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The expression and activity of thioredoxin reductase 1 splice variants v1 and v2 regulate the expression of genes associated with differentiation and adhesion.

Nalvarte I, Damdimopoulos AE, Rüegg J, Spyrou G - Biosci. Rep. (2015)

Bottom Line: The mammalian redox-active selenoprotein thioredoxin reductase (TrxR1) is a main player in redox homoeostasis.Overexpression of these two splice forms resulted in distinctive effects on various aspects of cellular functions including gene regulation patterns, alteration of growth rate, migration and morphology and susceptibility to selenium-induced toxicity.These data suggest that both TXNRD1_v1 and TXNRD1_v2 have distinct roles in differentiation, possibly by altering the expression of the genes associated with differentiation, and further emphasize the importance in distinguishing each unique action of different TrxR1 splice forms, especially when studying the gene silencing or knockout of TrxR1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Nutrition, Karolinska Institute, SE-141 83 Huddinge, Sweden ivan.nalvarte@ki.se.

No MeSH data available.


Related in: MedlinePlus

Expression of TXNRD1_v1 and TXNRD1_v2(A) Comparison between TXNRD1_v1 and v2 splicing variants. In contrast with TXNRD1_v1, TXNRD1v_2 has 52 extra N-terminal amino acids, encompassing a LXXLL consensus sequence (NR-Box) (underlined). Peptide sequence used for antibody production is shown in bold. Active sites are represented by CVNVGC motifs. Sec, selenocysteine. (B) Western blot analysis of TXNRD1_v1 and TXNRD1_v2 in cell lines. Hsp-90 was used as a loading control. (C) Western blot analysis of TXNRD1_v1 and TXNRD1_v2 in HEK-293 cells overexpressing either of the isoforms upon incubation with or without of 0.2 μM Se for 3 days. (D) TrxR1 activity and (E) real-time qPCR analysis in empty vector control, TXNRD1_v1 or TXNRD1_v2 overexpressing HEK-293 cells with or without 0.2 μM Se treatment for 3 days. Each bar represent mean for at least three independent experiments completed in duplicates and error bars correspond to S.D., *P<0.05, **P<0.01, ***P<0.001 using Student's ttest.
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Figure 1: Expression of TXNRD1_v1 and TXNRD1_v2(A) Comparison between TXNRD1_v1 and v2 splicing variants. In contrast with TXNRD1_v1, TXNRD1v_2 has 52 extra N-terminal amino acids, encompassing a LXXLL consensus sequence (NR-Box) (underlined). Peptide sequence used for antibody production is shown in bold. Active sites are represented by CVNVGC motifs. Sec, selenocysteine. (B) Western blot analysis of TXNRD1_v1 and TXNRD1_v2 in cell lines. Hsp-90 was used as a loading control. (C) Western blot analysis of TXNRD1_v1 and TXNRD1_v2 in HEK-293 cells overexpressing either of the isoforms upon incubation with or without of 0.2 μM Se for 3 days. (D) TrxR1 activity and (E) real-time qPCR analysis in empty vector control, TXNRD1_v1 or TXNRD1_v2 overexpressing HEK-293 cells with or without 0.2 μM Se treatment for 3 days. Each bar represent mean for at least three independent experiments completed in duplicates and error bars correspond to S.D., *P<0.05, **P<0.01, ***P<0.001 using Student's ttest.

Mentions: The TXNRD1_v1 and TXNRD1_v2 antibodies were generated by immunizing rabbits with purified inactive TXNRD1_v1 and with a TXNRD1_v2 specific peptide (KQRKIGGHGPTLKAY, Figure 1A) respectively and purified as previously described (20). The cells from 75 cm2 culture flasks were trypsinated, pelleted and washed once with ice-cold phosphate buffered saline (PBS), pH 7.4. The cells were freeze thawed once, resuspended in 0.25 M sucrose, 10 mM Tris/base pH 7.2, 2 mM EDTA and 0.1 mM PMSF (Sigma) and homogenized using a tight-fit glass-glass homogenizer. The homogenates were then centrifuged at 25000 g, 4°C for 30 min to remove cellular debris. The extracts were separated on SDS/PAGE, transferred to a nitrocellulose membrane (GE Life Sciences) and probed with TXNRD1_v1 antibody [17], TXNRD1_v2 antibody [20], fibronectin antibody, cadherin antibody or Hsp-90 antibody (all from Sigma). The bound antibodies were visualized using secondary anti-mouse or anti-rabbit horseradish peroxidase linked secondary antibody (GE Life Sciences) and ECL detection kit (GE Life Sciences).


The expression and activity of thioredoxin reductase 1 splice variants v1 and v2 regulate the expression of genes associated with differentiation and adhesion.

Nalvarte I, Damdimopoulos AE, Rüegg J, Spyrou G - Biosci. Rep. (2015)

Expression of TXNRD1_v1 and TXNRD1_v2(A) Comparison between TXNRD1_v1 and v2 splicing variants. In contrast with TXNRD1_v1, TXNRD1v_2 has 52 extra N-terminal amino acids, encompassing a LXXLL consensus sequence (NR-Box) (underlined). Peptide sequence used for antibody production is shown in bold. Active sites are represented by CVNVGC motifs. Sec, selenocysteine. (B) Western blot analysis of TXNRD1_v1 and TXNRD1_v2 in cell lines. Hsp-90 was used as a loading control. (C) Western blot analysis of TXNRD1_v1 and TXNRD1_v2 in HEK-293 cells overexpressing either of the isoforms upon incubation with or without of 0.2 μM Se for 3 days. (D) TrxR1 activity and (E) real-time qPCR analysis in empty vector control, TXNRD1_v1 or TXNRD1_v2 overexpressing HEK-293 cells with or without 0.2 μM Se treatment for 3 days. Each bar represent mean for at least three independent experiments completed in duplicates and error bars correspond to S.D., *P<0.05, **P<0.01, ***P<0.001 using Student's ttest.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4660583&req=5

Figure 1: Expression of TXNRD1_v1 and TXNRD1_v2(A) Comparison between TXNRD1_v1 and v2 splicing variants. In contrast with TXNRD1_v1, TXNRD1v_2 has 52 extra N-terminal amino acids, encompassing a LXXLL consensus sequence (NR-Box) (underlined). Peptide sequence used for antibody production is shown in bold. Active sites are represented by CVNVGC motifs. Sec, selenocysteine. (B) Western blot analysis of TXNRD1_v1 and TXNRD1_v2 in cell lines. Hsp-90 was used as a loading control. (C) Western blot analysis of TXNRD1_v1 and TXNRD1_v2 in HEK-293 cells overexpressing either of the isoforms upon incubation with or without of 0.2 μM Se for 3 days. (D) TrxR1 activity and (E) real-time qPCR analysis in empty vector control, TXNRD1_v1 or TXNRD1_v2 overexpressing HEK-293 cells with or without 0.2 μM Se treatment for 3 days. Each bar represent mean for at least three independent experiments completed in duplicates and error bars correspond to S.D., *P<0.05, **P<0.01, ***P<0.001 using Student's ttest.
Mentions: The TXNRD1_v1 and TXNRD1_v2 antibodies were generated by immunizing rabbits with purified inactive TXNRD1_v1 and with a TXNRD1_v2 specific peptide (KQRKIGGHGPTLKAY, Figure 1A) respectively and purified as previously described (20). The cells from 75 cm2 culture flasks were trypsinated, pelleted and washed once with ice-cold phosphate buffered saline (PBS), pH 7.4. The cells were freeze thawed once, resuspended in 0.25 M sucrose, 10 mM Tris/base pH 7.2, 2 mM EDTA and 0.1 mM PMSF (Sigma) and homogenized using a tight-fit glass-glass homogenizer. The homogenates were then centrifuged at 25000 g, 4°C for 30 min to remove cellular debris. The extracts were separated on SDS/PAGE, transferred to a nitrocellulose membrane (GE Life Sciences) and probed with TXNRD1_v1 antibody [17], TXNRD1_v2 antibody [20], fibronectin antibody, cadherin antibody or Hsp-90 antibody (all from Sigma). The bound antibodies were visualized using secondary anti-mouse or anti-rabbit horseradish peroxidase linked secondary antibody (GE Life Sciences) and ECL detection kit (GE Life Sciences).

Bottom Line: The mammalian redox-active selenoprotein thioredoxin reductase (TrxR1) is a main player in redox homoeostasis.Overexpression of these two splice forms resulted in distinctive effects on various aspects of cellular functions including gene regulation patterns, alteration of growth rate, migration and morphology and susceptibility to selenium-induced toxicity.These data suggest that both TXNRD1_v1 and TXNRD1_v2 have distinct roles in differentiation, possibly by altering the expression of the genes associated with differentiation, and further emphasize the importance in distinguishing each unique action of different TrxR1 splice forms, especially when studying the gene silencing or knockout of TrxR1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biosciences and Nutrition, Karolinska Institute, SE-141 83 Huddinge, Sweden ivan.nalvarte@ki.se.

No MeSH data available.


Related in: MedlinePlus