 deficient HeLa and EMT6 cells was still able to increase HIF-1 hypoxic activity. These results indicate that Txn1 is not essential for HIF-1 hypoxic stabilization or activity, that its overex-pression can increase HIF-1 hypoxic activity, and that this effect is observed regardless of TR1 status. Thus, regulation of HIF-1 by the thioredoxin system depends on the specific levels of this system’s major components.

SUMO Modification of NZFP Mediates Transcriptional Repression through TBP Binding

김미진,이병재,Zifan Chen,심명섭,이명숙,김지은,권영은,유택진,김진영,방제영,Bradley A. Carlson,설재홍,Dolph L. Hatfield 한국분자세포생물학회 2013 Molecules and cells Vol.35 No.1

The negatively regulating zinc finger protein (NZFP) is an essential transcription repressor required for early devel-opment during gastrulation in Xenopus laevis. In this study, we found that NZFP interacts with the small ubiq-uitin-like modifier (SUMO) conjugation E2 enzyme, Ubc9, and contains three putative SUMO conjugation sites. Studies with NZFP mutants containing mutations at the putative SUMO conjugation sites showed that these sites were able to be modified independently with SUMO. NZFP was found to be localized in the same nuclear bodies with SUMO-1. However, sumoylation of NZFP did not play a role either in the translocation of NZFP into the nucleus or on nuclear body formation. While wild type NZFP showed significant transcriptional repression, SUMO-conjugation site mutants manifested a decrease in transcriptional repression activity which is reversely proportional to the amount of sumoylation. The sumoylation defective mutant lost its TBP binding activity, while wild type NZFP interacted with TBP and inhibited transcription complex formation. These results strongly suggest that the sumoylation of NZFP facilitates NZFP to bind to TBP and the NZFP/TBP complex then represses the transcription of the target gene by in-hibiting basal transcription complex formation.