Aminoglycoside-driven biosynthesis of selenium-deficient Selenoprotein P

Selenoprotein biosynthesis relies on the co-translational insertion of selenocysteine in response to UGA codons. Aminoglycoside antibiotics interfere with ribosomal function and may cause codon misreading. We hypothesized that biosynthesis of the selenium (Se) transporter selenoprotein P (SELENOP) is particularly sensitive to antibiotics due to its ten in frame UGA codons. As liver regulates Se metabolism, we tested the aminoglycosides G418 and gentamicin in hepatoma cell lines (HepG2, Hep3B and Hepa1-6) and in experimental mice. In vitro, SELENOP levels increased strongly in response to G418, whereas expression of the glutathione peroxidases GPX1 and GPX2 was marginally affected. Se content of G418-induced SELENOP was dependent on Se availability, and was completely suppressed by G418 under Se-poor conditions. Selenocysteine residues were replaced mainly by cysteine, tryptophan and arginine in a codon-specific manner. Interestingly, in young healthy mice, antibiotic treatment failed to affect Selenop biosynthesis to a detectable degree. These findings suggest that the interfering activity of aminoglycosides on selenoprotein biosynthesis can be severe, but depend on the Se status, and other parameters likely including age and general health. Focused analyses with aminoglycoside-treated patients are needed next to evaluate a possible interference of selenoprotein biosynthesis by the antibiotics and elucidate potential side effects

Regulation of selenoproteins after synthetic selenocompound exposure and during the acute phase inflammatory response

Selen ist ein essentielles Spurenelement, das über den Einbau als 21-ste proteinogene Aminosäure Selenocystein einer kleinen Gruppe von Selenoproteinen den Namen gibt und als Bestandteil des aktiven Zentrums dieser Proteine die enzymatische Katalyse bestimmter Reaktionen ermöglicht. In der Evolution haben sich eine komplexe Synthesemaschinerie zur Biosynthese von Selenoproteinen und besondere Regulationsmechanismen zur Kontrolle der Expressionsraten entwickelt. Selenoproteine sind von besonderer Bedeutung für den Energiestoffwechsel, die Schilddrüse und das Immunsystem. In der Klinik wird Selen als Supplement zur Unterstützung des Immunsystems und zum Ausgleich des Selenverlustes bei Sepsis eingesetzt. Allerdings sind die molekularen Mechanismen des Selenverlustes in Sepsis nicht verstanden, und es herrscht Uneinigkeit über die Wirkung verschiedener Formen selenhaltiger Supplemente. Diese beiden Punkte stehen im Zentrum meiner Arbeit. In einem murinen Sepsis- Modell wurde durch Endotoxin eine Akutphasenreaktion hervorgerufen. Es zeigte sich eine streng hierarchische Verteilung des verfügbaren Selens auf die Biosynthese der unterschiedlichen Selenoproteine, die darüber hinaus auch geschlechterspezifisch war. Der Selentransporter SePP erwies sich als stabiler Selenbiomarker und als Negativ-Akutphase-Protein, während das ER-residente Selenoprotein SepS als Positiv-Akutphase-Protein eingeordnet werden konnte. Auf molekularer Ebene konnten mit der Regulation geschwindigkeitsbestimmender Proteine des Selenmetabolismus und der Charakterisierung von Hypoxie als starker Modulator der Biosynthese wichtige Mechanismen identifiziert werden, die zu einer Verschiebung der Selenoprotein-Expression in schweren Erkrankungen beitragen. Diese Studien zur Grundlage des Selenstoffwechsels wurden durch Versuche zur Intervention mit neuen selenhaltigen Präparaten ergänzt. Hier konnte mit der Gruppe der Imidoselenocarbamate eine Substanzklasse charakterisiert werden, die therapeutisch vielversprechende Effekte spezifisch in Tumorzellen induzierte und als besonders aktive Induktoren der selenabhängigen Dejodasen wirkt, ohne dabei toxisch zu sein. Zusammenfassend wurden in dieser Arbeit somit neue klinisch relevante Regulationsmechanismen der Expression von Selenoproteinen charakterisiert und mit den Imidoselenocarbamaten eine vielversprechende Substanzklasse für eine zukünftige pharmakologische Intervention identifiziert.The view on selenium (Se) changed from a toxic substance to an essential trace element with the discovery of the 21st aminoacid selenocysteine (Sec). Se exerts its biological function as part of Sec-containing selenoproteins. Sec synthesis is mediated by a complex molecular machinery and depends on an optimal selenium status. Se is essential for cellular functions and energy metabolism, thyroid gland and immune system. Se research gained interest by being linked to several diseases through animal and clinical studies. Se is a supplement in the clinics to support the immune system and replenish loss of Se in critical illness. Still, the function of several selenoproteins and the underlying physiological mechanisms controlling Se metabolism are not completely understood or have not even been explored. The present work includes experimental studies on the role and regulation of Se and selenoproteins in inflammatory diseases. To this end, an endotoxin (LPS)-challenged mouse model with different Se status was analysed, and Se- containing carbamates were characterized in tumor and non-transformed cells. Using LPS-injected mice as sepsis model, our work highlighted a hierarchical distribution of the available Se for the biosyntheses of the selenoproteome. In addition, these regulatory principles acted in a sex-specific way. The secreted SePP proved as stabile Se biomarker in blood. In inflammation, SePP qualified as a hepatic negative acute phase protein whereas SepS was regulated as a positive acute phase protein. SepS is located in the ER membrane and is part of the protein quality control and therefore reduces cellular ER stress. The second part of my work based on the interesting phenomenon that humans in Se deficient areas are diagnosed more often with cancer than others. In fact, several studies indicate an anticarcinogenic effect of Se. The relevant selenoproteins and physiological mechanisms are unknown. Comparative studies with the chemical class of imidoselenocarbamates using tumor-derived and non- transformed cells showed different effects on translation of different selenoproteins. The specific modulation of the selenoproteome expressed reflects substance- and cell-specific effects of selenocarbamates on specific selenoproteins, but not a general activation of the selenoprotein biosynthesis machinery. Especially methyl-carrying selenocarbamates proved to be efficient modulators. Moreover, this substance class proved to be effective modulators of deiodinase activities without showing toxic effects. A further characterization of the interaction of selenocompounds with specific selenoproteins and their role in inflammation, cancer and the immune system is needed to provide a better insight into underlying mechanisms needed to optimize Se supplementation

Hypoxia reduces and redirects selenoprotein biosynthesis

Selenium deficiency constitutes a risk factor for the incidence and negative course of severe diseases including sepsis, stroke, autoimmune diseases or cancer. In this study, hypoxia is identified as a powerful stimulus to redirect selenoprotein biosynthesis causing reduced selenoprotein P expression and diminished selenium export from hepatocytes in favour of increased biosynthesis of the essential protective intracellular phospholipid hydroperoxide glutathione peroxidase GPX4. Specifically, hypoxia decreases transcript concentrations of central factors controlling selenium and selenocysteine metabolism including selenophosphate synthetase-2, phosphoseryl-tRNASerSec kinase and selenocysteine lyase, which are all proven to be rate-limiting enzymes in selenoprotein biosynthesis. These effects are paralleled by a general decline of selenoprotein expression; however, not all selenoproteins are affected to the same extent by hypoxia, and GPX4 constitutes an exception as its expression becomes slightly increased. Supplemental selenium is able to overcome the hypoxia-dependent down regulation of selenoprotein expression in our cell culture model system, supporting the concept of using selenium as an adjuvant treatment option in severe diseases. Although it remains to be tested whether these effects constitute a hepatocyte-specific response, the selenium-dependent decline of selenoprotein P biosynthesis under hypoxic conditions may explain the progressive selenium deficit developing in severe diseases.Peer Reviewe

Hypoxia reduces and redirects selenoprotein biosynthesis

Selenium deficiency constitutes a risk factor for the incidence and negative course of severe diseases including sepsis, stroke, autoimmune diseases or cancer. In this study, hypoxia is identified as a powerful stimulus to redirect selenoprotein biosynthesis causing reduced selenoprotein P expression and diminished selenium export from hepatocytes in favour of increased biosynthesis of the essential protective intracellular phospholipid hydroperoxide glutathione peroxidase GPX4. Specifically, hypoxia decreases transcript concentrations of central factors controlling selenium and selenocysteine metabolism including selenophosphate synthetase-2, phosphoseryl-tRNASerSec kinase and selenocysteine lyase, which are all proven to be rate-limiting enzymes in selenoprotein biosynthesis. These effects are paralleled by a general decline of selenoprotein expression; however, not all selenoproteins are affected to the same extent by hypoxia, and GPX4 constitutes an exception as its expression becomes slightly increased. Supplemental selenium is able to overcome the hypoxia-dependent down regulation of selenoprotein expression in our cell culture model system, supporting the concept of using selenium as an adjuvant treatment option in severe diseases. Although it remains to be tested whether these effects constitute a hepatocyte-specific response, the selenium-dependent decline of selenoprotein P biosynthesis under hypoxic conditions may explain the progressive selenium deficit developing in severe diseases.Peer Reviewe

Selenoprotein P status correlates to cancer-specific mortality in renal cancer patients.

Selenium (Se) is an essential trace element for selenoprotein biosynthesis. Selenoproteins have been implicated in cancer risk and tumor development. Selenoprotein P (SePP) serves as the major Se transport protein in blood and as reliable biomarker of Se status in marginally supplied individuals. Among the different malignancies, renal cancer is characterized by a high mortality rate. In this study, we aimed to analyze the Se status in renal cell cancer (RCC) patients and whether it correlates to cancer-specific mortality. To this end, serum samples of RCC patients (n = 41) and controls (n = 21) were retrospectively analyzed. Serum Se and SePP concentrations were measured by X-ray fluorescence and an immunoassay, respectively. Clinical and survival data were compared to serum Se and SePP concentrations as markers of Se status by receiver operating characteristic (ROC) curve and Kaplan-Meier and Cox regression analyses. In our patients, higher tumor grade and tumor stage at diagnosis correlated to lower SePP and Se concentrations. Kaplan-Meier analyses indicated that low Se status at diagnosis (SePP<2.4 mg/l, bottom tertile of patient group) was associated with a poor 5-year survival rate of 20% only. We conclude that SePP and Se concentrations are of prognostic value in RCC and may serve as additional diagnostic biomarkers identifying a Se deficit in kidney cancer patients potentially affecting therapy regimen. As poor Se status was indicative of high mortality odds, we speculate that an adjuvant Se supplementation of Se-deficient RCC patients might be beneficial in order to stabilize their selenoprotein expression hopefully prolonging their survival. However, this assumption needs to be rigorously tested in prospective clinical trials

Box Plot analysis of serum SePP concentration in RCC patients and controls.

<p>RCC patients were stratified using pathologic tumor characteristics <b>A</b>) non metastatic versus metastatic <b>B</b>) G1/G2 versus G3/G4 and <b>C</b>) pT₁/pT₂ versus pT₃/pT_4. The corresponding significance levels between groups are given in the graph.</p

Serum concentrations of Se and SePP in RCC patients^* and controls.

*<p>Patients were classified by diagnosis; the serum data are presented as median, range, and percentile.</p>#<p>Mann-Whitney U-Test.</p