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Human NCU-G1 can function as a transcription factor and as a nuclear receptor co-activator.

Steffensen KR, Bouzga M, Skjeldal F, Kasi C, Karahasan A, Matre V, Bakke O, Guérin S, Eskild W - BMC Mol. Biol. (2007)

Bottom Line: NCU-G1 was found to be a highly conserved nuclear protein rich in proline with a molecular weight of approximately 44 kDa.In man, NCU-G1 was found to be widely expressed at the mRNA level with especially high levels detected in prostate, liver and kidney.NCU-G1 was found to activate transcription from this promoter and required presence of the footprint 1 element.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biosciences, University of Oslo, Norway. knut.steffensen@biosci.ki.se

ABSTRACT

Background: Novel, uncharacterised proteins represent a challenge in biochemistry and molecular biology. In this report we present an initial functional characterization of human kidney predominant protein, NCU-G1.

Results: NCU-G1 was found to be a highly conserved nuclear protein rich in proline with a molecular weight of approximately 44 kDa. It is localized on chromosome 1 and consists of 6 exons. Analysis of the amino acid sequence revealed no known transcription activation domains or DNA binding regions, however, four nuclear receptor boxes (LXXLL), and four SH3-interaction motives in addition to numerous potential phosphorylation sites were found. Two nuclear export signals were identified, but no nuclear localization signal. In man, NCU-G1 was found to be widely expressed at the mRNA level with especially high levels detected in prostate, liver and kidney. Electrophoretic mobility shift analysis showed specific binding of NCU-G1 to an oligonucleotide representing the footprint 1 element of the human cellular retinol-binding protein 1 gene promoter. NCU-G1 was found to activate transcription from this promoter and required presence of the footprint 1 element. In transiently transfected Drosophila Schneider S2 cells, we demonstrated that NCU-G1 functions as a co-activator for ligand-activated PPAR-alpha, resulting in an increased expression of a CAT reporter gene under control of the peroxisome proliferator-activated receptor-alpha responsive acyl-CoA oxidase promoter.

Conclusion: We propose that NCU-G1 is a dual-function protein capable of functioning as a transcription factor as well as a nuclear receptor co-activator.

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Related in: MedlinePlus

Human NCU-G1 functions as a ligand dependent and NR-box dependent co-activator for PPAR-alpha in S2 cells. Panel A: S2 cells were transiently transfected with 250 ng pAc5.1V5/His-LacZ, 350 ng of pACO-CAT, and increasing amounts of pMT-hPPARalpha, in the presence or absence of 10 ng of pAc-hNCU-G1. Twelve hrs after transfection WY14643 was added to 20 μM. The cells were harvested 56 hrs after transfection. CAT was assayed by ELISA and β-galactosidase by enzyme activity. The data represent relative average values (pg CAT/unit β-galactosidase/hr +/- SE) from three independent experiments carried out in triplicates. Panel B: S2 cells were transiently transfected with 250 ng pAc5.1V5/His-LacZ, 350 ng of pACO-CAT, together with pAc-hNCU-G1, pMT-hPPAR-alpha and WY14643 as indicated. The cells were treated and presented as in panel A. Panel C: S2 cells were transiently transfected with 250 ng pAc5.1V5/His-LacZ, 350 ng of pACO-CAT, 150 ng pMT-hPPAR-alpha as indicated and 10 ng of pAc-hNCU-G1, either wild-type or mutated as indicated in the figure. All cells were treated with 20 μM WY14643. The cells were harvested and data presented as in panel A.
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Figure 5: Human NCU-G1 functions as a ligand dependent and NR-box dependent co-activator for PPAR-alpha in S2 cells. Panel A: S2 cells were transiently transfected with 250 ng pAc5.1V5/His-LacZ, 350 ng of pACO-CAT, and increasing amounts of pMT-hPPARalpha, in the presence or absence of 10 ng of pAc-hNCU-G1. Twelve hrs after transfection WY14643 was added to 20 μM. The cells were harvested 56 hrs after transfection. CAT was assayed by ELISA and β-galactosidase by enzyme activity. The data represent relative average values (pg CAT/unit β-galactosidase/hr +/- SE) from three independent experiments carried out in triplicates. Panel B: S2 cells were transiently transfected with 250 ng pAc5.1V5/His-LacZ, 350 ng of pACO-CAT, together with pAc-hNCU-G1, pMT-hPPAR-alpha and WY14643 as indicated. The cells were treated and presented as in panel A. Panel C: S2 cells were transiently transfected with 250 ng pAc5.1V5/His-LacZ, 350 ng of pACO-CAT, 150 ng pMT-hPPAR-alpha as indicated and 10 ng of pAc-hNCU-G1, either wild-type or mutated as indicated in the figure. All cells were treated with 20 μM WY14643. The cells were harvested and data presented as in panel A.

Mentions: We chose to use a reporter gene construct expressing CAT under control of the rat acyl-CoA oxidase gene promoter [32], a well-known PPAR-alpha target gene [33]. We first determined the optimal level of PPAR-alpha to be co-expressed with NCU-G1 in S2 cells. As shown in figure 5A, expression of CAT was very low when PPAR-alpha was expressed only in the presence of the PPAR-alpha agonist WY14643, nor did expression of NCU-G1 lead to any increase in reporter gene expression. When co-expressed, however, a strong induction of reporter gene expression was observed, indicating that NCU-G1 may function as a co-activator for PPAR-alpha. Maximal induction using 150 ng of the PPAR-alpha expressing vector increased reporter gene expression by 11-fold.


Human NCU-G1 can function as a transcription factor and as a nuclear receptor co-activator.

Steffensen KR, Bouzga M, Skjeldal F, Kasi C, Karahasan A, Matre V, Bakke O, Guérin S, Eskild W - BMC Mol. Biol. (2007)

Human NCU-G1 functions as a ligand dependent and NR-box dependent co-activator for PPAR-alpha in S2 cells. Panel A: S2 cells were transiently transfected with 250 ng pAc5.1V5/His-LacZ, 350 ng of pACO-CAT, and increasing amounts of pMT-hPPARalpha, in the presence or absence of 10 ng of pAc-hNCU-G1. Twelve hrs after transfection WY14643 was added to 20 μM. The cells were harvested 56 hrs after transfection. CAT was assayed by ELISA and β-galactosidase by enzyme activity. The data represent relative average values (pg CAT/unit β-galactosidase/hr +/- SE) from three independent experiments carried out in triplicates. Panel B: S2 cells were transiently transfected with 250 ng pAc5.1V5/His-LacZ, 350 ng of pACO-CAT, together with pAc-hNCU-G1, pMT-hPPAR-alpha and WY14643 as indicated. The cells were treated and presented as in panel A. Panel C: S2 cells were transiently transfected with 250 ng pAc5.1V5/His-LacZ, 350 ng of pACO-CAT, 150 ng pMT-hPPAR-alpha as indicated and 10 ng of pAc-hNCU-G1, either wild-type or mutated as indicated in the figure. All cells were treated with 20 μM WY14643. The cells were harvested and data presented as in panel A.
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Figure 5: Human NCU-G1 functions as a ligand dependent and NR-box dependent co-activator for PPAR-alpha in S2 cells. Panel A: S2 cells were transiently transfected with 250 ng pAc5.1V5/His-LacZ, 350 ng of pACO-CAT, and increasing amounts of pMT-hPPARalpha, in the presence or absence of 10 ng of pAc-hNCU-G1. Twelve hrs after transfection WY14643 was added to 20 μM. The cells were harvested 56 hrs after transfection. CAT was assayed by ELISA and β-galactosidase by enzyme activity. The data represent relative average values (pg CAT/unit β-galactosidase/hr +/- SE) from three independent experiments carried out in triplicates. Panel B: S2 cells were transiently transfected with 250 ng pAc5.1V5/His-LacZ, 350 ng of pACO-CAT, together with pAc-hNCU-G1, pMT-hPPAR-alpha and WY14643 as indicated. The cells were treated and presented as in panel A. Panel C: S2 cells were transiently transfected with 250 ng pAc5.1V5/His-LacZ, 350 ng of pACO-CAT, 150 ng pMT-hPPAR-alpha as indicated and 10 ng of pAc-hNCU-G1, either wild-type or mutated as indicated in the figure. All cells were treated with 20 μM WY14643. The cells were harvested and data presented as in panel A.
Mentions: We chose to use a reporter gene construct expressing CAT under control of the rat acyl-CoA oxidase gene promoter [32], a well-known PPAR-alpha target gene [33]. We first determined the optimal level of PPAR-alpha to be co-expressed with NCU-G1 in S2 cells. As shown in figure 5A, expression of CAT was very low when PPAR-alpha was expressed only in the presence of the PPAR-alpha agonist WY14643, nor did expression of NCU-G1 lead to any increase in reporter gene expression. When co-expressed, however, a strong induction of reporter gene expression was observed, indicating that NCU-G1 may function as a co-activator for PPAR-alpha. Maximal induction using 150 ng of the PPAR-alpha expressing vector increased reporter gene expression by 11-fold.

Bottom Line: NCU-G1 was found to be a highly conserved nuclear protein rich in proline with a molecular weight of approximately 44 kDa.In man, NCU-G1 was found to be widely expressed at the mRNA level with especially high levels detected in prostate, liver and kidney.NCU-G1 was found to activate transcription from this promoter and required presence of the footprint 1 element.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Biosciences, University of Oslo, Norway. knut.steffensen@biosci.ki.se

ABSTRACT

Background: Novel, uncharacterised proteins represent a challenge in biochemistry and molecular biology. In this report we present an initial functional characterization of human kidney predominant protein, NCU-G1.

Results: NCU-G1 was found to be a highly conserved nuclear protein rich in proline with a molecular weight of approximately 44 kDa. It is localized on chromosome 1 and consists of 6 exons. Analysis of the amino acid sequence revealed no known transcription activation domains or DNA binding regions, however, four nuclear receptor boxes (LXXLL), and four SH3-interaction motives in addition to numerous potential phosphorylation sites were found. Two nuclear export signals were identified, but no nuclear localization signal. In man, NCU-G1 was found to be widely expressed at the mRNA level with especially high levels detected in prostate, liver and kidney. Electrophoretic mobility shift analysis showed specific binding of NCU-G1 to an oligonucleotide representing the footprint 1 element of the human cellular retinol-binding protein 1 gene promoter. NCU-G1 was found to activate transcription from this promoter and required presence of the footprint 1 element. In transiently transfected Drosophila Schneider S2 cells, we demonstrated that NCU-G1 functions as a co-activator for ligand-activated PPAR-alpha, resulting in an increased expression of a CAT reporter gene under control of the peroxisome proliferator-activated receptor-alpha responsive acyl-CoA oxidase promoter.

Conclusion: We propose that NCU-G1 is a dual-function protein capable of functioning as a transcription factor as well as a nuclear receptor co-activator.

Show MeSH
Related in: MedlinePlus