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Nmnat1-Rbp7 Is a Conserved Fusion-Protein That Combines NAD+ Catalysis of Nmnat1 with Subcellular Localization of Rbp7.

Chen H, Babino D, Schoenbichler SA, Arkhipova V, Töchterle S, Martin F, Huck CW, von Lintig J, Meyer D - PLoS ONE (2015)

Bottom Line: We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production.Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization.HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology/CMBI, University of Innsbruck, Technikerstrasse 25, 6020, Innsbruck, Austria.

ABSTRACT
Retinol binding proteins (Rbps) are known as carriers for transport and targeting of retinoids to their metabolizing enzymes. Rbps are also reported to function in regulating the homeostatic balance of retinoid metabolism, as their level of retinoid occupancy impacts the activities of retinoid metabolizing enzymes. Here we used zebrafish as a model to study rbp7a function and regulation. We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production. The data are consistent with a Nodal-dependent coordination of the allocation of retinoid precursors to processing enzymes with the catalysis of retinoic acid formation. Further, we describe a novel nmnat1-rbp7 transcript encoding a fusion of Rbp7 and the NAD+ (Nicotinamide adenine dinucleotide) synthesizing enzyme Nmnat1. We show that nmnat1-rbp7 is conserved in fish, mouse and chicken, and that in zebrafish regulation of nmnat1-rbp7a is distinct from that of rbp7a and nmnat1. Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization. HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme. These studies, taken together with previously documented NAD+ dependent interaction of RBPs with ER-associated enzymes of retinal catalysis, implicate functions of this newly described NMNAT1-Rbp7 fusion protein in retinol oxidation.

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Complementary regulation of rbp7a and aldh1a2 by Nodal signaling.[A-F] Lateral views of whole mount in situ stains of rbp7a in wild type embryos at 4hpf [A, C] 6hpf [B,D], 9hpf [E] and 10hpf [F]; [C-D] vibratome sections of 4hpf and 6hpf embryos. rbp7a signals are found in marginal cell [A-D], YSL, and in forerunner cells (arrowhead in [D-F]). [G-J] Marginal expression of aldh1a2 is strongly reduced in 6hpf MZsur [H] and MZoep [I] embryos; [J] RT-qPCR verified reduced expression levels of aldh1a2 in MZsur, MZoep as compared to wild type embryos. [K-P] Whole mount in situ hybridization for rbp7a (arrow heads) and goosecoid (gsc) (white asterisks) performed at 6 and 8 hpf in Msur+/- as control [K, N], MZsur [L, O] and MZoep [M, P] embryos. Note that expression levels of rbp7a around the YSL were similar among the gsc positive embryos and the gsc negative MZsur [B, E] and MZoep [C, F] mutants. [Q-S] rbp7a expression at 24hpf. Arrows mark rbp7a signals in the posterior midbrain that were present in MZsur [R] and MZoep embryos [S] but not in control embryo [Q]. [T] RT-qPCR results for rbp7a in of 6hpf and 24hpf embryos. Graphed is the mean and SEM from triplicate experiments. Error bars indicated the SEM. Unpaired T-test was used to test the significance (*P<0.05).
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pone.0143825.g001: Complementary regulation of rbp7a and aldh1a2 by Nodal signaling.[A-F] Lateral views of whole mount in situ stains of rbp7a in wild type embryos at 4hpf [A, C] 6hpf [B,D], 9hpf [E] and 10hpf [F]; [C-D] vibratome sections of 4hpf and 6hpf embryos. rbp7a signals are found in marginal cell [A-D], YSL, and in forerunner cells (arrowhead in [D-F]). [G-J] Marginal expression of aldh1a2 is strongly reduced in 6hpf MZsur [H] and MZoep [I] embryos; [J] RT-qPCR verified reduced expression levels of aldh1a2 in MZsur, MZoep as compared to wild type embryos. [K-P] Whole mount in situ hybridization for rbp7a (arrow heads) and goosecoid (gsc) (white asterisks) performed at 6 and 8 hpf in Msur+/- as control [K, N], MZsur [L, O] and MZoep [M, P] embryos. Note that expression levels of rbp7a around the YSL were similar among the gsc positive embryos and the gsc negative MZsur [B, E] and MZoep [C, F] mutants. [Q-S] rbp7a expression at 24hpf. Arrows mark rbp7a signals in the posterior midbrain that were present in MZsur [R] and MZoep embryos [S] but not in control embryo [Q]. [T] RT-qPCR results for rbp7a in of 6hpf and 24hpf embryos. Graphed is the mean and SEM from triplicate experiments. Error bars indicated the SEM. Unpaired T-test was used to test the significance (*P<0.05).

Mentions: The Nodal and RA signaling pathways are two key regulators of embryonic patterning. While bidirectional interactions between these pathways are well documented, the molecular details have not been fully characterized. Recently, rbp7a has been suggested to contribute to the RA/Nodal interaction network [24]. Consistent with this notion, we had previously identified rbp7a in screen for Nodal regulated genes [35]. Detailed analyses of embryonic rbp7a expression revealed the first specific rpb7a signals in marginal cells of the late blastula and early gastrula embryo (Fig 1A–1D). During gastrulation, expression was restricted to the YSL and forerunner cells (Fig 1B and 1D–1F). As the early marginal expression of rbp7a displayed similarities with that of aldh1A2 [36], encoding the key enzyme for early embryonic retinal to RA conversion, we speculated that these genes may be similarly regulated. Previous studies in mice reported a role of Nodal signaling and in particular of the transcription factor Foxh1 in controlling activation of aldh1A2 [37]. Consistent with a similar cross-talk between Nodal/FoxH1 and RA-signaling in zebrafish, we found that aldh1A2 expression is strongly reduced in both Nodal-signaling deficient MZoep mutants and MZsur/foxH1 mutants during gastrulation (Fig 1G–1J). In contrast, whole mount in situ hybridization analyses for rbp7a expression revealed a very similar pattern in control and mutant embryos at 6 and 8 hpf (Fig 1K–1P). The embryos showed weak signals in the YSL and in case of control and MZsur stronger signals in forerunner cells (arrow heads, asterisk mark co-stained gsc signals, which was used to distinguish embryos derived form cross of a homozygous female and a heterozygous male). Corresponding forerunner signals were missing in MZoep embryos as they lack these cells (Fig 1M and 1P). While rbp7a signals could not be detected in whole mount mRNA analyses of 24hpf wildtype embryos (Fig 1Q), MZsur and MZoep mutants both showed an ectopic signal in the posterior midbrain (Fig 1R and 1S). Quantitative RT-PCR analyses revealed an increase, rather than decrease, in rbp7a transcripts in 6hpf MZsur and Mzoep embryos as compared to control embryos (Fig 1T). Increased expression was even more striking at 24 hpf when rbp7a mRNA increased more than 10 and 20 fold in MZsur and MZoep mutants, respectively (Fig 1T). The data demonstrate opposite effects of Nodal FoxH1-signaling on rbp7a and aldh1a2 expression.


Nmnat1-Rbp7 Is a Conserved Fusion-Protein That Combines NAD+ Catalysis of Nmnat1 with Subcellular Localization of Rbp7.

Chen H, Babino D, Schoenbichler SA, Arkhipova V, Töchterle S, Martin F, Huck CW, von Lintig J, Meyer D - PLoS ONE (2015)

Complementary regulation of rbp7a and aldh1a2 by Nodal signaling.[A-F] Lateral views of whole mount in situ stains of rbp7a in wild type embryos at 4hpf [A, C] 6hpf [B,D], 9hpf [E] and 10hpf [F]; [C-D] vibratome sections of 4hpf and 6hpf embryos. rbp7a signals are found in marginal cell [A-D], YSL, and in forerunner cells (arrowhead in [D-F]). [G-J] Marginal expression of aldh1a2 is strongly reduced in 6hpf MZsur [H] and MZoep [I] embryos; [J] RT-qPCR verified reduced expression levels of aldh1a2 in MZsur, MZoep as compared to wild type embryos. [K-P] Whole mount in situ hybridization for rbp7a (arrow heads) and goosecoid (gsc) (white asterisks) performed at 6 and 8 hpf in Msur+/- as control [K, N], MZsur [L, O] and MZoep [M, P] embryos. Note that expression levels of rbp7a around the YSL were similar among the gsc positive embryos and the gsc negative MZsur [B, E] and MZoep [C, F] mutants. [Q-S] rbp7a expression at 24hpf. Arrows mark rbp7a signals in the posterior midbrain that were present in MZsur [R] and MZoep embryos [S] but not in control embryo [Q]. [T] RT-qPCR results for rbp7a in of 6hpf and 24hpf embryos. Graphed is the mean and SEM from triplicate experiments. Error bars indicated the SEM. Unpaired T-test was used to test the significance (*P<0.05).
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pone.0143825.g001: Complementary regulation of rbp7a and aldh1a2 by Nodal signaling.[A-F] Lateral views of whole mount in situ stains of rbp7a in wild type embryos at 4hpf [A, C] 6hpf [B,D], 9hpf [E] and 10hpf [F]; [C-D] vibratome sections of 4hpf and 6hpf embryos. rbp7a signals are found in marginal cell [A-D], YSL, and in forerunner cells (arrowhead in [D-F]). [G-J] Marginal expression of aldh1a2 is strongly reduced in 6hpf MZsur [H] and MZoep [I] embryos; [J] RT-qPCR verified reduced expression levels of aldh1a2 in MZsur, MZoep as compared to wild type embryos. [K-P] Whole mount in situ hybridization for rbp7a (arrow heads) and goosecoid (gsc) (white asterisks) performed at 6 and 8 hpf in Msur+/- as control [K, N], MZsur [L, O] and MZoep [M, P] embryos. Note that expression levels of rbp7a around the YSL were similar among the gsc positive embryos and the gsc negative MZsur [B, E] and MZoep [C, F] mutants. [Q-S] rbp7a expression at 24hpf. Arrows mark rbp7a signals in the posterior midbrain that were present in MZsur [R] and MZoep embryos [S] but not in control embryo [Q]. [T] RT-qPCR results for rbp7a in of 6hpf and 24hpf embryos. Graphed is the mean and SEM from triplicate experiments. Error bars indicated the SEM. Unpaired T-test was used to test the significance (*P<0.05).
Mentions: The Nodal and RA signaling pathways are two key regulators of embryonic patterning. While bidirectional interactions between these pathways are well documented, the molecular details have not been fully characterized. Recently, rbp7a has been suggested to contribute to the RA/Nodal interaction network [24]. Consistent with this notion, we had previously identified rbp7a in screen for Nodal regulated genes [35]. Detailed analyses of embryonic rbp7a expression revealed the first specific rpb7a signals in marginal cells of the late blastula and early gastrula embryo (Fig 1A–1D). During gastrulation, expression was restricted to the YSL and forerunner cells (Fig 1B and 1D–1F). As the early marginal expression of rbp7a displayed similarities with that of aldh1A2 [36], encoding the key enzyme for early embryonic retinal to RA conversion, we speculated that these genes may be similarly regulated. Previous studies in mice reported a role of Nodal signaling and in particular of the transcription factor Foxh1 in controlling activation of aldh1A2 [37]. Consistent with a similar cross-talk between Nodal/FoxH1 and RA-signaling in zebrafish, we found that aldh1A2 expression is strongly reduced in both Nodal-signaling deficient MZoep mutants and MZsur/foxH1 mutants during gastrulation (Fig 1G–1J). In contrast, whole mount in situ hybridization analyses for rbp7a expression revealed a very similar pattern in control and mutant embryos at 6 and 8 hpf (Fig 1K–1P). The embryos showed weak signals in the YSL and in case of control and MZsur stronger signals in forerunner cells (arrow heads, asterisk mark co-stained gsc signals, which was used to distinguish embryos derived form cross of a homozygous female and a heterozygous male). Corresponding forerunner signals were missing in MZoep embryos as they lack these cells (Fig 1M and 1P). While rbp7a signals could not be detected in whole mount mRNA analyses of 24hpf wildtype embryos (Fig 1Q), MZsur and MZoep mutants both showed an ectopic signal in the posterior midbrain (Fig 1R and 1S). Quantitative RT-PCR analyses revealed an increase, rather than decrease, in rbp7a transcripts in 6hpf MZsur and Mzoep embryos as compared to control embryos (Fig 1T). Increased expression was even more striking at 24 hpf when rbp7a mRNA increased more than 10 and 20 fold in MZsur and MZoep mutants, respectively (Fig 1T). The data demonstrate opposite effects of Nodal FoxH1-signaling on rbp7a and aldh1a2 expression.

Bottom Line: We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production.Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization.HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme.

View Article: PubMed Central - PubMed

Affiliation: Institute of Molecular Biology/CMBI, University of Innsbruck, Technikerstrasse 25, 6020, Innsbruck, Austria.

ABSTRACT
Retinol binding proteins (Rbps) are known as carriers for transport and targeting of retinoids to their metabolizing enzymes. Rbps are also reported to function in regulating the homeostatic balance of retinoid metabolism, as their level of retinoid occupancy impacts the activities of retinoid metabolizing enzymes. Here we used zebrafish as a model to study rbp7a function and regulation. We find that early embryonic rbp7a expression is negatively regulated by the Nodal/FoxH1-signaling pathway and we show that Nodal/FoxH1 activity has the opposite effect on aldh1a2, which encodes the major enzyme for early embryonic retinoic acid production. The data are consistent with a Nodal-dependent coordination of the allocation of retinoid precursors to processing enzymes with the catalysis of retinoic acid formation. Further, we describe a novel nmnat1-rbp7 transcript encoding a fusion of Rbp7 and the NAD+ (Nicotinamide adenine dinucleotide) synthesizing enzyme Nmnat1. We show that nmnat1-rbp7 is conserved in fish, mouse and chicken, and that in zebrafish regulation of nmnat1-rbp7a is distinct from that of rbp7a and nmnat1. Injection experiments in zebrafish further revealed that Nmnat1-Rbp7a and Nmnat1 have similar NAD+ catalyzing activities but a different subcellular localization. HPLC measurements and protein localization analysis highlight Nmnat1-Rbp7a as the only known cytoplasmic and presumably endoplasmic reticulum (ER) specific NAD+ catalyzing enzyme. These studies, taken together with previously documented NAD+ dependent interaction of RBPs with ER-associated enzymes of retinal catalysis, implicate functions of this newly described NMNAT1-Rbp7 fusion protein in retinol oxidation.

Show MeSH