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Übersichten New publications originating from the SeSPP 1087
New publications originating from the SeSPP 1087 J Chromatogr A. 2007 Jul 6;1155(2):180-6. Epub 2007 Feb 1. Studies on the selenoproteome in the cultured cells of lung and trachea by gel electrophoretic techniques Bukalis K, Wolf C, Behne D, Kyriakopoulos A. Abstract: In the present studies radiotracer techniques have been combined with biochemical separation procedures to investigate the selenium-containing
proteins in the culture cells of the lung, trachea and their subcellular fractions. Subcellular separation of the lung and trachea tissues has been achieved
by differential ultracentrifugation. The selenium-containing proteins in these compartments have been investigated by labeling of lung and trachea cultured
cells in vitro with Se-75, gel electrophoretic separation of the proteins and autoradiographic detection of the tracer. The protein separation by gel electrophoresis
using mono-dimensional (1D)- and two-dimensional (2D)-SDS-PAGE has been successfully applied for the selenium research. It has resulted in the detection of
a large number of selenium-containing proteins. Two-dimensional gel electrophoresis (2-DE) was also helpful in the identification of the proteins of interest
according to their molecular mass and isoelectric point. In this way more than 30 selenium-containing proteins could be distinguished in the lung and trachea
samples. Some of them such as Gpx1, Trx1, SelP, SelT and Sel15 could be identified by means of immunoassays, their molecular weight and pI values and
localized in the cellular compartments. J Mol Biol. 2007 Jun 29;370(1):116-27. Epub 2007 Apr 24. The Structure of Human Thioredoxin Reductase 1 Provides Insights into C-terminal Rearrangements During Catalysis Fritz-Wolf K, Urig S, Becker K. Abstract: Human thioredoxin reductase (hTrxR) is a homodimeric flavoprotein crucially involved in the regulation of cellular redox reactions, growth
and differentiation. The enzyme contains a selenocysteine residue at its C-terminal active site that is essential for catalysis. This redox center
is located on a flexible arm, solvent-exposed and reactive towards electrophilic inhibitors, thus representing a target for antitumor drug development.
During catalysis reducing equivalents are transferred from the cofactor NADPH to FAD, then to the N-terminal active site cysteine residues and from
there to the flexible C-terminal part of the other subunit to be finally delivered to a variety of second substrates at the molecule's surface. Here we report
the first crystal structure of hTrxR1 (Sec-->Cys) in complex with FAD and NADP(+) at a resolution of 2.8 A. From the crystals three different conformations
of the carboxy-terminal arm could be deduced. The predicted movement of the arm is facilitated by the concerted action of the three side-chain residues
of N418, N419 and W407, which act as a guiding bar for the C-terminal sliding process. As supported by previous kinetic data, the three visualized
conformations might reflect different stages in enzymatic catalysis. Comparison with other disulfide reductases including human glutathione reductase
revealed specific inhibitor binding sites in the intersubunit cavity of hTrxR that can be exploited for structure-based inhibitor development. Eur Heart J. 2007 Jun 13; [Epub ahead of print] Selene, the goddess of the moon: does she shine on men only? Schomburg, L. Crit Care Med. 2007 Mar;35(3):995-6; author reply 996-7. Selenium in intensive care (SIC) study: the XX files are still unresolved. Schomburg, L. J Mol Biol. 2007 Jan 26;365(4):1033-46. Epub 2006 Oct 13. The thioredoxin specificity of Drosophila GPx: a paradigm for a peroxiredoxin-like mechanism of many glutathione peroxidases. Maiorino M, Ursini F, Bosello V, Toppo S, Tosatto SC, Mauri P, Becker K, Roveri A, Bulato C, Benazzi L, De Palma A, Flohé L. Abstract: Some members of the glutathione peroxidase (GPx) family have been reported to accept thioredoxin as reducing substrate. However, the
selenocysteine-containing ones oxidise thioredoxin (Trx), if at all, at extremely slow rates. In contrast, the Cys homolog of Drosophila melanogaster
exhibits a clear preference for Trx, the net forward rate constant, k'(+2), for reduction by Trx being 1.5x10(6) M(-1) s(-1), but only 5.4 M(-1) s(-1) for
glutathione. Like other CysGPxs with thioredoxin peroxidase activity, Drosophila melanogaster (Dm)GPx oxidized by H(2)O(2) contained an intra-molecular
disulfide bridge between the active-site cysteine (C45; C(P)) and C91. Site-directed mutagenesis of C91 in DmGPx abrogated Trx peroxidase activity, but
increased the rate constant for glutathione by two orders of magnitude. In contrast, a replacement of C74 by Ser or Ala only marginally affected activity
and specificity of DmGPx. Furthermore, LC-MS/MS analysis of oxidized DmGPx exposed to a reduced Trx C35S mutant yielded a dead-end intermediate
containing a disulfide between Trx C32 and DmGPx C91. Thus, the catalytic mechanism of DmGPx, unlike that of selenocysteine (Sec)GPxs, involves formation
of an internal disulfide that is pivotal to the interaction with Trx. Hereby C91, like the analogous second cysteine in 2-cysteine peroxiredoxins, adopts the role of a
"resolving" cysteine (C(R)). Molecular modeling and homology considerations based on 450 GPxs suggest peculiar features to determine Trx specificity: (i) a
non-aligned second Cys within the fourth helix that acts as C(R); (ii) deletions of the subunit interfaces typical of tetrameric GPxs leading to flexibility of the
C(R)-containing loop. Based of these characteristics, most of the non-mammalian CysGPxs, in functional terms, are thioredoxin peroxidases. J Mol Biol. 2007 Jun 29;370(1):116-27. Epub 2007 Apr 24. Neuronal and ependymal expression of selenoprotein P in the human brain. Scharpf M, Schweizer U, Arzberger T, Roggendorf W, Schomburg L, Köhrle J. Abstract: Selenoprotein P (SePP) is central to selenium (Se) metabolism in the mammalian organism. Human SePP contains 10 Se atoms
that are covalent constituents of the polypeptide chain incorporated as the rare amino acid selenocysteine (Sec). Since hepatocytes
secrete SePP into plasma, SePP is commonly regarded as a Se transport protein, although SePP mRNA is expressed in many organs.
Gene targeting of SePP in mice leads to neurological dysfunction resulting from Se deficiency and associated reduction of selenoenzyme
activities in the brain. However, more recent data revealed that isolated hepatic SePP deficiency does not alter brain Se levels, suggesting
a role for SePP locally expressed in the brain. Some of the best characterized and most abundant selenoenzymes, glutathione peroxidases,
thioredoxin reductases, and methionine sulfoxide reductase B, play major roles in the cellular defense against reactive oxygen species.
Therefore, it was hypothesized that reduced brain Se bioavailability may be involved in the pathogenesis of neurodegenerative disease and
normal ageing. We present evidence that human CSF contains SePP and that the human brain expresses SePP mRNA. Moreover, SePP-like
immunoreactivity localizes to neurons and ependymal cells and thus appears strategically situated for maintenance and control of Se-dependent
anti-oxidative defense systems. Ann N Y Acad Sci. 2007 Jan;1096:179-83 The role of selenite on microglial migration. Dalla Puppa L, Savaskan NE, Bräuer AU, Behne D, Kyriakopoulos A. Abstract: Oxidative brain damage, such as excitotoxicity and stroke, leads to primary neuronal destruction. The primary damage is
further potentiated by macrophages and microglial cells, which are attracted and invade into the zone of damage resulting in
secondary neuronal death. Since the essential trace element selenium has anti-inflammatory properties, we analyzed the effects
of selenium on these inflammatory cells. Here, we show that the essential trace element selenium abrogates the stress-induced
migration of microglial cells. Thus, the antimigratory effects of selenium may attenuate the secondary cell death cascade by
preventing microglial invasion. Ann N Y Acad Sci. 2007 Jan;1095:467-72 Effects of selenium diet on expression of selenoproteins in the lung of the rat Bukalis K, Alber D, Bukalis G, Behne D, Kyriakopoulos A. Abstract: In the present article the radiotracer techniques have been combined with biochemical separation procedures to investigate
the effects of changes in the selenium status on the expression of the selenium-containing proteins in the lung and their subcellular
fractions. Subcellular separation of the lung has been achieved by differential ultracentrifugation. The selenium-containing proteins
in these compartments have been investigated by labeling of rats in vivo with (75)Se, gel electrophoretic separation of the proteins,
and autoradiographic detection of the tracer. In the lung of the selenium-deficient animals, the selenium administered was used
predominantly to restore the levels of the selenoproteins, while in the lung of the selenium-sufficient animals most of the selenium
retained was incorporated into the glutathione peroxidase. Also, higher activity of this enzyme has been found in the lung of the
selenium-sufficient animals. The differences in the specific incorporation of the element in the selenium deficiency into different
compounds suggested that there are different metabolic pathways for selenium, strongly dependent on its status. Ann N Y Acad Sci. 2007 Jan;1095:300-4 Protein expression in the tissues of the cardiovascular system of the rat under selenium deficiency and adequate conditions. Kyriakopoulos A, Richter A, Pohl T, Wolf C, Grbavac I, Plotnikov A, Kühbacher M, Bertelsmann H, Behne D. Abstract: We investigate the effects of selenium in tissues of the cardiovascular system. In order to examine the expression of the heart and aorta proteins
(part of them) in the homogenate of selenium-deficient (Se(-)) and selenium control rats (Se(+)), two-dimensional electrophoresis was used by means
of giga gels (30 x 35 cm). After electrophoresis, the protein expression pattern of the (Se(-)) gel and (Se(+)) gel was compared. The evaluation of the
protein difference was implemented by means of a computer program suitable for the analysis of protein separated by the two-dimensional electrophoresis.
In this way more than 2000 proteins a gel (heart) were detected and more than 1900 protein spots were detected in the aorta fraction. Ten significant
differences were found between the gel of (Se(+)) and (Se(-)) heart homogenate of the rat and more than 15 significant differences between the gel of
(Se(+)) and (Se(-)) of the aorta. By means of MALDI-MS-ESI-MS some of these proteins with different expression levels were not determined until now.
Of those, three proteins were detected as the alpha myosin heavy chain (alpha-MHC), myosin light chain 1 and 2 (MLC 1 and 2), and the mitochondrial
enzyme creatinine kinase. First results suggest that selenium deficiency affects myocardial energy metabolism and contractile proteins. Ann N Y Acad Sci. 2007 Jan;1095:204-8 Is the distribution of selenium and zinc in the sublocations of spermatozoa regulated? Bertelsmann H, Sieme H, Behne D, Kyriakopoulos A. Abstract: In the sperm nuclei, of mammalian species selenium has been found only in the form of sperm nuclei glutathione peroxidase (snGPx) where
it is most likely bound to the chromatin of spermatozoa. Over 80% of selenium in sperm is bound to the selenoprotein phospholipid hydroperoxide
glutathione peroxidase (PHGPx) in the midpiece of rat sperm. Zinc in sperm is mainly contained in the outer dense fiber (ODF) proteins of the flagella
of mammalian spermatozoa. In the sperm nuclei, zinc is predominately located in the chromatin to the protamine proteins. In order to investigate if the
insertion of zinc and selenium in sperm chromatin is regulated, the element concentrations were determined in equine spermatozoa and purified sperm nuclei.
We found a significant positive correlation between the selenium concentration in equine spermatozoa and sperm nuclei. The same finding was obtained
for the zinc concentration in spermatozoa and sperm nuclei. The results assume that the distribution of selenium and zinc in spermatozoa is regulated by
cell signaling pathways and in this way determining the selenium and zinc amount in the chromatin of spermatozoa. Optimization of spatiotemporal gene inactivation in mouse heart by oral application of tamoxifen citrate. Kiermayer C, Conrad M, Schneider M, Schmidt J, Brielmeier M. Abstract: Inducible and tissue-specific gene inactivation in mice has become a powerful tool to bypass embryonic and postnatal lethality of knockout mice.
The most frequently used inducible system is based on Cre recombinase fused to either one or two mutated estrogen receptor ligand binding domains,
thus rendering Cre function tamoxifen-dependent. To achieve Cre-mediated inactivation of a given gene, 4-OH tamoxifen (4-OHT) dissolved either in alcohol
and/or oil is usually administered by repeated intraperitoneal (i.p.) injections. Since this procedure imposes considerable stress on mice, we compared
the effect of tamoxifen citrate, mixed into a standard mouse diet at different concentrations, with that of i.p. administration of 4-OHT on Cre-mediated,
heart-specific inactivation of thioredoxin reductase 2. Here we show that tamoxifen citrate in the chow was equally effective as 4-OHT given i.p.
Oral tamoxifen administration is thus a convenient and cost-saving way for gene induction, and, most importantly, it reduces stress and avoids
adverse effects in mice. Endocrinology. 2006 Dec;147(12):5883-92. Epub 2006 Sep 7. Selenium-dependent pre- and posttranscriptional mechanisms are responsible for sexual dimorphic expression of selenoproteins in murine tissues. Riese C, Michaelis M, Mentrup B, Götz F, Köhrle J, Schweizer U, Schomburg L. Abstract: Important enzymes for thyroid hormone metabolism, antioxidative defense, and intracellular redox control contain selenocysteine (Sec) in their
active centers. Expression of these selenoproteins is tightly controlled, and a sex-specific phenotype is observed on disturbance of selenium (Se)
transport in mice. Therefore, we analyzed Se concentrations and expression levels of several selenoproteins including type I iodothyronine
deiodinase (Dio1) and glutathione peroxidase (GPx) isozymes in male and female mice. On regular lab chow, serum Se levels were comparable,
but serum GPx3 activity was higher in females than males (1.3-fold). Selenoprotein P (SePP) mRNA levels were higher in livers (1.3-fold) and lower
in kidneys (to 31%) in female compared with male mice. Orchidectomy alleviated the sex-specific differences in SePP mRNA amounts, indicating
modulatory effects of androgens on SePP expression. Female mice expressed higher levels of Dio1 mRNA in kidney (2.6-fold) and liver (1.4-fold) in
comparison with male mice. This sexual dimorphic expression of Dio1 mRNA was paralleled by increased Dio1 activity in female kidney (1.8-fold)
but not in liver in which males expressed higher Dio1 activity (2.8-fold). Interestingly, Se deficiency decreased Dio1 activity more effectively in males
than females, and resulting hepatic enzyme levels were then comparable between the sexes. At the same time, the sex-specific difference of Dio1
activity widened in kidney. Orchidectomy or estradiol treatment of ovariectomized females impacted stronger on renal than hepatic Dio1 expression.
Thus, we conclude that Se-dependent posttranscriptional mechanisms are operational that affect either translational efficiency or Dio1 stability in a
sex- and tissue-specific manner. Eur J Endocrinol. 2006 Dec;155(6):807-12. Selenium and goiter prevalence in borderline iodine sufficiency Brauer VF, Schweizer U, Köhrle J, Paschke R. Abstract: DESIGN: Selenium (Se) is required for the biosynthesis of selenocysteine-containing proteins. Several selenoenzymes, e.g. glutathione
peroxidases and thioredoxin reductases, are expressed in the thyroid. Selenoenzymes of the deiodinase family regulate the levels of thyroid
hormones. For clinical investigators, it is difficult to determine the role of Se in the etiology of (nodular-)goiter, because there are considerable
variations of Se concentrations in different populations as reflected by dietary habits, bioavailability of Se compounds, and racial differences.
Moreover, most previous clinical trials which investigated the influence of Se on thyroid volume harbored a bias due to the coexistence of
severe iodine deficiency in the study populations. METHODS: Therefore, we investigated the influence of Se on thyroid volume in an area with
borderline iodine sufficiency. First, we investigated randomly selected probands for urinary iodine (UI) and creatinine excretion in spot urine
samples and determined the prevalence of goiter and thyroid nodules by high-resolution ultrasonography. After this, we determined urinary Se
excretion (USe) in probands with goiter as well as in matched probands without goiter. Adjustments between the two compared groups were
made for age, gender, history of thyroid disorders, smoking, and UI excretion. RESULTS: The mean USe and UI rates of all 172 probands
were 24 micro g Se/l or 27 micro g Se/g creatinine and 96 micro g I/l or 113 micro g I/g creatinine indicating borderline selenium
(20-200 micro g/l) and iodine (100-200 micro g/l) sufficiency of the study population. Probands with goiter (n=89) showed significantly higher
USe levels than probands with normal thyroid volume (n=83; P < 0.05). USe rates were not influenced by present smoking or pregnancy.
CONCLUSIONS: In our investigation, USe was not an independent risk factor for the development of goiter. The higher USe in probands
with goiter in comparison with probands with normal thyroid volume is most likely a coincidence. Se does not significantly influence thyroid
volume in borderline iodine sufficiency because the iodine status is most likely the more important determinant. Am J Respir Cell Mol Biol. 2006 Dec;35(6):639-50. Epub 2006 Jun 22. Glutathione peroxidase 2, the major cigarette smoke-inducible isoform of GPX in lungs, is regulated by Nrf2. Singh A, Rangasamy T, Thimmulappa RK, Lee H, Osburn WO, Brigelius-Flohé R, Kensler TW, Yamamoto M, Biswal S. Abstract: Disruption of NF-E2-related factor (Nrf2), a redox-sensitive basic leucine zipper transcription factor, causes early-onset and more severe emphysema due to
chronic cigarette smoke. Nrf2 determines the susceptibility of lungs to cigarette smoke-induced emphysema in mice through the transcriptional induction of
numerous antioxidant genes. The lungs of Nrf2-/- mice have higher oxidative stress as evident from the increased levels of lipid peroxidation (4-hydroxy-2-nonenal)
and oxidative DNA damage (7,8-dihydro-8-Oxo-2'deoxyguanosine) in response to cigarette smoke. Glutathione peroxidases (GPX) are the primary antioxidant
enzymes that scavenge hydrogen peroxide and organic hydroperoxides. Among the five GPX isoforms, expression of GPX2 was significantly induced at both
mRNA and protein levels in the lungs of Nrf2+/+ mice, in response to cigarette smoke. Activation of Nrf2 by specific knock down of the cytosolic inhibitor of Nrf2,
Keap1, by small inhibitory RNA (siRNA) upregulated the expression of GPx2, whereas Nrf2 siRNA down-regulated the expression of GPX2 in lung epithelial cells.
An ARE sequence located in the 5' promoter-flanking region of exon 1 that is highly conserved between mouse, rat, and human was identified.
Mutation of this ARE core sequence completely abolished the activity of promoter-reporter gene construct. The binding of Nrf2 to the GPX2 antioxidant response
element was confirmed by chromatin immunoprecipation, electrophoretic mobility shift assays, and site-directed mutagenesis. This study shows that
GPX2 is the major oxidative stress-inducible cellular GPX isoform in the lungs, and that its basal as well as inducible expression is dependent on Nrf2. Semin Cancer Biol. 2006 Dec;16(6):452-65. Epub 2006 Sep 26. On the potential of thioredoxin reductase inhibitors for cancer therapy Urig S, Becker K. Abstract: Thioredoxin reductase (TrxR)-as part of a major thiol regulating system-allows redox metabolism to adjust to cellular requirements.
Therefore, changes at the redox level reflect as a pars pro toto changes concerning the entire cell. Three different TrxR isoenzymes, TrxR1 as cytosolic,
TrxR2 as mitochondrial, and TrxR3 as testis-specific thiol regulator are known. All three enzymes contain a reactive and solvent accessible selenocysteine
residue which is located on a flexible C-terminal arm of the protein. This selenocysteine is essentially involved in the catalytic cycle of TrxR and thus
represents an attractive binding site for inhibitors. Many tumor cells have elevated TrxR levels and TrxR has been shown to play a major role in drug
resistance. Inhibition of TrxR and its related redox reactions may thus contribute to a successful single, combinatory or adjuvant cancer therapy.
A great number of effective natural and synthetic TrxR inhibitors are now available possessing antitumor potential ranging from induction of oxidative
stress to cell cycle arrest and apoptosis. This article summarizes the present knowledge on the potential of TrxR inhibitors and TrxR as anticancer drug target. Chembiochem. 2006 Nov;7(11):1649-52. Mutational studies confirm the catalytic triad in the human selenoenzyme thioredoxin reductase predicted by molecular modeling Gromer S, Wessjohann LA, Eubel J, Brandt W. Biol Chem. 2006 Oct-Nov;387(10-11):1329-35. Glutathione peroxidases and redox-regulated transcription factors Brigelius-Flohé R. Abstract: Analysis of the selenoproteome identified five glutathione peroxidases (GPxs) in mammals: cytosolic GPx (cGPx, GPx1), phospholipid
hydroperoxide GPx (PHGPX, GPx4), plasma GPx (pGPX, GPx3), gastrointestinal GPx (GI-GPx, GPx2) and, in humans, GPx6, which is restricted
to the olfactory system. GPxs reduce hydroperoxides to the corresponding alcohols by means of glutathione (GSH). They have long been considered
to only act as antioxidant enzymes. Increasing evidence, however, suggests that nature has not created redundant GPxs just to detoxify hydroperoxides.
cGPx clearly acts as an antioxidant, as convincingly demonstrated in GPx1-knockout mice. PHGPx specifically interferes with NF-kappaB activation by
interleukin-1, reduces leukotriene and prostanoid biosynthesis, prevents COX-2 expression, and is indispensable for sperm maturation and embryogenesis.
GI-GPx, which is not exclusively expressed in the gastrointestinal system, is upregulated in colon and skin cancers and in certain cultured cancer cells.
GI-GPx is a target for Nrf2, and thus is part of the adaptive response by itself, while PHGPx might prevent cancer by interfering with inflammatory pathways.
In conclusion, cGPx, PHGPx and GI-GPx have distinct roles, particularly in cellular defence mechanisms. Redox sensing and redox regulation of metabolic
events have become attractive paradigms to unravel the specific and in part still enigmatic roles of GPxs. Anal Biochem. 2006 Sep 1;356(1):148-50. Epub 2006 May 11. Single-step purification of specific tRNAs by hydrophobic tagging Kothe U, Paleskava A, Konevega AL, Rodnina MV. Free Radic Res. 2006 Sep;40(9):936-43. Selenoprotein P protects endothelial cells from oxidative damage by stimulation of glutathione peroxidase expression and activity Steinbrenner H, Bilgic E, Alili L, Sies H, Brenneisen P. Abstract: A major fraction of the essential trace element selenium circulating in human blood plasma is present as selenoprotein P (SeP).
As SeP associates with endothelial membranes, the participation of SeP in selenium-mediated protection against oxidative damage was investigated,
using the human endothelial cell line Ea.hy926 as a model system. Hepatocyte-derived SeP prevented tert-butylhydroperoxide (t-BHP)-induced oxidative
cell death of Ea.hy926 cells in a similar manner as did sodium selenite, counteracting a t-BHP-induced loss of cellular membrane integrity.
Protection was detected after at least 10 h of SeP supplementation and it peaked at 24 h. SeP time-dependently stimulated the expression
of cytosolic glutathione peroxidase (cGPx) and increased the enzymatic activities of glutathione peroxidase (GPx) and thioredoxin reductase (TR).
The cGPx inhibitor mercaptosuccinate as well as the gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine counteracted the
SeP-mediated protection, while the TR inhibitors cisplatin and auranofin had no effect. The presented data suggest that selenium supplementation
by SeP prevents oxidative damage of human endothelial cells by restoring expression and enzymatic activity of GPx. Andrologia. 2006 Aug;38(4):152-7. Failure of phospholipid hydroperoxide glutathione peroxidase expression in oligoasthenozoospermia and mutations in the PHGPx gene Diaconu M, Tangat Y, Böhm D, Kühn H, Michelmann HW, Schreiber G, Haidl G, Glander HJ, Engel W, Nayernia K Abstract: Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein belonging to the family of glutathione peroxidases. PHGPx has
long been considered a major antioxidant that, in cooperation with vitamin E, protects biomembranes. To determine the expression pattern of PHGPx
mRNA in human, quantitative RT-polymerase chain reaction (PCR) analyses using RNA from different embryonal and adult tissues were performed.
A predominant expression was found in testes. In spermatozoa, PHGPx was found to be localized in the mid-piece of spermatozoa. We studied the
relationship between spermatozoa PHGPx expression, mutations in PHGPx gene and human oligoasthenozoospermia, a defect in which both the
number and the motility of spermatozoa are significantly below normal. Spermatozoa specimens from 45 infertile males were analysed for fertility-related
parameters according to World Health Organisation and were classified as suffering from oligoasthenozoospermia. Two patients (4.44%) showed no
expression of PHGPx and in nine patients (20.00%), a reduced expression of the enzyme was observed. DNA sequences of various regions of the
PHGPx gene (coding, 5'flanking region and intron 1) from these patients and 58 fertile volunteers were analysed for mutations by PCR amplification
and direct sequencing. Sequence data revealed no cause/effect relationship for any of the variants. From these data it can be concluded that
oligoasthenozoospermia is associated with a decrease in the level of expression of PHGPx in the spermatozoa of some infertile men (24.44%),
but is not linked to mutations in PHGPx gene. Free Radic Res. 2006 Aug;40(8):775-87. Part of the series: from dietary antioxidants to regulators in cellular signaling and gene regulation. Sulforaphane and selenium, partners in adaptive response and prevention of cancer Brigelius-Flohé R. Abstract: The association of decreased cancer risk with intake of cruciferous vegetables and selenium is stronger than that reported for fruits and vegetables
in general. An active constituent in cruciferae is sulforaphane. Chemopreventive effects of both, sulforaphane and selenium have been attributed to an
antioxidant action which certainly is too simplicistic. Sulforaphane induces via activation of the Nrf2/Keap1 system phase 2 enzymes that protect against
carcinogens and oxidants. Induced enzymes comprise the selenoproteins thioredoxin reductase-1 (TrxR1) and gastrointestinal glutathione peroxidase
(GI-GPx, GPx2), which contain antioxidant response elements (ARE) in their promoter regions. Translational realisation of the enhanced transcripts
depends on adequate selenium supply, which explains the synergism of Nrf2 activators and selenium. Regarding tumorigenesis the role of TrxR1 is
ambiguous: it is essential for fast tumor cell growth but also diminishes vascularisation of tumors. The anticarcinogenic role of GI-GPx is evident from
enhanced gastrointestinal tumor formation in gpx2/gpx1 double KO mice. J Biol Chem. 2006 Jul 14;281(28):19655-64. Epub 2006 May 9. The role of phospholipid hydroperoxide glutathione peroxidase isoforms in murine embryogenesis Borchert A, Wang CC, Ufer C, Schiebel H, Savaskan NE, Kuhn H. Abstract: Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a selenocysteine-containing enzyme, and three different isoforms
(cytosolic, mitochondrial, and nuclear) originate from the GPx4 gene. Homozygous GPx4-deficient mice die in utero at midgestation,
since they fail to initiate gastrulation and do not develop embryonic cavities. To investigate the biological basis for embryonic lethality,
we first explored expression of the GPx4 in adult murine brain and found expression of the protein in cerebral neurons. Next, we profiled
mRNA expression during the time course of embryogenesis (embryonic days 6.5-17.5 (E6.5-17.5)) and detected mitochondrial and cytosolic
mRNA species at high concentrations. In contrast, the nuclear isoform was only expressed in small amounts. Cytosolic GPx4 mRNA was
present at constant levels (about 100 copies per 1000 copies of glyceraldehyde-3-phosphate dehydrogenase mRNA), whereas nuclear and
mitochondrial isoforms were down-regulated between E14.5 and E17.5. In situ hybridization indicated expression of GPx4 isoforms in all
developing germ layers during gastrulation and in the somite stage in the developing central nervous system and in the heart. When we
silenced expression of GPx4 isoforms during in vitro embryogenesis using short interfering RNA technology, we observed that knockdown
of mitochondrial GPx4 strongly impaired segmentation of rhombomeres 5 and 6 during hindbrain development and induced cerebral apoptosis.
In contrast, silencing expression of the nuclear isoform led to retardations in atrium formation. Taken together, our data indicate specific
expression of GPx4 isoforms in embryonic brain and heart and strongly suggest a role of this enzyme in organogenesis. These findings
may explain in part intrauterine lethality of GPx4 knock-out mice. FEBS Lett. 2006 Jun 26;580(15):3595-600. Epub 2006 May 23. Truncated mutants of human thioredoxin reductase 1 do not exhibit glutathione reductase activity Urig S, Lieske J, Fritz-Wolf K, Irmler A, Becker K Abstract: The substrate spectrum of human thioredoxin reductase (hTrxR) is attributed to its C-terminal extension of 16 amino acids carrying a
selenocysteine residue. The concept of an evolutionary link between thioredoxin reductase and glutathione reductase (GR) is presently
discussed and supported by the fact that almost all residues at catalytic and substrate recognition sites are identical. Here, we addressed the
question if a deletion of the C-terminal part of TrxR leads to recognition of glutathione disulfide (GSSG), the substrate of GR. We introduced
mutations at the putative substrate binding site to enhance GSSG binding and turnover. However, none of these enzyme species accepted
GSSG as substrate better than the full length cysteine mutant of TrxR, excluding a role of the C-terminal extension in preventing GSSG binding.
Furthermore, we show that GSSG binding at the N-terminal active site of TrxR is electrostatically disfavoured. Free Radic Biol Med. 2006 May 1;40(9):1513-23. Epub 2006 Jan 13 Involvement of selenoprotein P in protection of human astrocytes from oxidative damage Steinbrenner H, Alili L, Bilgic E, Sies H, Brenneisen P. Abstract: Selenoprotein P (SeP) is a highly glycosylated, selenium-rich plasma protein. Aside from its role as selenium carrier protein, an antioxidative function
of SeP has been suggested. Astrocytes, which detoxify reactive oxygen species in the brain, were described as potential target cells of SeP.
We investigated the expression of SeP in human astrocytes and its involvement in the protection of these cells against tert-butyl hydroperoxide (t-BHP)-induced
oxidative damage. We show that primary human astrocytes and the human astrocytoma cell line MOG-G-CCM express SeP as an unglycosylated protein,
which is not secreted. SeP expression in astrocytes is constitutive. Preincubation of astrocytes with hepatocyte-derived SeP mimicks the protective effect
of low-molecular-weight selenocompounds such as sodium selenite or selenomethionine against oxidative damage, shielding astrocytes from t-BHP-induced
cytotoxicity. Selenium supplementation of astrocytes counteracts oxidative stress via an increase in expression and activity of the selenoenzyme cytosolic
glutathione peroxidase (cGPx). Furthermore, specific downregulation of SeP expression by small interfering RNA decreases cell viability of human astrocytes
and makes them more susceptible to t-BHP-induced cytotoxicity. Our results implicate an antioxidant activity of constitutively expressed SeP in
selenium-deficient astrocytes, while during adequate selenium supply the enhanced protection against oxidative stress is exerted by cGPx. Free Radic Biol Med. 2006 Mar 1;40(5):763-78. Epub 2005 Oct 19. Antiglioma activity of 2,2':6',2"-terpyridineplatinum(II) complexes in a rat model--effects on cellular redox metabolism Ahmadi R, Urig S, Hartmann M, Helmke BM, Koncarevic S, Allenberger B, Kienhoefer C, Neher M, Steiner HH, Unterberg A, Herold-Mende C, Becker K. Abstract: The mammalian thioredoxin system, comprising the selenoenzyme thioredoxin reductase (TrxR) and the 12-kDa protein thioredoxin (Trx), is implicated in thiol-mediated antioxidant defense and redox regulatory processes including transcriptional control, DNA synthesis, and apoptosis. Cell proliferation supported by the thioredoxin system can be suppressed by TrxR inhibition. In this study, we assessed the effects of the potent hTrxR inhibitors 4-mercaptopyridine (4'-chloro-2,2':6',2"-terpyridine)platinum nitrate (I(23)2N) and 2-mercaptopyridine (4'-chloro-2,2':6',2"-terpyridine)platinum nitrate (I(25)2N) on glioblastoma in a rat model. These compounds show no or little cross-resistance with cisplatin and are thus of great clinical interest. Triple intravenous application of 25-35 mg/kg of the compounds led to a significant decrease of tumor growth as determined by magnetic resonance imaging. Metabolic as well as redox parameters in the blood of the animals were not altered. However, TrxR activity was significantly decreased in the tumor tissue, and redox parameters-including glutathione concentrations, total antioxidant status, and the activities of different antioxidant enzymes-showed tissue-specific variations. As indicated by different apoptotic markers, the antitumor activity of I(23)2N is not mediated by the induction of programmed cell death but rather by hTrxR inhibition and DNA intercalation leading to cell cycle arrest. Gene Expr Patterns. 2006 Jan 31; Embryonic expression profile of phospholipid hydroperoxide glutathione peroxidase Schneider M, Vogt Weisenhorn DM, Seiler A, Bornkamm GW, Brielmeier M, Conrad M Abstract: The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is indispensable for murine embryonic
development; yet, the cellular mechanisms leading to embryonic death around gastrulation are still unclear. To investigate
PHGPx expression patterns during embryogenesis, we performed a detailed analysis that revealed a complex expression
profile. Up to embryonic day 9.5, PHGPx was ubiquitously expressed, which was, albeit to a lower extent, maintained throughout
later stages of embryogenesis. Notably, strong expression was frequently observed in epithelial tissue. A transient increase in
PHGPx expression was detected in developing tissues, suggesting a crucial role for PHGPx in proliferation and differentiation.
By semi-quantitative RT-PCR analysis we observed that the cytosolic form of PHGPx was present in embryonic and somatic
tissues whereas the mitochondrial and nuclear forms were detectable only in testicular tissue. This strongly suggests that it
is the cytosolic form of PHGPx that is indispensable for embryonic development. Received on March 16, 2005; accepted for publication on January 7, 2006. © AlphaMed Press 1066-5099 doi: 10.1634/stemcells.2005-0117 Stem Cells Express, published online January 19, 2006; doi:10.1634/stemcells.2005-0117 Copyright © 2006 AlphaMed Press Selenium supplementation restores the antioxidative capacity and prevents cell damage in bone marrow stromal cells in vitro Regina Ebert1, Matthias Ulmer1, Sabine Zeck1, Jutta Meissner-Weigl1, Doris Schneider1, Helga Stopper2,
Nicole Schupp2, Moustapha Kassem3 and Franz Jakob1 Abstract: Bone marrow stromal cells (BMSC) and other cell populations derived from mesenchymal precursors are
developed for cell based therapeutic strategies and undergo cellular stress during ex vivo procedures. Reactive oxygen
species (ROS) of cellular and environmental origin are involved in redox signalling, cumulative cell damage, senescence
and tumour development. Selenium dependent (glutathione peroxidases (GPx), thioredoxin reductases (TrxR)) and independent
(superoxide dismutases (SOD) and catalase (CAT)) enzyme systems regulate cellular ROS steady state levels. SOD process
superoxide anion to hydrogen peroxide, which is subsequently neutralized by GPx and CAT, TrxR neutralizes other ROS like
peroxinitrite. Primary BMSC and telomerase-immortalized human mesenchymal stem cells (hMSC-TERT) express GPx 1-3,
TrxR1 and 2, SOD1 and 2 and CAT. We show here that in standard cell cultures (5-10% FCS, selenite 5-10 nM) the activity of
antioxidative selenoenzymes is impaired in hMSC-TERT and BMSC. Under these conditions the superoxide anion processing
enzyme SOD1 is not sufficiently stimulated by an ROS load. Resulting oxidative stress favors generation of micronuclei in BMSC.
Supplementation of selenite (100 nM) restores basal GPx and TrxR activity, rescues basal and ROS-stimulated SOD1 mRNA
expression and activity, reduces ROS accumulation in hMSC-TERT and micronuclei generation in BMSC. In conclusion
BMSC in routine cell culture have low antioxidative capacity and are subjected to oxidative stress as indicated by the generation of
micronuclei. Selenite supplementation of BMSC cell cultures appears to be an important countermeasure to restore their
antioxidative capacity and to reduce cell damage in the context of tissue engineering and transplantation procedures. Endocrinology. 2006 Mar;147(3):1306-13. Epub 2005 Dec 1 Synthesis and Metabolism of Thyroid Hormones Is Preferentially Maintained in Selenium-Deficient Transgenic Mice Lutz Schomburg, Cornelia Riese, Marten Michaelis, Emine Griebert, Marc O. Klein, Remy Sapin, Ulrich Schweizer, and Josef Köhrle Abstract: The thyroid gland is rich in selenium (Se) and expresses a variety of selenoproteins that are involved in antioxidative defense and
metabolism of thyroid hormones (TH). Se deficiency impairs regular synthesis of selenoproteins and adequate TH metabolism. We
recently generated mice that lack the plasma Se carrier, selenoproteinP(SePP). SePP-knockout mice display decreased serum Se
levels and manifest growth defects and neurological abnormalities partly reminiscent of thyroid gland dysfunction or profound
hypothyroidism. Thus, we probed the TH axis in developing and adult SePP-knockout Mice. Surprisingly, expression of Se-dependent
5_-deiodinase type 1 was only slightly altered in liver, kidney, or thyroid at postnatal d 60, and 5_-deiodinase type 2 activity in brain
was normal in SePP-knockout mice. Thyroid gland morphology, thyroid glutathione peroxidase activity, thyroid Se concentration,
and serum levels of TSH, T4, or T3 were within normal range. Pituitary TSH_ transcripts and hepatic 5_-deiodinase type 1 mRNA levels
were unchanged, indicating regular T3 bioactivity in thyrotropes and hepatocytes. Cerebellar granule cell migration as a sensitive
indicator of local T3 action during development was undisturbed. Collectively, these findings demonstrate that low levels of serum Se
or SePP in the absence of other challenges do not necessarily interfere with regular functioning of the TH axis. 5_-deiodinase
isozymes are preferentially supplied, and Se-dependent enzymes in the thyroid are even less-dependent on serum levels of Se
or SePP than in brain. This indicates a top priority of the thyroid gland and its selenoenzymes with respect to the hierarchical
Se supply within the organism. Tetrahedron Lett. 47, 2006, in print. DOI 10.1016/j.tetlet.2005.11.101 Stereoselective synthesis of Boc-protected L-seleno- and tellurolanthionine, L-seleno- and tellurocystine and derivatives Alex Schneider, Oscar E.D. Rodrigues, Márcio Weber Paixão, Helmoz R. Appelt, Antonio L. Braga, Ludger A. Wessjohann
Chem Commun (Camb). 2006 Feb 7;(5):541-3. Epub 2005 Dec 15 One pot synthesis of selenocysteine containing peptoid libraries by Ugi multicomponent reactions in water Abbas M, Bethke J, Wessjohann LA Abstract: Selenocysteine containing peptoids and peptide-peptoid conjugates were synthesized by combinatorial Ugi-MCRs
(multicomponent reactions) in water: for the first time, an acetal (selenoacetal 2a) was used in Ugi-MCR to furnish selenocysteine
peptoids in one step as model compounds for selenocysteine peptides and proteins. Angew Chem Int Ed Engl. 2006 Feb 22;45(12):1881-1886 [Epub ahead of print] Undressing of Phosphine Gold(I) Complexes as Irreversible Inhibitors of Human Disulfide Reductases Sabine Urig 1, Karin Fritz-Wolf, Dr. 2, Régis Réau, Prof. Dr. 3, Christel Herold-Mende, Dr. 4, Katalin Tóth, Dr. 5, Elisabeth Davioud-Charvet, Dr. 6, Katja Becker, Prof. Dr. 1 Abstract: Phosphole-based p-conjugated organic systems are useful for optoelectronic applications since the phosphole ring is weakly
aromatic and possesses a central nucleophilic P atom.[1–3] Modification of their nucleophilic heteroatom by coordination to metal
centers may render these compounds promising candidates for chemotherapeutic applications. [4] Inhibition of disulfide reductases
contributes to the cytotoxic effects of anticancer compounds. [5, 6] Here, we report the effects of novel, highly potent,
phosphole-containing gold and platinum complexes on human glutathione reductase (hGR), thioredoxin reductase (hTrxR), and DNA,
as well as their growthinhibitory action on tumor cells. To date, hardly any structures of medicinally relevant gold (i)–protein complexes
have been reported. [7] Herein, we describe the first crystal structure of modified hGR with almost linear S-AuI-S-coordination in the
active site. Nucleic Acids Res. 2006 Jan 20;34(2):496-505. Print 2006 The Plasmodium selenoproteome Lobanov AV, Delgado C, Rahlfs S, Novoselov SV, Kryukov GV, Gromer S, Hatfield DL, Becker K, Gladyshev VN. Abstract: The use of selenocysteine (Sec) as the 21st amino acid in the genetic code has been described in all three major domains
of life. However, within eukaryotes, selenoproteins are only known in animals and algae. In this study, we characterized
selenoproteomes and Sec insertion systems in protozoan Apicomplexa parasites. We found that among these organisms,
Plasmodium and Toxoplasma utilized Sec, whereas Cryptosporidium did not. However, Plasmodium had no homologs of
known selenoproteins. By searching computationally for evolutionarily conserved selenocysteine insertion sequence (SECIS)
elements, which are RNA structures involved in Sec insertion, we identified four unique Plasmodium falciparum selenoprotein
genes. These selenoproteins were incorrectly annotated in PlasmoDB, were conserved in other Plasmodia and had no detectable
homologs in other species. We provide evidence that two Plasmodium SECIS elements supported Sec insertion into parasite and
endogenous selenoproteins when they were expressed in mammalian cells, demonstrating that the Plasmodium SECIS elements
are functional and indicating conservation of Sec insertion between Apicomplexa and animals. Dependence of the plasmodial
parasites on selenium suggests possible strategies for antimalarial drug development. Free Radic Biol Med. 2006 Jan 15;40(2):285-94. Epub 2005 Oct 17 Blocking tumor cell eicosanoid synthesis by GP x 4 impedes tumor growth and malignancy Heirman I, Ginneberge D, Brigelius-Flohe R, Hendrickx N, Agostinis P, Brouckaert P, Rottiers P, Grooten J. Abstract: Using tumor cell-restricted overexpression of glutathione peroxidase 4 (GP x 4), we investigated the contribution of tumor cell
eicosanoids to solid tumor growth and malignant progression in two tumor models differing in tumorigenic potential. By lowering
cellular lipid hydroperoxide levels, GP x 4 inhibits cyclooxygenase (COX) and lipoxygenase (LOX) activities. GP x 4 overexpression
drastically impeded solid tumor growth of weakly tumorigenic L929 fibrosarcoma cells, whereas B16BL6 melanoma solid tumor growth
was unaffected. Yet, GP x 4 overexpression did markedly increase the sensitivity of B16BL6 tumors to angio-destructive TNF-alpha
therapy and abolished the metastatic lung colonizing capacity of B16BL6 cells. Furthermore, the GP x 4-mediated suppression of
tumor cell prostaglandin E(2) (PGE(2)) production impeded the induction of COX-2 expression by the tumor stress conditions
hypoxia and inflammation. Thus, our results reflect a PGE(2)-driven positive feedback loop for COX-2 expression in tumor cells.
This was further supported by the restoration of COX-2 induction capacity of GP x 4-overexpressing L929 tumor cells when cultured
in the presence of exogenous PGE(2). Thus, although COX-2 expression and eicosanoid production may be enabled by PGE(2)
from the tumor microenvironment, our results demonstrate the predominant tumor cell origin of protumoral eicosanoids, promoting
solid tumor growth of weakly tumorigenic tumors and malignant progression of strongly tumorigenic tumors. IUBMB Life. 2005 Nov;57(11):737-44 New insights into the physiological actions of selenoproteins from genetically modified mice Schweizer U, Schomburg L. Abstract: Selenium (Se) is an essential trace element in mammals. Dietary Se restriction or conditions of Se malabsorption
lead to deficiency syndromes or exacerbate established diseases in humans and in many animal models. It is assumed
that most, if not all, physiological actions of Se are mediated by selenocysteine (Sec) containing proteins. However, the
exact role of particular selenoproteins for certain molecular pathways, for the metabolism of nutrients, hormones or cellular
components and for the development and adaptive responses of the organism have often remained elusive. Through the use
of transgenic animals, it becomes increasingly feasible to interfere specifically with the expression of single selenoproteins
in certain tissues or at certain times. While some transgenic animals exhibit phenotypes that were expected from biochemical
studies, in other instances the observed effects were a surprise in view of earlier hypotheses. Biochemistry. 2005 Oct 11;44(40):13315-27 Structural and functional investigation of a putative archaeal selenocysteine synthase Kaiser JT, Gromadski K, Rother M, Engelhardt H, Rodnina MV, Wahl MC Abstract: Bacterial selenocysteine synthase converts seryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec) for selenoprotein biosynthesis.
The identity of this enzyme in archaea and eukaryotes is unknown. On the basis of sequence similarity, a conserved open reading
frame has been annotated as a selenocysteine synthase gene in archaeal genomes. We have determined the crystal structure of the
corresponding protein from Methanococcus jannaschii, MJ0158. The protein was found to be dimeric with a distinctive domain
arrangement and an exposed active site, built from residues of the large domain of one protomer alone. The shape of the dimer is
reminiscent of a substructure of the decameric Escherichia coli selenocysteine synthase seen in electron microscopic projections.
However, biochemical analyses demonstrated that MJ0158 lacked affinity for E. coli seryl-tRNA(Sec) or M. jannaschii seryl-tRNA(Sec),
and neither substrate was directly converted to selenocysteinyl-tRNA(Sec) by MJ0158 when supplied with selenophosphate.
We then tested a hypothetical M. jannaschii O-phosphoseryl-tRNA(Sec) kinase and demonstrated that the enzyme converts
seryl-tRNA(Sec) to O-phosphoseryl-tRNA(Sec) that could constitute an activated intermediate for selenocysteinyl-tRNA(Sec)
production. MJ0158 also failed to convert O-phosphoseryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). In contrast, both archaeal
and bacterial seryl-tRNA synthetases were able to charge both archaeal and bacterial tRNA(Sec) with serine, and E. coli
selenocysteine synthase converted both types of seryl-tRNA(Sec) to selenocysteinyl-tRNA(Sec). These findings demonstrate that
a number of factors from the selenoprotein biosynthesis machineries are cross-reactive between the bacterial and the archaeal
systems but that MJ0158 either does not encode a selenocysteine synthase or requires additional factors for activity. Anim Reprod Sci. 2005 Oct;89(1-4):212-5 Selenium contents in equine semen and semen fractions and their relations with chromatin integrity and foal birthing rate Bertelsmann H, Bollwein H, Sieme H, Alber D, Kyriakopoulos A, Behne D PubMed Mol Cell Biol. 2005 Jun;25(12):4914-23 The GI-GPx gene is a target for Nrf2 Banning A, Deubel S, Kluth D, Zhou Z, Brigelius-Flohe R Abstract: The gastrointestinal glutathione peroxidase (GI-GPx, GPx2) is a selenoprotein that was suggested to act as
barrier against hydroperoxide absorption but has also been implicated in the control of inflammation and malignant
growth. In CaCo-2 cells, GI-GPx was induced by t-butyl hydroquinone (tBHQ) and sulforaphane (SFN), i.e., "antioxidants"
known to activate the "antioxidant response element" (ARE) via electrophilic thiol modification of Keap1 in the Nrf2/Keap1
system. The functional significance of a putative ARE in the GI-GPx promoter was validated by transcriptional activation of
reporter gene constructs upon exposure to electrophiles (tBHQ, SFN, and curcumin) or overexpression of Nrf2 and by reversal
of these effects by mutation of the ARE in the promoter and by overexpressed Keap1. Binding of Nrf2 to the ARE sequence in
authentic gpx2 was corroborated by chromatin immunoprecipitation. Thus, the presumed natural antioxidants sulforaphane and
curcumin may exert their anti-inflammatory and anticarcinogenic effects not only by induction of phase 2 enzymes but also by
the up-regulation of the selenoprotein GI-GPx. Mol Aspects Med. 2005 Aug-Oct;26(4-5):256-67 Selenium, oxidative stress, and health aspects Brenneisen P, Steinbrenner H, Sies H Abstract: Metabolic processes which generate oxidants and antioxidants are governed by genetic disposition
as well as environmental factors. Changes in lifestyle, including increased environmental pollution, sun exposure,
and dietary habits modify the challenge of the organism by reactive oxygen species. Defence mechanisms are reinforced
by increasing dietary intake of antioxidants and micronutrients such as vitamins and selenium (Se). Se deficiency has been
recognized to promote some disease states. Epidemiological findings link a lowered Se status to neurodegenerative and
cardiovascular diseases as well as to increased cancer risk. While evidence exists to suggest that additional selenocompounds
would be beneficial in some health conditions, results from future intervention trials are needed to substantiate the argument for
increasing Se intake. Several pieces of the puzzle concerning the molecular mechanisms underlying the reactive oxygen
species-triggered disease state and intervention by enzymatic antioxidants have been elucidated. A novel concept of protection
of stromal cells against the dominating influence of tumor cells in tumor-stroma interaction by selenocompounds and other
antioxidants is presented herein, which may translate into therapeutic strategies in chemoprevention of tumor invasion. Org Biomol Chem. 2005 Oct 7;3(19):3564-9. Epub 2005 Aug 31 Combining benzo[d]isoselenazol-3-ones with sterically hindered alicyclic amines and nitroxides: enhanced activity as glutathione peroxidase mimics Kalai T, Mugesh G, Roy G, Sies H, Berente Z, Hideg K Neue Publikationen von Mitgliedern des DFG-Schwerpunktprogramms Selenoproteine und neue Übersichten zum Thema des Schwerpunkts: S. R. Stapleton J. R. Arthur M. Persson Moschos P. D. Whanger J. Köhrle G. N. Schrauzer S. R. Stapleton Kono H, Arteel GE, Rusyn I I, Sies H, Thurman RG. Gladilin S, Bidmon HJ, Divanach A, Arteel GE,Witte OW, Zilles K, Sies H. Arteel GE, Franken S, Kappler J, Sies H. Buchczyk DP, Briviba K, Hartl FU, Sies H. Assmann A, Bonifacic M, Briviba K, Sies H, Asmus KD. Jacob C, Arteel GE, Kanda T, Engman L, Sies H. Sharov VS, Briviba K, Sies H. Endocrine Reviews. Published September 20, 2005 as doi:10.1210/er.2001-0034 Selenium, the Thyroid, and the Endocrine System J. Köhrle*, F. Jakob+, B. Contempré#, J.E. Dumont# Abstract: Recent identification of new selenocysteine-containing proteins has revealed relationships between the two trace elements selenium and
iodine and the hormone network. Several selenoproteins participate in the protection of thyrocytes from damage by H2O2 produced for thyroid
hormone biosynthesis. Iodothyronine deiodinases are selenoproteins contributing to systemic or local thyroid hormone homeostasis. The selenium
content in endocrine tissues (thyroid, adrenals, pituitary, testes, ovary) is higher than in many other organs. Nutritional selenium depletion results
in retention while selenium repletion is followed by a rapid accumulation of selenium in endocrine tissues, reproductive organs and the brain.
Selenproteins such as thioredoxin reductases constitute the link between the selenium metabolism and the regulation of transcription by redox
sensitive ligand-modulated nuclear hormone receptors. Hormones and growth factors regulate the expression of selenoproteins and, conversely,
selenium supply modulates hormone actions. Selenoproteins are involved in bone metabolism as well as functions of the endocrine pancreas and
adrenal glands. Furthermore, spermatogenesis depends on adequate selenium supply, whereas selenium excess may impair ovarian function.
Comparative analysis of the genomes of several life forms reveals that higher mammals contain a limited number of identical genes encoding
newly detected selenocysteine-containing proteins. Biochemical and Biophysical Research Communications 337 (2005) 641–647 Hepatic deiodinase activity is dispensable for the maintenance of normal circulating thyroid hormone levels in mice Florian Streckfuß a,b,1, Inka Hamann b,1, Lutz Schomburg b, Marten Michaelis b, Remy Sapin c, Marc O. Klein d, Josef Ko¨ hrle b, Ulrich Schweizer a,b,* Abstract: Thyroid hormone (TH) homeostasis depends on peripheral activation and inactivation of iodothyronines by selenoenzymes of the deiodinase
(Dio) family. We genetically inactivated hepatic selenoenzyme expression, including Dio1, in order to determine the contribution of hepatic Dio to
circulating TH levels. Serum levels of TSH, total T4, and total T3 were not di.erent from controls. We measured Dio1 and Dio2 in kidney, skeletal muscle,
heart, brown adipose tissue, and brain, but did not .nd compensatory up-regulation in these tissues. Finally, we determined expression in the liver of the
following T3 target genes: Spot14, a-glycerophosphate dehydrogenase (aGPD), and malic enzyme (ME). On the transcript level, both Spot14 and aGPD
were reduced in Dio-de.cient liver to about 60–70% of controls. However, mRNA and activity of ME were signi.cantly increased in the same mice.
Together, our results indicate that hepatic Dio1 activity is not absolutely required to sustain the euthyroid state in mice. THYROID Volume 15, Number 8, 2005 Selenium and the Control of Thyroid Hormone Metabolism Josef Köhrle Abstract: Thyroid hormone synthesis, metabolism and action require adequate availability of the essential trace elements iodine and selenium, which
affect homeostasis of thyroid hormone–dependent metabolic pathways. The three selenocysteine-containing iodothyronine deiodinases
constitute a novel gene family. Selenium is retained and deiodinase expression is maintained at almost normal levels in the thyroid gland, the brain
and several other endocrine tissues during selenium deficiency, thus guaranteeing adequate local and systemic levels of the active thyroid hormone T3.
Due to their low tissue concentrations and their mRNA SECIS elements deiodinases rank high in the cellular and tissue-specific hierarchy of selenium
distribution among various selenoproteins. While systemic selenium status and expression of abundant selenoproteins (glutathione peroxidase or
selenoprotein P) is already impaired in patients with cancer, disturbed gastrointestinal resorption, unbalanced nutrition or patients requiring intensive
care treatment, selenium-dependent deiodinase function might still be adequate. However, disease-associated alterations in proinflammatory cytokines,
growth factors, hormones and pharmaceuticals modulate deiodinase isoenzyme expression independent from altered selenium status and might thus
pretend causal relationships between systemic selenium status and altered thyroid hormone metabolism. Limited or inadequate supply of both trace elements,
iodine and selenium, leads to complex rearrangements of thyroid hormone metabolism enabling adaptation to unfavorable conditions. Thyroid. 2005 May;15(5):405-16 Selenoprotein Expression in Hürthle Cell Carcinomas and in the Human Hürthle Cell Carcinoma Line XTC.UC1 Menth M, Schmutzler C, Mentrup B, Hoang-Vu C, Takahashi K, Honjoh T, Köhrle J. Abstract: Hurthle cell carcinomas (HTC) are characterized by mitochondrial amplification and enhanced oxygen metabolism. To clarify if defects in enzymes scavenging reactive oxygen species are involved in the pathogenesis of HTC, we analyzed selenium (Se)-dependent expression of various detoxifying selenoproteins in the HTC cell line XTC.UC1. Glutathione peroxidase and thioredoxin reductase activity was found both in cell lysates and conditioned media of XTC.UC1 cells and was increased by Na(2)SeO(3). Western blot analysis demonstrated the presence of thioredoxin reductase both in cell lysates and conditioned media and of glutathione peroxidase 3 in conditioned media. Type I 5'-deiodinase, another selenoprotein that catalyzes thyroid hormone metabolism, was detectable only in cell lysates by enzyme assay and Western blot, and responded to stimulation by both Na(2)SeO(3) and retinoic acid. A selenoprotein P signal was detected in conditioned media by Western blot, but was not enhanced by Na(2)SeO(3) treatment. In situ hybridization revealed glutathione peroxidase mRNAs in HTC specimen; glutathione peroxidase 3 mRNA levels were reduced. These data suggest adequate expression and Se-dependent regulation of a couple of selenoproteins involved in antioxidant defense and thyroid hormone metabolism in XTC.UC1 cells, so far giving no evidence of a role of these proteins in the pathogenesis of HTCs. PubMed Nat Genet. 2005 Oct 16; [Epub ahead of print] Mutations in SECISBP2 result in abnormal thyroid hormone metabolism. Alexandra M Dumitrescu1, Xiao-Hui Liao2, Mohamed S Y Abdullah3, Joaquin Lado-Abeal2, 9, Fathia Abdul Majed4, Lars C Moeller2, Gerard Boran5,
Lutz Schomburg6, Roy E Weiss2 & Samuel Refetoff2, 7, 8 Abstract: Incorporation of selenocysteine (Sec), through recoding of the UGA stop codon, creates a unique class of proteins. Mice lacking tRNA(Sec)
die in utero, but the in vivo role of other components involved in selenoprotein synthesis is unknown, and Sec incorporation defects have not been
described in humans. Deiodinases (DIOs) are selenoproteins involved in thyroid hormone metabolism. We identified three of seven siblings with clinical
evidence of abnormal thyroid hormone metabolism. Their fibroblasts showed decreased DIO2 enzymatic activity not linked to the DIO2 locus. Systematic
linkage analysis of genes involved in DIO2 synthesis and degradation led to the identification of an inherited Sec incorporation defect, caused by a
homozygous missense mutation in SECISBP2 (also called SBP2). An unrelated child with a similar phenotype was compound heterozygous with respect
to mutations in SECISBP2. Because SBP2 is epistatic to selenoprotein synthesis, these defects had a generalized effect on selenoproteins. Incomplete
loss of SBP2 function probably causes the mild phenotype. PubMed Nat Genet. 2005 Oct 9; [Epub ahead of print] Genetic variation in selenoprotein S influences inflammatory response. Joanne E Curran1, 6, Jeremy B M Jowett2, 3, 6, Kate S Elliott2, 6, Yuan Gao4, Kristi Gluschenko2, Jianmin Wang2, Dalia M Abel Azim5,
Guowen Cai1, Michael C Mahaney1, Anthony G Comuzzie1, Thomas D Dyer1, Ken R Walder3, 4, Paul Zimmet2, 3, Jean W MacCluer1, Greg R Collier3, 4,
Ahmed H Kissebah5, 6 & John Blangero1, 3, 6 1 Southwest Foundation for Biomedical Research, San Antonio, Texas 78227, USA. Abstract: Chronic inflammation has a pathological role in many common diseases and is influenced by both genetic and environmental factors. Here we assess
the role of genetic variation in selenoprotein S (SEPS1, also called SELS or SELENOS), a gene involved in stress response in the endoplasmic reticulum
and inflammation control. After resequencing SEPS1, we genotyped 13 SNPs in 522 individuals from 92 families. As inflammation biomarkers, we measured
plasma levels of IL-6, IL-1beta and TNF-alpha. Bayesian quantitative trait nucleotide analysis identified associations between SEPS1 polymorphisms and all
three proinflammatory cytokines. One promoter variant, -105G --> A, showed strong evidence for an association with each cytokine (multivariate P = 0.0000002). Functional analysis of this polymorphism showed that the A variant significantly impaired SEPS1 expression after exposure to endoplasmic reticulum stress agents (P = 0.00006). Furthermore, suppression of SEPS1 by short interfering RNA in macrophage cells increased the release of IL-6 and TNF-alpha. To investigate further the significance of the observed associations, we genotyped -105G --> A in 419 Mexican American individuals from 23 families for replication. This analysis confirmed a significant association with both TNF-alpha (P = 0.0049) and IL-1beta (P = 0.0101). These results provide a direct mechanistic link between SEPS1 and the production of inflammatory cytokines and suggest that SEPS1 has a role in mediating inflammation. Biochemical Journal Immediate Publication 7 Jan 2005 as manuscript BJ20041973 Hepatically-derived selenoprotein P is a key factor for kidney but not for brain selenium supply Ulrich Schweizer, Florian Streckfuß, Paco Pelt, Bradley A. Carlson, Dolph L. Hatfield, Josef Köhrle, Lutz Schomburg Neurobiology of Selenium, Neuroscience Research Center, Institute for Experimental Endocrinology, Institute of Cell Biology and Neurobiology,
Center for Anatomy, Charite University Medical School Berlin, Germany. Abstract: Liver-specific inactivation of Trsp, the gene for selenocysteine tRNA, removes selenoprotein P (SePP) from plasma causing selenium levels
and glutathione peroxidase activities to drop drastically in plasma and kidney. However, unlike in SePP knockout mice, brain selenium levels
remain unaffected and neurological defects do not occur. Thus, SePP fulfils a transport function in plasma and a hitherto unexpected essential role in brain. J Nutr. 2004 Apr;134(4):707-10 The neurobiology of selenium: lessons from transgenic mice Schweizer U, Schomburg L, Savaskan NE Neurobiology of Selenium, Neuroscience Research Center, Institute for Experimental Endocrinology, Institute of Cell Biology and Neurobiology,
Center for Anatomy, Charite University Medical School Berlin, Germany. Abstract: The brain represents a privileged organ with respect to selenium (Se) supply and retention. It contains high amounts of this essential trace element,
which is efficiently retained even in conditions of Se deficiency. Accordingly, no severe neurological phenotype has been reported for animals exposed to
Se-depleted diets. They are, however, more susceptible to neuropathological challenges. Recently, gene disruption experiments supported a pivotal role
for different selenoproteins in brain function. Using these and other transgenic models, longstanding questions concerning the preferential supply of Se to
the brain and the hierarchy among the different selenoproteins are readdressed. Given that genes for at least 25 selenoproteins have been identified in the
human genome, and most of these are expressed in the brain, their specific roles for normal brain function and neurological diseases remain to be elucidated. Biochem J. 2004 Feb 15;378(Pt 1):21-6 Efficient selenium transfer from mother to offspring in selenoprotein-P-deficient mice enables dose-dependent rescue of phenotypes associated with selenium deficiency Schweizer U, Michaelis M, Kohrle J, Schomburg L Neurobiologie des Selens, Neurowissenschaftliches Forschungszentrum, Charite-Universitatsmedizin Berlin, Charite Campus Mitte,
Schumannstrasse 20/21, D-10117 Berlin, Germany Abstract: Mice deficient in selenoprotein P exhibit a disturbed selenium distribution and reduced activities of other selenoenzymes and display defects
in growth and motor co-ordination. We have normalized selenoenzyme activities and rescued the phenotype of mutant mice by supplementing
their nursing mothers with sodium selenite. Our results indicate that selenium from inorganic sources can be transferred efficiently via mother's
milk to the developing offspring in a form that is both highly bioavailable by target tissues and yet sufficiently safe to prevent overdosages. Nutr Cancer. 2003;46(2):125-30. S Selenium supplementation enhances low selenium levels and stimulates glutathione peroxidase activity in peripheral blood and distal colon mucosa in past and present carriers of colon adenomas. Al-Taie OH, Seufert J, Karvar S, Adolph C, Mork H, Scheurlen M, Kohrle J, Jakob F Medizinische Poliklinik, Abteilung Molekulare Innere Medizin, Universität Würzburg, Germany Abstract: Selenoproteins such as glutathione peroxidases (GPx), thioredoxin reductases (TrxR), and selenoprotein P (SePP) contain molecular
selenium in form of selenocysteines within their active center. They are involved in the defense of reactive oxygen species, which otherwise
may cause DNA damage and alterations of protein function. Selenium intake has been linked to colon carcinogenesis in epidemiological and
interventional studies. In a double-blinded, placebo-controlled trial, we demonstrate that carriers of colon adenomas present with low basal serum
levels of selenium and plasma glutathione peroxidase (pGPx) activity before treatment, but both parameters can be normalized by interventional
selenium supplementation. GPx activity in colon mucosa was enhanced in the verum group, albeit this had only borderline significance. No change
of activity was observed for mucosal TrxR activity on selenium supplementation. In summary, our results confirm the existence of low selenium levels
in patients prone to colon adenomas and show that by selenium supplementation this can be normalized. If prospective trials confirm that selenium
supplementation reduces colon cancer incidence rates, it may be concluded that selenium supplementation should be recommended for patients at risk. J Biol Chem. 2003 Jan 24;278(4):2571-80. Epub 2002 Nov 08 Regulation of expression of the phospholipid hydroperoxide/sperm nucleus glutathione peroxidase gene Borchert A, Savaskan NE, Kühn H. Institute of Biochemistry, Humboldt University Medical School Charite, Monbijoustrasse 2, 10117 Berlin, Germany Abstract: A sperm nucleus glutathione peroxidase (snGPx), which is closely related to the phospholipid hydroperoxide glutathione peroxidase (phGPx), was recently discovered in late spermatids. Both GPx isoforms originate from a joint ph/snGPx gene, but their N-terminal peptides are encoded by alternative first exons. The expression of the two enzymes is differentially regulated in various cells, but little is known about the regulatory mechanisms. To explore the tissue-specific regulation of expression of the two isoenzymes, we first investigated their tissue distribution. Whereas phGPx is expressed at low levels in many organs, snGPx was only detected in testis, kidney, and in the human embryonic kidney cell line HEK293. Subcellular fractionation studies and immunoelectron microscopy revealed a cytosolic localization. To explore the mechanistic reasons for the differential expression pattern, we first tested the activity of the putative phGPx and snGPx promoters. The 5'-flanking region of the joint ph/snGPx gene exhibits strong promoter activity. In contrast, the putative snGPx promoter, which comprises 334 bp of intronic sequences, lacks major promoter activity. However, it strongly suppresses the activity of the ph/snGPx promoter. These data suggest negative regulatory elements in the first intron of the ph/snGPx gene, and DNase protection assays revealed the existence of several protein-binding sites. The corresponding trans-regulatory proteins (SP1, ERG1, GATA1, SREBP1, USF1, and CREBP1) were identified, and in vivo binding of EGR1 and SREBP1 was shown by chromatin immunoprecipitation. These data indicate for the first time somatic expression of the snGPx and provide evidence for the existence of intronic negative cis-regulatory elements in the ph/snGPx gene. Our failure to detect an alternative snGPx promoter suggests that transcription of the ph/snGPx gene may be regulated by a joint basic promoter. The decision, which GPx isoform is expressed in a given cell, appears to be made by alternative splicing of a joint primary transcript. Nucleic Acids Res. 2003 Aug 1;31(15):4293-303 Functional characterization of cis- and trans-regulatory elements involved in expression of phospholipid hydroperoxide glutathione peroxidase Ufer C, Borchert A, Kühn H. Institute of Biochemistry, Humboldt University Medical School Charite, Monbijoustrasse 2, 10117 Berlin, Germany Abstract: Phospholipid hydroperoxide glutathione peroxidase (phGPx) is a member of the seleno glutathione peroxidase family that is comprised of five selenoproteins capable of reducing hydroperoxy lipids to the corresponding alcohols. The enzyme has been implicated in antioxidative defense, but its high expression level in testicular tissue suggests a more specific function during sperm maturation. The phGPx is encoded for by a joint sperm nucleus/phGPx gene (sn/phGPx) and can be expressed as a mitochondrial or cytosolic isoform. Although sn/phGPx genes have been cloned from various mammalian species expression regulation of the enzyme has not been studied in detail. We investigated the 5'-flanking region of the murine sn/phGPx gene and observed basic promoter activity in a 200 bp region localized immediately upstream of the translational initiation site of the cytosolic isoform (3'-ATG). DNase protection assays indicated the presence of five distinct protein-binding regions and electrophoretic mobility shift assays and supershift experiments revealed binding of stimulating protein 1 (SP1), nuclear factor Y (NF-Y) and members of the SMAD family. Site-directed mutagenesis of the consensus binding sequences abolished in vitro transcription factor binding. Expression of reporter genes was most effectively impaired when SP1/SP3 and NF-Y binding site-deficient constructs were tested. Chromatin immunoprecipitation suggested the in vivo relevance of these transcription factors. Our data indicate that the basic phGPx promoter constitutes a 200 bp oligonucleotide, which is localized immediately upstream of the 3'-ATG and involves functional SP1/SP3, NF-Y and SMAD binding sites. The corresponding trans-regulatory proteins may contribute to differential expression regulation of the mitochondrial and cytosolic phGPx isoforms. J Biol Chem. 2003 Sep 5;278(36):34286-90. Epub 2003 Jun 20 Distinct promoters determine alternative transcription of gpx-4 into phospholipid-hydroperoxide glutathione peroxidase variants Maiorino M, Scapin M, Ursini F, Biasolo M, Bosello V, Flohe L. Department of Biological Chemistry, Viale G. Colombo 3, University of Padova, I-35121 Padova, Italy Abstract: A nuclear variant of phospholipid-hydroperoxide glutathione peroxidase (PHGPx, GPx-4) was considered to be derived from alternative pre-mRNA
splicing in testis and to regulate sperm maturation. The genomic sequence of rat gpx-4 was established and investigated in respect to expression into
the cytosolic, mitochondrial, and nuclear forms of PHGPx. In silico analysis suggested the presence of two distinct promoter regions, the upstream
one leading to transcripts translating into cPHGPx or mPHGPx and the downstream one yielding nPHGPx. The promoter activity of both regions was
verified by luciferase-based reporter constructs in A7r5 and H9c2 cells. The data reveal that the formation of nPHGPx is due to alternative transcription and
not to alternative splicing. Transcripts encoding nPHGPx were most abundant in testis although not restricted to this organ. This observation points to a
general role of the nuclear PHGPx variant in regulating cell division. Biol Reprod. 2003 Apr;68(4):1134-41. Epub 2002 Nov 27 Genetic Variations of gpx-4 and Male Infertility in Humans Maiorino M, Bosello V, Ursini F, Foresta C, Garolla A, Scapin M, Sztajer H, Flohe L. Department of Biological Chemistry, University of Padova, I-35121 Padova, Italy Abstract: Phospholipid hydroperoxide glutathione peroxidase (PHGPx), the product of gpx-4, is the major selenoprotein in sperm and is considered essential for fertilization because of its multiple roles in spermatogenesis, such as hydroperoxide detoxification, formation of the mitochondrial capsule, and chromatin condensation. Genomic DNA sequences of 3.148 kilobases covering the whole gpx-4 and its flanking regions were amplified from 63 men using the polymerase chain reaction and were analyzed for polymorphisms by direct sequencing. A total of 23 variant sites were detected; 2 were present only in control men (proven fathers; n = 21) and 10 were common to fertile controls and infertile patients (n = 42). A further 11 variant sites were seen in five of the infertile men only. Four of the gpx-4 variants were considered irrelevant to GPx-4-related fertility problems because they occurred homozygously in controls. The majority of the remaining variant sites are also of questionable relevance because they are located in introns or, as third base exchanges, do not affect the protein sequence. However, one of the exon variations leads to an Ala93-Thr exchange that reduces activity in a porcine GPx-4 homologue. Two detected promoter variations were shown by reporter gene constructs to affect transcription in somatic cell lines. These results indicate that gpx-4 polymorphism cannot generally account for the correlation of PHGPx content of sperm and fertility-related parameters, but further examination of this gene as a potential cause of infertility in particular cases is warranted. Andrologia Volume 35 Issue 1 Page 6 - February 2003 PHGPx is the mitochondrial capsule selenoprotein of mammalian sperm (Poster Presentation) L. Flohé , C. Foresta , A. Garolla , A. Roveri , F. Ursini , J. Wissing and M. Maiorino Abstract: Introduction: Selenium is highly concentrated in the mitochondrial capsule of spermatozoa. The trace element is
thought to be contained in a structural protein 'MCS', thereby assuring structural and functional sperm integrity. Reinvestigation of the chemical
nature of MCS revealed that the mitochondrial capsule consisted of at least 50% of enzymatically inactive, oxidatively cross-linked phospholipid
hydroperoxide glutathione peroxidase (PHGPx) (Ursini et al., 1999), a selenoprotein that is abundantly expressed as an active peroxidase in round
spermatids (Maiorino et al., 1998). PHGPx activity can be rescued from the capsule material by reductive treatment. Methods: The clinical relevance of sperm PHGPx was investigated in a pilot trial correlating rescued PHGPx
activity (Roveri et al., 2002) with fertility-related parameters in infertile subjects (n = 75) and healthy volunteers (n = 37). Genomic PHGPx DNA
was amplified by PCR from white blood cells of selected subjects and sequenced. Results: Also in human sperm, the inactive form accounted for more than 95% of total PHGPx. The Sperm PHGPx
content was inversely correlated with sperm count, morphological alterations and motility, but less markedly with vitality. PHGPx gene polymorphism
was frequent in both controls and infertile subjects. Conclusions:Low PHGPx content of sperm is associated with impaired male fertility. The reasons for decreased PHGPx
content in infertile subjects remain elusive. Biochem J. 2003 Mar 1;370(Pt 2):397-402 Gene disruption discloses role of selenoprotein P in selenium delivery to target tissues Schomburg L, Schweizer U, Holtmann B, Flohe L, Sendtner M, Kohrle J. Institut fur Experimentelle Endokrinologie, Universitatsklinikum Charite, Schumannstrasse 20/21, 10117 Berlin, Germany Abstract: Selenoprotein P (SePP), the major selenoprotein in plasma, has been implicated in selenium transport, selenium detoxification or antioxidant defence. We generated SePP-knockout mice that were viable, but exhibited reduced growth and developed ataxia. Selenium content was elevated in liver, but low in plasma and other tissues, and selenoenzyme activities changed accordingly. Our data reveal that SePP plays a pivotal role in delivering hepatic selenium to target tissues. Angew Chem Int Ed Engl. 2003 Oct 13;42(39):4742-58 Sulfur and selenium: the role of oxidation state in protein structure and function Jacob C, Giles GI, Giles NM, Sies H. School of Chemistry, University of Exeter, Stocker Road, Exeter EX4 4QD, UK Abstract: Sulfur and selenium occur in proteins as constituents of the amino acids cysteine, methionine, selenocysteine, and selenomethionine. Recent research underscores that these amino acids are truly exceptional. Their redox activity under physiological conditions allows an amazing variety of posttranslational protein modifications, metal free redox pathways, and unusual chalcogen redox states that increasingly attract the attention of biological chemists. Unlike any other amino acid, the "redox chameleon" cysteine can participate in several distinct redox pathways, including exchange and radical reactions, as well as atom-, electron-, and hydride-transfer reactions. It occurs in various oxidation states in the human body, each of which exhibits distinctive chemical properties (e.g. redox activity, metal binding) and biological activity. The position of selenium in the periodic table between the metals and the nonmetals makes selenoproteins ideal catalysts for many biological redox transformations. It is therefore apparent that the chalcogen amino acids cysteine, methionine, selenocysteine, and selenomethionine exhibit a unique biological chemistry that is the source of exciting research opportunities. Org Biomol Chem. 2003 Aug 21;1(16):2848-52 Selenenyl iodide: a new substrate for mammalian thioredoxin reductase Mugesh G, Klotz LO, du Mont WW, Becker K, Sies H. Institut fur Biochemie und Molekularbiologie I, Heinrich-Heine-Universität, Düsseldorf, Postfach 101007, D-40001 Düsseldorf, Germany Abstract: Areneselenenyl iodide stabilised by internal chelation has been synthesized and evaluated as a substrate of thioredoxin reductase (TrxR).
The reactivity of TrxR obtained from human placenta towards selenenyl iodide was found to be much higher than that of the E. coli enzyme,
indicating the essential nature of a selenocysteine residue in the active site of the human enzyme. The addition of thioredoxin (Trx) significantly enhanced
the TrxR-catalysed reduction of selenenyl iodide 1. These studies on the reduction of a selenenyl iodide by the thioredoxin system suggest that stable
selenenyl iodides could be new substrates for human TrxR. The Trx system could act as a cofactor for iodothyronine deiodinase by reducing the selenenyl
iodide intermediate in the second-half of the deiodinase catalytic cycle to regenerate the active site. The TrxR-catalysed reduction of 1 was not inhibited
by the anti-thyroid drug, PTU, suggesting that the involvement of the Trx system in the deiodinase cycle may be responsible for the insensitivity of certain
deiodinases towards clinically useful thiourea drugs. Tetrahedron Lett. 2003, 44 (36), 6911-6913. Synthesis of N,N-disubstituted selenoamides by O/Se-exchange with Selenium-Lawesson´s reagent John Bethke, Konstantin Karaghiosoff and Ludger A. Wessjohann a Leibniz-Institute of Plant Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany Abstract: The selenium analogue of Lawesson's reagent, [PhP(Se)(μ-Se)]2 is an effective reagent for synthesizing N,N-disubstituted selenoamides.
The reaction is carried out under mild conditions (room temperature) and affords the selenoamide in higher yield than using other selenation reagents. Antioxid. Redox Signal. 5, 205-215 Selenium-dependent enzymes in endothelial cell function Brigelius-Flohé R., Banning A., Schnurr K. Department of Vitamins and Atherosclerosis, German Institute of Human Nutrition Abstract: Glutathione peroxidases and thioredoxin reductases are the main selenoproteins expressed by endothelial cells. These enzymes reduce hydroperoxides, their role in endothelial cell physiology, however, by far exceeds prevention of oxidative damage. Reactive oxygen and nitrogen species, especially superoxide, hydroperoxides, and nitric oxide, are crucial signaling molecules in endothelial cells. Their production is regulated by vascular NAD(P)H oxidases and the endothelial nitric oxide synthase. Their metabolism and physiological functions are coordinated by glutathione peroxidases and the thioredoxin/thioredoxin reductase system. Endothelial selenoproteins are involved in the regulation of the vascular tone by maintaining the superoxide anion/nitric oxide balance, of cell adhesion by controlling cell adhesion molecule expression, of apoptosis via inhibition/activation of apoptosis signal-regulating kinase-1, and of eicosanoid production by controlling the activity of cyclooxygenases and lipoxygenases. Accordingly, they regulate inflammatory processes and atherogenesis. The underlying mechanisms are various and differ between individual selenoproteins. Scavenging of hydroperoxides not only prevents oxidative damage, but also interferes with signaling cascades and enzymes involved. Modulation of proteins by hydroperoxide-driven thiol/disulfide exchange is a novel mechanism that needs to be further investigated. A better understanding of the complex interplay of selenoproteins in regulating endothelial cell functions will help to develop a rationale for an improvement of health by an optimum selenium supply. Biol Chem. 2003 Apr;384(4):609-17 Recruitment of the interleukin-1 receptor (IL-1RI) associated kinase IRAK to the IL 1RI is redox regulated. Böl, G.-F., Jurrmann, N. and Brigelius-Flohé, R. Department of Vitamins and Atherosclerosis, German Institute of Human Nutrition Abstract: Interleukin-1 signaling is initiated by recruitment of adapter proteins and kinases to the type I interleukin-1 receptor (IL-1RI). It is modulated by accompanying redox processes at various levels, such as (auto-) phosphorylation of the IL-1RI-associated kinase IRAK, the phosphorylation of IkappaB and translocation and transcriptional activity of NF-kappaB. Here we demonstrate that the thiol-modifying agents diamide, menadione, and phenylarsine oxide (PAO) block the recruitment of IRAK to the receptor without inhibiting kinase activity in the immunoprecipitated IL-1RI complex in the human epithelial cell line ECV304 and the murine T cell line EL-4. Inhibition of IRAK receptor association by menadione is reversible in a GSH-dependent manner, while the PAO effect proved to be irreversible. Phospholipid hydroperoxide glutathione peroxidase attenuates inhibition by menadione. Recruitment correlates with the presence of thiol groups in IRAK that were available for IAIT-labeling. We conclude that recruitment of IRAK to the IL-1RI is redox regulated by the glutathione system, a reduced status being a prerequisite for an appropiate IL-1 response. Int J Cancer. 2003 Jun 20;105(3):300-4 Glutathione peroxidase isoforms as part of the local antioxidative defense system in normal and Barrett's esophagus Mörk H, Scheurlen M, Al-Taie O, Zierer A, Kraus M, Schottker K, Jakob F, Köhrle J. Medizinische Poliklinik, University of Wurzburg, Germany Abstract: The development of an oesophageal adenocarcinoma arising in Barrett's mucosa is associated with a multistep process of genetic lesions that may be
triggered by persistent oxidative damage. The glutathione peroxidase isoforms pGPx and GI-GPx, which were identified recently in the mucosa of the
esophagus, may play a role as defense factors to prevent such oxidative injury. To determine alterations of the expression of pGPx and GI-GPx in
Barrett's mucosa as compared to primary and regenerative squamous epithelium. Biopsy samples of oesophageal mucosa of patients with
Barrett's esophagus (n = 12), patients with squamous restoration after thermal ablation (n = 10), and healthy controls (n = 5) were analyzed for pGPx
and GI-GPx mRNA expression by Northern blot and for glutathione peroxidase activity by enzymatic assay. Squamous regeneration was Copyright2003 Wiley-Liss, Inc. Biol Chem. 2003 Apr;384(4):575-88 Versatility of selenium catalysis in PHGPx unraveled by LC/ESI-MS/MS Pierluigi Mauri¹, Louise Benazzi¹, Leopold Flohé², Matilde Maiorino³, Piero G. Pietta¹, Sandra Pilawa², Antonella Roveri³ and Fulvio Ursini³,◊ ¹ Institute for Biomedical Technologies, National Research Council, Milano, Italy Abstract: Phospholipid hydroperoxide glutathione peroxidase (PHGPx; EC 1.11.1.12), being a broad-spectrum thiol-dependent peroxidase, deserves renewed interest as a regulatory factor in various signaling cascades and as a structural protein in sperm. A first attempt is presented to identify catalytic intermediates and derivatives of the selenoprotein by liquid chromatography coupled to electrospray tandem mass spectrometry (LC/ESI-MS/MS) and to explain observed specificities by molecular modeling. The ground state enzyme E proved to correspond to positions 3 - 170 of the deduced porcine sequence with selenium being present as selenocysteine at position 46. The selenenic acid form, which is considered to be the first catalytic intermediate F formed by reaction with hydroperoxide, could not be identified. The second catalytic intermediate G was detected as Se-glutathionylated enzyme. This intermediate also proved to be generated in the reverse reaction where the active site selenol interacts with glutathione disulfide (GSSG). According to molecular models, specific binding of reduced glutathione (GSH) and of GSSG is inter alia facilitated by electrostatic attraction of Lys-48 and Lys-125. Polymerization of PHGPx is obtained under oxidizing conditions in the absence of low molecular weight thiols. Analysis of MS spectra revealed that the process is due to a selective reaction of Sec-46 with Cys-148’ resulting in linear polymers representing dead-end intermediates G'. FT Docking of PHGPx molecules allowed reactions of Sec-46 with either Cys-66’ , Cys-107’ , Cys-168’ or Cys-148’, the latter option being most likely as judged by the number of proposed intermediates with reasonable hydrogen bonds, interaction energies and interface areas. It is concluded that the same catalytic principles, depending on conditions, can drive the seemingly diverse actions of PHGPx, i. e. hydroperoxide reduction, GSSG reduction, S-derivatization and self-incorporation into biological structures. Key words: Biochem J. 2003 Mar 1;370(Pt 2):397-402 Gene disruption discloses role of selenoprotein P in selenium delivery to target tissues Schomburg L, Schweizer U, Holtmann B, Flohé L, Sendtner M, Köhrle J. Institut für Experimentelle Endokrinologie, Universitatsklinikum Charite, Schumannstrasse 20/21, 10117 Berlin, Germany Abstract: Selenoprotein P (SePP), the major selenoprotein in plasma, has been implicated in selenium transport, selenium detoxification or antioxidant defence. We generated SePP-knockout mice that were viable, but exhibited reduced growth and developed ataxia. Selenium content was elevated in liver, but low in plasma and other tissues, and selenoenzyme activities changed accordingly. Our data reveal that SePP plays a pivotal role in delivering hepatic selenium to target tissues. Biol. Chem., Vol. 384, pp. 11 - 18, January 2003, Copyright © 3’UTRs of Glutathione Peroxidases Differentially Affect Selenium-Dependent mRNA Stability and Selenocysteine Incorporation Efficiency Cordula Müller¹, Kirstin Wingler² and Regina Brigelius-Flohé ¹, ³,◊ ¹ German Institute of Human Nutrition, Department of Vitamins and Atherosclerosis, Potsdam-Rehbrücke, Germany Abstract: Selenoprotein mRNAs are particular in several aspects. They contain a specific secondary structure intheir 3´UTR, called Secis (selenocysteine inserting sequence), which is indispensable for selenocysteine incorporation, and they are degraded under selenium- limiting conditions according to their ranking in the hierarchy of selenoproteins. In the familiy of selenium- dependent glutathione peroxidases (GPx) the ranking is GI-GPx ϒ ⇑PHGPx › cGPx = pGPx. This phenomenon was studied by mutually combining the coding regions of GI-GPx, PHGPx and cGPx with their 3´UTRs. HepG2 cells were stably transfected with the resulting constructs. Expression of glutathione peroxidases was estimated by activity measurement and Western blotting, the selenium-dependent mRNA stability by real-time PCR. Whereas 3´UTRs from stable PHGPx and GI-GPx could be exchanged without loss of stability, they were not able to stabilize cGPx mRNA. cGPx 3´UTR rendered GI-GPx and PHGPx mRNA unstable. Thus, cGPx mRNA contains seleniumresponsive instability elements in both the translated and the untranslated region, which cannot be compensated by one of the stable homologs. Stabilizing efficiency of an individual GPx 3´UTR did not correlate with the efficiency of selenocysteine incorporation. PHGPx 3´UTR was equally effective as cGPx 3´UTR in enhancing GPx activity in all constructs, while GI-GPx 3´UTR showed a markedly lower efficacy. We conclude that different mRNA sequences and/or RNA-binding proteins might regulate mRNA stability and translation of selenoprotein mRNA. Key words: Arch Microbiol 179: 116-130 (2003) Molecular and biochemical characterization of two tungsten and selenium-containing formate dehydrogenases from Eubacterium acidaminophilum that are associated with components of an iron-only hydrogenase Andrea Graentzdoerffer - David Rauh - Andreas Pich¹ - Jan R. Andreesen Institut für Mikrobiologie, Martin-Luther-Universität Halle-Wittenberg Abstract: Two gene clusters encoding similar formate dehydrogenases(FDH) were identified in Eubacterium acidaminophilum. Each cluster is composed of one gene coding for a catalytic subunit(fdhA-I, fdhA-II) and one for an electron transferring subuni(fdhB-I, fdhB-II). Both fdhA genes contain a TGA codon for selenocysteine incorporation and the encoded proteins harbour five putative iron-sulfur clusters in their N-terminal region. Both FdhB subunits resemble the N-terminal region of FdhA on the amino acid level containing the five putative iron-sulfur clusters. Four genes supposedly encoding the subunits of an iron-only hydrogenase are located upstream of FDH gene cluster I. By sequence comparison, HymA and HymB are predicted to contain one and four iron sulfur clusters, respectively, the latter protein also binding sites for FMN and NAD(P). Thus, HymA and HymB seem to represent electron-transferring subunits, HymC the putative catalytic subunit containing motifs for four iron sulfur clusters and one H-cluster specific for Fe-only hydrogenases. HymD has six predicted transmembrane helices and this subunit might be an integral membrane protein. Viologen-dependent FDH activity was purified from serine-grown cells of E. acidaminophilum and the purified protein complex contained four subunits, FdhA and FdhB encoded by FDH gene cluster II and HymA and HymB identified after determination of their N-terminal sequences. Thus, this complex might represent the supposedly most simple type of a formate hydrogen lyase. The purified formate dehydrogenase fraction contained iron, tungsten, a pterin cofactor and zinc, but no molybdenum. FDH-II had a two-fold higher Km-value for formate (0.37 mM) than FDH-I and catalyzed also a CO2 reduction to formate. RT-PCR pointed to an increased expression of FDH-II in serine-grown cells, supporting the actual isolation of this FDH isoform. The fdhA-I gene was expressed as inactive protein in Escherichia coli. The in frame UGA codon for a selenocysteine incorporation was read in the heterologous system only as stop codon although its potential SECIS element exhibited a quite high similarity to that of the E. coli FDH. Key words: Thyroid. 2002 Oct;12(10):867-78 The impact of iron and selenium deficiencies on iodine and thyroid metabolism: Zimmermann MB, Köhrle J. Laboratory for Human Nutrition, Swiss Federal Institute of Technology, Zurich, Switzerland Institut für Experimentelle Endokrinologie, Universitatsklinikum Charite, Schumannstrasse 20/21, 10117 Berlin, Germany Summary: Several minerals and trace elements are essential for normal thyroid hormone metabolism, e.g., iodine, iron, selenium, and zinc. Coexisting deficiencies of these elements can impair thyroid function. Iron deficiency impairs thyroid hormone synthesis by reducing activity of heme-dependent thyroid peroxidase. Iron-deficiency anemia blunts and iron supplementation improves the efficacy of iodine supplementation. Combined selenium and iodine deficiency leads tomyxedematous cretinism. The normal thyroid gland retains high selenium concentrations even under conditions of inadequate selenium supply and expresses many of the known selenocysteine-containing proteins. Among these selenoproteinsare the glutathione peroxidase, deiodinase, and thioredoxine reductase families of enzymes. Adequate selenium nutrition supports efficient thyroid hormone synthesis and metabolism and protects the thyroid gland from damage by excessive iodide exposure. In regions of combined severe iodine and selenium deficiency, normalization of iodine supply is mandatory before initiation of selenium supplementation in order to prevent hypothyroidism. Selenium deficiency and disturbed thyroid hormone economy may develop under conditions of special dietary regimens such as long-term total parenteral nutrition, phenylketonuria diet, cystic fibrosis, or may be the result of imbalanced nutrition in children, elderly people, or sick patients. Eur J Hum Genet. 2002 Sep;10(9):499-504 A complex DNA-repeat structure within the Selenoprotein P promoter contains a functionally relevant polymorphism and is genetically unstable under conditions of mismatch repair deficiency Al-Taie OH, Seufert J, Mork H, Treis H, Mentrup B, Thalheimer A, Starostik P, Abel J, Scheurlen M, Köhrle J, Jakob F. Medizinische Poliklinik der Universitat Würzburg, Germany. Summary: Epidemiological data, animal studies and interventional studies provide evidence for a potential chemopreventive effect of selenium during development of colorectal cancer. The human glycoprotein Selenoprotein P (SeP) contains up to 50% of plasma selenium content. SeP is expressed in the gastrointestinal tract and the liver, where its expression is downregulated by various proinflammatory cytokines (Il1beta, TGFbeta, IFNgamma). Previously, we have demonstrated dramatically reduced SeP expression in human colon adenomas. Here, we have identified a complex (A)4-C-(A)4-GG-(A)8-GCT-(TC)5-(T)17 (bp -429 to bp - 477) repeat structure within the SeP promoter and we have analysed this regulatory DNA sequence with respect to polymorphisms, genomic instability and functional relevance to promoter activity. As opposed to the (TC)5 variant we identified a novel (TC)3 polymorphism within this repeat in the general population, which conferred significantly reduced basal promoter activity to reporter gene constructs in HepG2 cells. Allelic distribution of this (TC)(n) element was similar in colon carcinoma patients and healthy controls. Additionally, we observed genetic instability within the (T)17 repeat motif in colon cancers of the mutator phenotype. This instability of the (T)17 repeat had no effect on basal promoter activity in reporter gene assays. In conclusion, we characterised a complex repeat structure within the SeP promoter that may be of functional relevance to SeP gene expression. Further studies on the effect of different SeP promoter genotypes on SeP protein expression and disease susceptibility are needed. Eur J Endocrinol. 2002 Aug;147(2):263-8 Expression of 5'-deiodinase enzymes in normal pituitaries and in various human pituitary adenomas. Baur A, Buchfelder M, Köhrle J Abteilung fur Molekulare Innere Medizin und Klinische Forschergruppe der Summary: OBJECTIVE: Local 5'-deiodination of l-thyroxine (T(4)) to active thyroid hormone 3,3',5-tri-iodothyronine (T(3)) catalyzed by the two 5'-deiodinase enzymes (D1 and D2) regulates various T(3)-dependent functions in the anterior pituitary and has been well studied in rodents. Only limited information about deiodinase expression and its cellular distribution in human anterior pituitaries is available. DESIGN: We examined 5'-deiodinase enzyme activities in pituitary adenomas (18 non-functioning, seven TSH-producing, one GH- and TSH-producing, five GH-producing, eight prolactin (PRL)-producing, two adenomas each from patients with Cushing's disease and Nelson's syndrome) and three normal anterior pituitaries. METHODS: Activities were measured as release of (125)I(-) from tyrosyl-ring labeled reverse T(3) with or without propylthiouracil, a potent inhibitor of D1 which does not influence D2 activities. RESULTS: Most of the adenomas and normal tissues expressed both isoenzymes, with D2 activity higher than D1. In a few tissues D1 activity was higher than D2 and some tissues did not express D1 activity at all. Highest activities of both enzymes were found in TSH- and PRL-producing adenomas but absolute activities and the D1/D2 ratio were variable in the same kind of tumor in different patients. CONCLUSION: The finding that all examined tissues expressed 5'-deiodinase activity, most of them expressing both isoenzymes, implies that both enzymes are still active in tumors and that local deiodination is important for the function and feedback regulation of human anterior pituitary. Exp Clin Endocrinol Diabetes. 2002 Jun;110(4):166-70 Thyroid hormone receptors and type I iodothyronine 5'-deiodinase activity of human thyroid toxic adenomas and benign cold nodules Brtko J, Bobalova J, Podoba J, Schmutzler C, Köhrle J. Institute of Experimental Endocrinology, Slovak Academy of Sciences, 833 06 Summary: The majority of thyroid adenomas are of clonal origin. In a subset of toxic adenomas (TAs) and cold nodules (CNs) activating mutations in the thyrotropin (TSH) receptor or G s -alpha gene may explain the altered functions in these benign tumours. The present study was undertaken to investigate the status of functional thyroid hormone receptors, major thyroid hormone signal mediators, in both the human TAs and CNs in comparison with a normal thyroid tissue from the same patient. Electrophoretic mobility shift assays using a DR4 ("direct repeats" 4), a thyroid hormone responsive element (TRE) of human type I iodothyronine 5'-deiodinase demonstrated the DNA-binding of thyroid hormone receptors (TRs) in thyroid tissue nuclear extracts. A significant increase (p < 0.05) in the functional binding properties of TRs to the DR4 thyroid hormone responsive element was found in TAs when compared to normal thyroid tissue. Contrary, a marked diminution in the TR-TRE complex formation was found in CNs in comparison with normal thyroid tissue. In addition, functional activity of the iodothyronine 5'-deiodinase (5'DI) was analyzed in benign tumours, thyroid TAs and CNs in comparison with that of normal thyroid tissue. A significantly increased (p < 0.01) activity of 5'DI was demonstrated in TAs, and in contrast, decreased values of the enzyme activity were found in CNs when compared to a normal tissue. From the data it is suggested that both the status of TR-TRE complex formation and the activity of the 5'DI may be altered in benign tumours of human thyroid gland. Eur J Endocrinol. 2002 Apr;146(4):559-66 Proinflammatory cytokines inhibit the expression and function of human type I 5'-deiodinase in HepG2 hepatocarcinoma cells Jakobs TC, Mentrup B, Schmutzler C, Dreher I, Köhrle J. Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA Summary: OBJECTIVE: The sick euthyroid syndrome in critically ill patients without primary disease of the thyroid gland is characterised by low serum total triiodothyronine (T3), normal to elevated thyroxine (T4), elevated reverse T3 (rT3) and normal TSH levels. The aim of this work was to clarify if impaired T4 and rT3 5'-deiodination is an underlying mechanism. DESIGN AND METHODS: We analysed the effect of the human recombinant proinflammatory cytokines interleukin (IL)-6 and IL-1beta, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on human type I 5'-iodothyronine deiodinase (5'DI) enzyme activity in the human hepatocarcinoma cell line HepG2, i.e. in a homologous human system. Furthermore, we analysed transcriptional effects of the cytokines by transient transfection assays using the luciferase or chloramphenicol acetyltransferase (CAT) reporter genes under the control of 1480 nucleotides of the human 5'DI promoter. RESULTS: IL-6 at 500 pg/ml and TNF-alpha at 25 ng/ml had no significant effect, whereas 100 ng/ml IFN-gamma or 10 ng/ml IL-1beta reduced 5'DI enzyme activity to 77.9 and 59.5% of control values. IFN-gamma did not alter, IL-6 and TNF-alpha moderately decreased (in the case of IL-6 only in the CAT system), and IL-1beta (0.01-10 ng/ml) dose-dependently inhibited 5'DI promoter activity to a minimum of 38.1%. CONCLUSION: IL-1beta inhibited both 5'DI enzyme and promoter activity and, thus, may exert its effect on thyroid hormone metabolism at least partially through direct inhibition of hepatic 5'DI gene transcription. Methods Enzymol. 2002;347:125-67 Iodothyronine deiodinases Köhrle J. Division of Molecular Internal Medicine, Medizinische Poliklinik, University of Würzburg, D-97070 Würzburg, Germany Introduction: Thyroid hormones are essential for normal development, growth and metabolic functions in higher vertebrates. The thyroid gland produces and secretes mainly the prohormone L-thyroxine, which is further metabolized by three deiodinase enzymes (5´DI., 5´DII and 5DIII) to yield thyromimetically active hormones and inactive metabolites or derivatives with regulatory properties in thyroid hormone economy. The essential trace element iodine is required for thyroid hormone synthesis (Dobson1), and iodine contributes 65 % of the mass of L-thyroxine. A further essential trace element, selenium, is absolutely required for thyroid hormone synthesis and metabolism of thyroid hormones (Köhrle2). Probably a co-evolution occurred during terrestrial life of higher vertebrates, using both trace elements, which are deficient in wide areas of most continents due to postglacial erosion. Recently, the deiodinase enzyme family has been characterized in more detail, cDNA and genomic clones have been analyzed, and mutants have been constructed to identify essential structural and functional elements in their genes, mRNAs and proteins which are required for expression and for enzymic thyroid hormone deiodination. Most of the effects of thyroid hormones are exerted by 3,3',5-triiodo-L-thyronine (T3), the 5'-deiodination product of T4. T3 is also secreted to a minor extent by the thyroid in iodine deficiency, where the relative T3 to T4 ratio secreted by the thyroid increases. T3 binds to three forms of T3 receptors, members of ligand dependent transcription factors of the c-erbA family (Zhang and Lazar3). Functional T3 receptors have recently also been identified in mitochondria in addition to the well established nuclear receptors (Casas et al. 4). Effects of iodothyronines, especially T4 and reverse-T3 (rT3), the 5-deiodination product of T4, have been observed at the plasma membrane level and are linked to reorganisation of the actin filament cytoskeleton (Leonard and Farwell5). So far, no human gene defects for one of the three deiodinase enzymes have been identified (Köhrle et al. 6; Toyoda et al. 7) but in a mouse strain a variant of the 5'DI gene with functional consequences on decreased expression levels have been found (Maia et al. 8). In vertebrates and especially in man, only severe selenium-deficiency leads to alterations in expression and function of deiodinases, and especially activity of 5´DI in liver, kidney and several other organs decreases whereas no major effects are observed in the thyroid, the central nervous system and several other endocrine organs. Mild selenium deficiency provokes no major and direct alterations of expression and function of 5´DI- and 5DIII. However, expression and function of the deiodinase isoenzymes is very sensitive to thyroid hormone status, various cytokines and growth factors, severe illness, reactive oxygen species, a variety of hormones and signaling compounds, circadian rhythm and pharmacological agents and therefore might be useful in sensor function of physiology and pathophysiology. Eur J Biochem. 2001 Dec;268(23):6176-81 Identification of an element within the promoter of human selenoprotein Presponsive to transforming growth factor-beta Mostert V, Wolff S, Dreher I, Köhrle J, Abel J. Medizinisches Institut fur Umwelthygiene an der Heinrich-Heine-Universität Summary: Selenoprotein P (SeP) is a plasma protein that contains up to 10 selenocysteine residues and accounts for about 50% of total selenium in human plasma. We have previously shown that SeP expression in the human liver cell line HepG2 is inhibited by transforming growth factor (TGF)-beta1 on a transcriptional level. Smad proteins are the transcriptional mediators of TGF-beta signalling and putative Smad-binding elements (SBE) comprising the core sequence CAGACA arepresent at two positions in the SeP promoter. The aim of our study was to investigate whether Smad molecules are involved in inhibition of SeP expression by TGF-beta1 and to locate the promoter region critical for this effect. As seen in electrophoretic-mobility-shift assays, TGF-beta1 treatment led to enhanced binding of nuclear proteins to a putative SBE from the SeP promoter. Overexpression of Smad 3 and 4, but not of Smad 2, resulted in a marked down-regulation of SeP mRNA expression. Similar effects were observed for luciferase expression under control of a human SeP-promoter construct. Deletion as well as point-mutation of putative SBEs led to a loss of promoter sensitivity towards TGF-beta1 treatment. Hence, we demonstrated an involvement of Smad 3 and 4 in transcriptional regulation of SeP by TGF-beta1 and we were able to identify the TGF-beta-responsive element in the SeP promoter. Biofactors. 2001;14(1-4):135-42 Modulation of selenoprotein P expression by TGF-beta(1) is mediated by Smad proteins Mostert V, Dreher I, Köhrle J, Wolff S, Abel J. Abteilung Experimentelle Toxikologie, Medizinisches Institut fur Umwelthygiene, Summary: Selenoprotein P (SeP) is a selenium-rich plasma protein which accounts for more than 50% this study, the effect of TGF-beta(1) on the expression of SeP in the human liver cell line HepG2 was investigated. Western analysis revealed a dose-dependent reduction of SeP content in cell supernatant. RT-PCR analysis of SeP-mRNA expression demonstrated a marked inhibition and a reporter gene under control of the SeP promoter was negatively regulated by TGF-beta(1). Smad proteins are the transcriptional mediators of TGF-beta signaling. A putative Smad-binding element (SBE) is present in the SeP promoter. In electrophoretic-mobility-shift assays, TGF-beta(1) enhanced the binding of nuclear proteins to this SBE. Overexpression of Smad3 and 4 resulted in a downregulation of SeP-promoter activity whereas deletion of the SBE led to a loss of TGF-beta(1) responsiveness. We conclude that SeP expression is modulated by the binding of Smad3/4 complexes to a functional SBE in the SeP promoter. J Endocrinol. 2000 Dec;167(3):505-15 Effects of proinflammatory cytokines on anterior pituitary 5'-deiodinase type I and type II Baur A, Bauer K, Jarry H, Köhrle J. Abteilung fur Molekulare Innere Medizin und Klinische Forschergruppe der Summary: Cytokines are locally produced in the anterior pituitary and act through para-/autocrine mechanisms to modulate cell growth and hormone production. 5'-Deiodinases type I (D1) and type II (D2) are also expressed in the anterior pituitary and play an integrative role in the regulation of hormone production and pituitary feedback. D1 activity is known to be regulated by proinflammatory cytokines in liver and thyroid. Therefore, we examined the effects of IL-1 beta, IL-6 and TNF alpha on 5'-deiodinase activities in reaggregates of rat anterior pituitaries and rat somatomammotroph GH3 cells cultured alone, or in a bicameral culture system together with the murine folliculo-stellate (FS) cell line TtT/Gf. In reaggregate cultures of rat anterior pituitaries IL-1 beta stimulated D1 and D2 dose-dependently and D2 activity was increased by TNF alpha. When GH3 cells were cocultured with TtT/Gf cells, D2 activities were 2.3-fold lower than in GH3 cells cultured alone. TNF alpha (50 ng/ml) and IL-6 (100 U/ml) stimulated D2 in GH3 cells when the cells were cultured alone and treated with these cytokines for 24 h. When TtT/Gf cells in the coculture model were treated with IL-1 beta, TNF alpha and IL-6, no effect on D1 or D2 activities in GH3 cells was observed. In male, adult rats a single LPS injection (i.p.) stimulated D2 and D1 activities in the anterior pituitary, and decreased liver D1 activities and serum TSH levels. In vitro, LPS stimulation of the coculture model of GH3 and FS cells also increased D1 activity. Electrophoretic mobility shift assays (EMSAs) revealed that IL-1 beta and TNF alpha activate the transcription factor NF kappa B in reaggregates of rat anterior pituitaries and in TtT/Gf cells cultured alone or cocultured with GH3 cells. Taken together, these findings imply that in anterior pituitary cells 5'-deiodinase activities are stimulated by locally produced cytokines in a para-/autocrine manner but cell types other than FS cells seem to mediate some of the effects. Nutr Cancer. 2000;37(1):108-16 Inverse mRNA expression of the selenocysteine-containing proteins GI-GPx and SeP in colorectal adenomas compared with adjacent normal mucosa Mork H, al-Taie OH, Bahr K, Zierer A, Beck C, Scheurlen M, Jakob F, Köhrle J. Summary: Four selenocysteine-containing proteins (gastrointestinal glutathione peroxidase, plasma glutathione peroxidase, selenoprotein P, and thioredoxin reductase-alpha) are expressed in the colonic mucosa. Because of their antioxidant functions, a protective role in colon carcinogenesis is discussed. The aim of this study was to elucidate an involvement of gastrointestinalselenoproteins during the adenoma-carcinoma sequence. Matched pairs of biopsies of colorectal adenomas and adjacent normal mucosa from 11 patients were analyzed for mRNA expression, protein expression, or enzyme activity of selenoproteins by Northern blot, Western blot, or enzymatic tests. All adenomas revealed a marked reduction of selenoprotein P and a variable increase of gastrointestinal glutathione peroxidase mRNA compared with adjacent tissue. Thioredoxin reductase-alpha and plasma glutathione peroxidase mRNA expression were not altered in adenomas. The Northern blot results were confirmed by Western blot analysis or enzyme activity measurement, respectively. We conclude that gastrointestinal glutathione peroxidase and selenoprotein P play a complementary role in the antioxidative cell defense along the adenoma-carcinoma sequence. It remains to be shown whether upregulation of gastrointestinal glutathione peroxidase in adenomas represents a compensatory mechanism to reduce susceptibility for oxidative damage resulting from the loss of selenoprotein P. Thioredoxin reductase as a pathophysiological factor and drug target Katja Becker, Stephan Gromer, R. Heiner Schirmer and Sylke Müller Eur J Biochem. 2000 Oct;267(20):6118-25 Summary: Human cytosolic thioredoxin reductase (TrxR), a homodimeric protein containing 1 selenocysteine and 1 FAD per subunit of 55 kDa, catalyses the NADPH-dependent reduction of thioredoxin disulfide and of numerous other oxidized cell constituents. As a general reducing enzyme with little substrate specificity, it also contributes to redox homeostasis and is involved in prevention, intervention and repair of damage caused by H2O2-based oxidative stress. Being a selenite-reducing enzyme as well as a selenol-containing enzyme, human TrxR plays a central role in selenium (patho)physiology. Both dietary selenium deficiency and selenium oversupplementation, a lifestyle phenomenon of our time, appear to interfere with the activity of TrxR. Selenocysteine 496 of human TrxR is a major target of the anti-rheumatic gold-containing drug auranofin, the formal Ki for the stoichiometric inhibition being 4 nM. The hypothesis that TrxR and extracellular thioredoxin play a pathophysiologic role in chronic diseases such as rheumatoid arthritis, Sjögren’s syndrom, AIDS, and certain malignancies, is substantiated by biochemical, virological, and clinical evidence. Reduced thioredoxin acts as an autocrine growth factor in various tumour diseases, as a chemoattractant, and it synergises with interleukins 1 and 2. The effects of anti-tumour drugs such as carmustine and cisplatin can be explained in part by the inhibition of TrxR. Consistently, high levels of the enzyme can support drug resistance TrxRs from different organisms such as Escherichia coli, Mycobacterium leprae, Plasmodium falciparum, Drosophila melanogaster, and man show a surprising diversity in their chemical mechanism of thioredoxin reduction. This is the basis for attempts to develop specific TrxR inhibitors as drugs against bacterial infections like leprosy and parasitic diseases like amebiasis and malaria. "The influence of the cytokines Il-1 ß and INFg on the expression of selenoproteins in the human hepatocarcinoma cell line Hep G2" K. Hesse-Bähr, I. Dreher and J. Köhrle Abstract: Metabolic labelling of HepG2 cells with 75 selenite indicated the expression of more than four selenoproteins in the 80 to 45 kDa range and more than four selenoproteins in the 24 to 14 kDa range. Although there were no significant changes in the total protein content, determined by the method of Bradford, the densitometric analysis of the autoradiograms showed a reduced expression of all labelled selenoproteins in the cell lysates of HepG2 after cytokine treatment for 24 hours. A stronger reduction was observed after treatment with Il-1 ß than with IFNg . Moreover, the inhibitory effects were more significant for those selenoproteins in the higher molecular mass range. Our data on the inhibition of selenoprotein synthesis and on repression of SeP promoter activity show that the expression of selenoproteins, especially of SeP, is influenced by acute phase reaction and pro-inflammatory reactions. Selenium in Biology, Facts and Medical Perspectives Josef Köhrle, Regina Brigelius-Flohé, August Böck, Roland Gärtner, Ortwin Meyer and Leopold Flohé Biological Chemistry (2000) 381: 849-864 Summary: Several decades after the discovery of selenium as an essential trace element in vertebrates approximately 20 eukaryotic and more than 15 prokaryotic selenoproteins containing the 21st proteinogenic amino acid selenocysteine have been identified, partially characterized or cloned in several species. Many of these proteins are involved in redox reactions with selenocysteine acting as an essential component of the catalytic cycle. Enzyme activity has been assigned to the glutathione peroxidase family, to the thioredoxin reductases, which were recently identified as selenoproteins, to the iodothyronine deiodinases, metabolizing thyroid hormones, and to the selenophosphate synthetase 2, which is involved in selenoprotein biosynthesis. Prokaryotic selenoproteins catalyze redox reactions and formation of selenoethers in (stress-induced) metabolism and energy production of E. coli, of the clostridial cluster XI and of other prokaryotes. Apart from the specific and complex biosynthesis of selenocysteine, selenium also reversibly binds to proteins, is incorporated into selenomethionine in bacteria, yeast and higher plants, or posttranslationally modifies a catalytically essential cysteine residue of CO dehydrogenase. Expression of individual eukaryotic selenoproteins exhibits high tissue specificity, depends on selenium availability, in some cases is regulated by hormones, and if impaired contributes to several pathological conditions. Disturbance of selenoprotein expression or function is associated with deficiency syndromes (Keshan and Kashin-Beck disease), might contribute to tumorigenesis and atherosclerosis, is altered in several bacterial and viral infections, and leads to infertility in male rodents. Ebselen: prospective therapy for cerebral ischaemia Michael Parnham & Helmut Sies PLIVA d.d., Research & Development, Prilaz baruna Filipovia 25, HR - 10 000 Zagreb, Croatia and Institut für Physiologische Chemie I, Heinrich-Heine-Universität, Postfach 101007, D - 40001 Düsseldorf, Germany Summary: Stroke occurs due to haemorrhage or occlusive injury and results in ischaemia and reperfusion injury. A variety of destructive mechanisms are involved including oxygen radical generation, calcium overload, cytotoxicity and apoptosis as well as the generation of inflammatory mediators. Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one (PZ 51, DR3305), is a mimic of GSH peroxidase which also reacts with peroxynitrite and can inhibit enzymes such as lipoxygenases, NO synthases, NADPH oxidase, protein kinase C and H+/K+-ATPase. Ebselen is in a late stage of development for the treatment of stroke. The molecular actions of ebselen contribute to its anti-inflammatory and anti-oxidant properties, which have been demonstrated in a variety of in vivo models. Numerous in vitro experiments using isolated LDL, liposomes, microsomes, isolated cells and organs have established that ebselen protects against oxidative challenge. Unlike many inorganic and aliphatic selenium compounds, ebselen has low toxicity as metabolism of the compound does not liberate the selenium moiety, which remains within the ring structure. Subsequent metabolism involves methylation, glucuronidation and hydroxylation. Experimental studies in rats and dogs have revealed that ebselen is able to inhibit both vasospasm and tissue damage in stroke models, which correlates with its inhibitory effects on oxidative processes. Results from randomised, placebo-controlled, double-blind clinical studies on the neurological consequences of acute ischaemic stroke, subarachnoid haemorrhage and acute middle cerebral artery occlusion, have revealed that ebselen significantly enhances outcome in patients who have experienced occlusive cerebral ischaemia of limited duration. The benefit achieved with ebselen is closely related to the rapidity with which the treatment is initiated, following the onset of the stroke attack. Safety and tolerability are good and no adverse effects have become apparent. Ebselen is currently at the pre-registration stage for subarachnoid haemorrhage and stroke in Japan. Published in: Exp. Opin. Invest. Drugs (2000) 9:607-619 The selenoenzyme family of deiodinase isozymes controls local thyroid hormone availability Josef Köhrle Summary: The human thyroid gland secretes thyroid hormones under the control of the hypothalamus-pituitary-periphery feedback axis already during the last fetal trimester and is the only endogenous source of thyroid hormones after postnatal weaning. The main secretory thyroid hormone product is L-thyroxine (T4) and to a varying extent 3,3',5-triiodo-L-thyronine (T3). The latter iodothyronine exerts most of the effects of thyroid hormones on development, differentiation and metabolic pathways via binding to ligand-modulated nuclear or mitochondrial T3-receptors. Direct hormone effects have also been described for T4 and its deiodination products T3 and 3,3’,5'-triiodothyronine (reverse-T3, rT3) at the plasma membrane and for 3,5-T2 at the mitochondrial level. T4 and its derivatives undergo extensive metabolic reactions in several tissues, organs and cells, the maiority of which are catalyzed by the selenoenzyme family of iodothyronine deiodinases [1]. The three deiodinase isozymes so far identified are expressed in tissue-specific and developmentally regulated patterns thus controlling local production and degradation of the thyromimetic hormone T3 and of the other lower iodinated iodothyronines. The deiodinases act as gatekeepers of access of T3 to its intracellular receptors and modulate local availability of T4 and the other iodothyronines for their interaction with cellular targets different from T3-receptors [2]. Type I iodothyronine 5'-deiodinase (5'DI) preferentially deiodinates T4 and reverse-T3 at the 5'-position of the phenolic ring, but also removes iodide from the 5-position of the tyrosyl-ring under certain reaction conditions. Type II 5'-deiodinase (5'DII) deiodinates T4 and with lower affinity also rT3 specifically at the phenolic ring in 5'-position. The type III iododthyronine 5-deiodinase (5DIII) removes iodide from the 5-position of the tyrosyl ring and thus catalyzes the inactivation of the thyromimetic activity of T4 and T3. Published in: Reviews in Endocrine and Metabolic Disorders (2000) 1:49-58 J Lab Med 1999; 23 (11): 594-599 "Selensupplementation durch Selenhefe und Natriumselenit: Analyse des Selenstatus sowie Risiken des Mangels und der Intoxikation" Selenium Supplementation by Selenium Yeast and Sodium Selenite: Analysis of the Selenium Status as well as Risks of Deficiency and Intoxication Kirsten Bähr, Ingeborg Dreher, J. Köhrle Zusammenfassung: Seitdem Selen als essentielles Spurenelement identifiziert wurde, sind verschiedene Studien zu den Fragen der Bestimmung des Selenstatus eines Individuums, der Grenzwerte für Selenmangel und Selenüberfluß, sowie der Auswirkungen der Selensubstitution an gesunden Individuen und Individuen unter verschiedenen pathologischen Zuständen durchgeführt worden. Trotzdem gibt es bis heute für die Selenzufuhr keine absoluten Richtwerte, sondern nur Empfehlungen aufgrund von Schätzwerten, die in Abhängigkeit von Studie und Land zwischen 20 und 600 µg/Tag variieren. Ursachen sind hierfür einmal die Durchführung der Studien unter verschiedenen Rahmenbedingungen, so daß ein direkter Vergleich der Studien untereinander häufig nicht möglich ist. Außerdem ist der Metabolismus des Selens bisher noch unvollständig aufgeklärt: Unterschiede in Bioverfügbarkeit, biologischer Wirkung und mögliche toxische Nebenwirkungen der verschiedenen Selenverbindungen sind unzureichend berücksichtigt. Die Bestimmung des Selenstatus des Individuums ist zudem dadurch erschwert, daß es keine einzelne Meßgröße gibt, die für eine zuverlässige, individuelle Diagnostik herangezogen werden kann. Kenntnisse über Intoxikationen durch Selen beruhen bisher auf Studien in Gebieten mit selenreichen Böden und akuten oder chronischen Vergiftungen. Zudem sind die klinischen Symptome einer Selenintoxikation wenig spezifisch. Trotzdem werden zunehmend freiverkäufliche Selenpräparate als Nahrungssupplemente zur Selbstmedikation auf dem Markt angeboten, da Selen ein essentielles Spurenelement ist und die Selenversorgung in Deutschland als niedrig eingestuft wird. Als Anhaltspunkt zur Bewertung der Risiken einer Selensubstitution soll die vorliegende Arbeit einen Überblick über den aktuellen Wissensstand geben. "Selenhefetabletten" als freiverkäufliche Präparate werden besonders berücksichtigt. Intoxikationen durch Selenhefetabletten und natriumselenithaltige Präparate sind bei bestimmungsgemäßem Gebrauch bei den auf dem deutschen Markt erhältlichen Konzentrationen nicht zu erwarten. Abstract: Since selenium has been identified as an essential trace element, numerous studies have been carried out concerning the determination of the individual selenium status and threshold levels of selenium deficiency or excess. Furthermore, the effect of selenium supplementation on healthy individuals or patients affected by different pathological conditions have been examined. Up to now, however, there are only recommendations about the safe daily intake levels which vary in the range of 20 to 600 µg/day, based on different study design or country where the study had been taken place. The selenium metabolism has not been elucidated entirely: differences in bioavailability, physiological effects or toxicity of various selenium compounds have not been clarified in detail. The determination of the individual selenium state remains difficult, as no single parameter can be applied to give reliable data on the whole body selenium supply. Information about selenium intoxication is restricted to studies in countries with high selenium concentrations in soil or to accidental acute or chronic intake of high selenium doses. Clinical symptoms of selenosis are not very specific. Nevertheless, the number of available compounds for food supplementation is steadily increasing, as more and more people are aware that selenium is an essential trace element and the selenium supply in Germany has been classified as low. This paper presents an overview about the current knowledge of the risks of selenium supplementation with special consideration of selenium yeast. Intoxication by the available selenium yeast or sodium selenite compounds is not likely if they are applied as recommended. The trace element selenium and the thyroid gland Josef Köhrle Summary: Aparat from the essential trace element iodine, which is the central constitutent of thyroid hormones, a second essential trace element, selenium, is required for appropriate thyroid hormone synthesis, activation and metabolism. The human thyroid gland has the highest selenium content per gramm tissue among all organs. Several selenocysteine-containing proteins resp. enzymes are functionally expressed in the thyroid, mainly in thyrocytes themselves: Three forms of glutathione peroxidases (cGPx, pGPx, and PH-GPx), the type I 5'-deiodinase, thioredoxin reductase and selenoprotein P. The thyroidal expression of type II 5'-deiodinase still is controversial. As thyrocytes produce H2O2 continuously throughout life an effective cell defense system against H2O2 and reactive oxygen intermediates derived therefrom is essential for maintenance of normal thyroid function and protection of the gland. In experimental animal models long-term and strong selenium deficiency leads to necrosis and fibrosis after high iodide loads. Combined iodide and selenium deficiency such as in central Zaire is thought to cause the myxedematous form of endemic cretinism. Inadequate selenium supply and prediagnostically low serum selenium levels are significantly correlated to the development of thyroid carcinoma and other tumors. Though selenium supply controls expression and translation of selenocysteine-con |