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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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Retinoid and NAD+ levels in mRNA in Morpholino injected embryos.[A] HPLC measurements show no significant changes in of ROL, RE and RA levels in 14hpf embryos injected with either RNA encoding Rbp7a, Nmnat1-Rbp7, or morpholinos blocking rbp7a translation (MO-ATG) and proper splicing of rbp7a exon 2 (MO-Δex2). For each dataset three independent treatments were performed and 100 embryos each were collected at 14hpf from each group of mRNA injected, morphant, and non-injected embryos. [B] NAD+ levels increase after over-expressions of nmnat1 and nmnat1-rbp7a fusion mRNA. Spectral peaks of HPLC measurements shows the peak characteristic of NAD+ reference. For each experiment, three times 100 embryos at 6hpf were collected in different treatment or wild type groups Error bars indicated the SEM (**P<0.01).
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pone.0143825.g004: Retinoid and NAD+ levels in mRNA in Morpholino injected embryos.[A] HPLC measurements show no significant changes in of ROL, RE and RA levels in 14hpf embryos injected with either RNA encoding Rbp7a, Nmnat1-Rbp7, or morpholinos blocking rbp7a translation (MO-ATG) and proper splicing of rbp7a exon 2 (MO-Δex2). For each dataset three independent treatments were performed and 100 embryos each were collected at 14hpf from each group of mRNA injected, morphant, and non-injected embryos. [B] NAD+ levels increase after over-expressions of nmnat1 and nmnat1-rbp7a fusion mRNA. Spectral peaks of HPLC measurements shows the peak characteristic of NAD+ reference. For each experiment, three times 100 embryos at 6hpf were collected in different treatment or wild type groups Error bars indicated the SEM (**P<0.01).

Mentions: To assess functional properties of Rbp7a and Nmnat1-Rbp7a, different in vivo gene-knock down and gain-of function experiments were conducted. Morpholino injection was used to either selectively block translation of rbp7a transcripts (MO-ATG) or to induce skipping of the rbp7a and nmnat1-rbp7a shared rbp7a-exon 2 (MO-Δex2), respectively (Fig 3, see also S1 Fig and Material and Methods). Based on the role of Rbp7 in delivering retinol to Lrat, we speculated that rbp7a morphants would show similar early patterning defects to lrabt morphants. In contrast to this prediction, injection of up to 4ng/embryo of both morpholinos resulted in morphologically normal looking 20hpf embryos. While higher morpholino amounts (8ng/embryo) caused severe neuronal and mesoderm patterning defects, majority of these defects was rescued by co-injection of p53 morpholinos (not shown). This suggests that Rbp7a and Nmant1-Rbp7a are dispensable for Lratb functions that higher morpholino amounts caused p53-dependent off target effects [38]. Furthermore, HPLC measurement of all-trans-retinol, retinyl ester and RA levels revealed no significant changes in 14 hpf control embryos and in 14 hpf embryos injected with either rbp7a or nmnat1-rbp7a mRNA, or with rbp7 morpholinos (Fig 4A and S2 Fig). This shows, that neither induction nor loss of Rpb7a function has a major effect on early embryonic retinoid homeostasis and RA signaling. Next we compared Nmnat1 and Nmnat1-rbp7a protein activities. Nmnat proteins convert nicotinamide mononucleotide (NMN) to NAD+, which serves as an electron carrier in numerous redox reactions, including the oxidation of retinol to retinal [11, 39]. Although synthesis of NMN is supposed to be the rate-limiting step in NAD+ synthesis [40, 41], we found that injection of either nmnat1 or nmnat1-rbp7a mRNAs (100 pg/embryo) caused a weak but significant increase in NAD+ levels as compared to control embryos (Fig 4B and S2 Fig). In three independent sets of HPLC measurement on 60% epiboly embryos, this increase ranged from 3% to 10% and 7% to 9% for nmnat1 and nmnat1-rbp7a injections, respectively, showing that both proteins have a similar capacity for NAD+ synthesis in the embryo.


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)

Retinoid and NAD+ levels in mRNA in Morpholino injected embryos.[A] HPLC measurements show no significant changes in of ROL, RE and RA levels in 14hpf embryos injected with either RNA encoding Rbp7a, Nmnat1-Rbp7, or morpholinos blocking rbp7a translation (MO-ATG) and proper splicing of rbp7a exon 2 (MO-Δex2). For each dataset three independent treatments were performed and 100 embryos each were collected at 14hpf from each group of mRNA injected, morphant, and non-injected embryos. [B] NAD+ levels increase after over-expressions of nmnat1 and nmnat1-rbp7a fusion mRNA. Spectral peaks of HPLC measurements shows the peak characteristic of NAD+ reference. For each experiment, three times 100 embryos at 6hpf were collected in different treatment or wild type groups Error bars indicated the SEM (**P<0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664474&req=5

pone.0143825.g004: Retinoid and NAD+ levels in mRNA in Morpholino injected embryos.[A] HPLC measurements show no significant changes in of ROL, RE and RA levels in 14hpf embryos injected with either RNA encoding Rbp7a, Nmnat1-Rbp7, or morpholinos blocking rbp7a translation (MO-ATG) and proper splicing of rbp7a exon 2 (MO-Δex2). For each dataset three independent treatments were performed and 100 embryos each were collected at 14hpf from each group of mRNA injected, morphant, and non-injected embryos. [B] NAD+ levels increase after over-expressions of nmnat1 and nmnat1-rbp7a fusion mRNA. Spectral peaks of HPLC measurements shows the peak characteristic of NAD+ reference. For each experiment, three times 100 embryos at 6hpf were collected in different treatment or wild type groups Error bars indicated the SEM (**P<0.01).
Mentions: To assess functional properties of Rbp7a and Nmnat1-Rbp7a, different in vivo gene-knock down and gain-of function experiments were conducted. Morpholino injection was used to either selectively block translation of rbp7a transcripts (MO-ATG) or to induce skipping of the rbp7a and nmnat1-rbp7a shared rbp7a-exon 2 (MO-Δex2), respectively (Fig 3, see also S1 Fig and Material and Methods). Based on the role of Rbp7 in delivering retinol to Lrat, we speculated that rbp7a morphants would show similar early patterning defects to lrabt morphants. In contrast to this prediction, injection of up to 4ng/embryo of both morpholinos resulted in morphologically normal looking 20hpf embryos. While higher morpholino amounts (8ng/embryo) caused severe neuronal and mesoderm patterning defects, majority of these defects was rescued by co-injection of p53 morpholinos (not shown). This suggests that Rbp7a and Nmant1-Rbp7a are dispensable for Lratb functions that higher morpholino amounts caused p53-dependent off target effects [38]. Furthermore, HPLC measurement of all-trans-retinol, retinyl ester and RA levels revealed no significant changes in 14 hpf control embryos and in 14 hpf embryos injected with either rbp7a or nmnat1-rbp7a mRNA, or with rbp7 morpholinos (Fig 4A and S2 Fig). This shows, that neither induction nor loss of Rpb7a function has a major effect on early embryonic retinoid homeostasis and RA signaling. Next we compared Nmnat1 and Nmnat1-rbp7a protein activities. Nmnat proteins convert nicotinamide mononucleotide (NMN) to NAD+, which serves as an electron carrier in numerous redox reactions, including the oxidation of retinol to retinal [11, 39]. Although synthesis of NMN is supposed to be the rate-limiting step in NAD+ synthesis [40, 41], we found that injection of either nmnat1 or nmnat1-rbp7a mRNAs (100 pg/embryo) caused a weak but significant increase in NAD+ levels as compared to control embryos (Fig 4B and S2 Fig). In three independent sets of HPLC measurement on 60% epiboly embryos, this increase ranged from 3% to 10% and 7% to 9% for nmnat1 and nmnat1-rbp7a injections, respectively, showing that both proteins have a similar capacity for NAD+ synthesis in the embryo.

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
Related in: MedlinePlus