Transcriptional and translational mechanisms of cytochrome b5 reductase isoenzyme generation in humans

2001 ◽  
Vol 355 (2) ◽  
pp. 529-535 ◽  
Author(s):  
Alena LEROUX ◽  
Luisa MOTA VIEIRA ◽  
Axel KAHN
Keyword(s):  
Cytochrome B5 ◽  
Protein Isoforms ◽  
In Vitro Translation ◽  
Alu Elements ◽  
Cytochrome B5 Reductase ◽  
Dynamic Events ◽  
Membrane Bound ◽  
Essential Enzyme ◽  
Translational Mechanisms

Cytochrome b5 reductase (b5R) is an essential enzyme that exists in soluble and membrane-bound isoforms, each with specific functions. In the rat, the two forms are generated from alternative transcripts differing in the first exons. In contrast, the biogenesis of b5R isoforms in the human is not yet well understood. In the present study we have detected three novel alternative exons, designated 1S, S′ and 1B, located between the first alternative exon 1M and the common second exon in the human b5R gene. Accordingly, multiple M-type, S-type and SS′-type and B-type transcripts are generated. All types of human b5R transcript are expressed ubiquitously. An analysis of in vitro translation products demonstrated an alternative use of different AUG initiators resulting in the production of various human b5R protein isoforms. Our results indicate that the organization of the 5′ region of the b5R gene is not conserved between rodents and humans. Insertion of Alu elements into the human b5R gene, in particular just upstream of the S/S′ region, could be responsible for dynamic events of gene rearrangement during evolution.

scholarly journals A single mRNA, transcribed from an alternative, erythroid-specific, promoter, codes for two non-myristylated forms of NADH-cytochrome b5 reductase

1992 ◽  
Vol 117 (5) ◽  
pp. 975-986 ◽  
Author(s):  
G Pietrini ◽  
D Aggujaro ◽  
P Carrera ◽  
J Malyszko ◽  
A Vitale ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mutational Analysis ◽  
Consensus Sequence ◽  
Cytochrome B5 ◽  
In Vitro Translation ◽  
Relative Molecular Mass ◽  
Specific Sequence ◽  
Cytochrome B5 Reductase ◽  
Nadh Cytochrome

Two forms of NADH-cytochrome b5 reductase are produced from one gene: a myristylated membrane-bound enzyme, expressed in all tissues, and a soluble, erythrocyte-specific, isoform. The two forms are identical in a large cytoplasmic domain (Mr approximately 30,000) and differ at the NH2-terminus, which, in the membrane form, is responsible for binding to the bilayer, and which contains the myristylation consensus sequence and an additional 14 uncharged amino acids. To investigate how the two differently targeted forms of the reductase are produced, we cloned a reductase transcript from reticulocytes, and studied its relationship to the previously cloned liver cDNA. The reticulocyte transcript differs from the liver transcript in the 5' non-coding portion and at the beginning of the coding portion, where the seven codons specifying the myristoylation consensus are replaced by a reticulocyte-specific sequence which codes for 13 non-charged amino acids. Analysis of genomic reductase clones indicated that the ubiquitous transcript is generated from an upstream "housekeeping" type promoter, while the reticulocyte transcript originates from a downstream, erythroid-specific, promoter. In vitro translation of the reticulocyte-specific mRNA generated two products: a minor one originating from the first AUG, and a major one starting from a downstream AUG, as indicated by mutational analysis. Both the AUGs used as initiation codons were in an unfavorable sequence context. The major, lower relative molecular mass product behaved as a soluble protein, while the NH2-terminally extended minor product interacted with microsomes in vitro. The generation of soluble reductase from a downstream AUG was confirmed in vivo, in Xenopus oocytes. Thus, differently localized products, with respect both to tissues and to subcellular compartments, are generated from the same gene by a combination of transcriptional and translational mechanisms.

scholarly journals Endogenous proteolysis of membrane-bound red cell cytochrome-b5 reductase in adults and newborns: its possible relevance to the generation of the soluble "methemoglobin reductase"

Blood ◽  
1983 ◽  
Vol 61 (5) ◽  
pp. 894-898
Author(s):  
D Choury ◽  
A Reghis ◽  
AL Pichard ◽  
JC Kaplan
Keyword(s):  
Current Knowledge ◽  
Reductase Activity ◽  
Cytochrome B5 ◽  
Red Cells ◽  
Cytochrome B5 Reductase ◽  
Calcium Dependent ◽  
Membrane Bound ◽  
Relative Deficiency ◽  
Low Activity

The problem of the low activity of so-called methemoglobin reductase in red cells from newborns was reinvestigated in view of our current knowledge of this enzyme, i.e., (1) its being cytochrome-b5 reductase and (2) its presence in two forms: soluble and membrane-bound. We found that red cells from cord blood and newborns exhibited a 50% decrease of soluble cytochrome-b5 reductase activity, whereas membrane-bound activity was in the adult range. Ghosts from these cells possessed diminished ability to solubilize membrane-bound cytochrome-b5 reductase in the course of in vitro auto-incubation. This autosolubilizing ability increased with age and reached adult level concomitantly with soluble cytochrome-b5 reductase activity at 6 mo. We conclude that the relative deficiency of soluble cytochrome-b5 reductase observed at birth is due to diminished post-translational processing of the membrane-bound enzyme during erythropoiesis of fetal cells. This processing is calcium-dependent related to calmodulin.

scholarly journals Endogenous proteolysis of membrane-bound red cell cytochrome-b5 reductase in adults and newborns: its possible relevance to the generation of the soluble "methemoglobin reductase"

Blood ◽  
1983 ◽  
Vol 61 (5) ◽  
pp. 894-898 ◽  
Author(s):  
D Choury ◽  
A Reghis ◽  
AL Pichard ◽  
JC Kaplan
Keyword(s):  
Current Knowledge ◽  
Reductase Activity ◽  
Cytochrome B5 ◽  
Red Cells ◽  
Cytochrome B5 Reductase ◽  
Calcium Dependent ◽  
Membrane Bound ◽  
Relative Deficiency ◽  
Low Activity

Abstract The problem of the low activity of so-called methemoglobin reductase in red cells from newborns was reinvestigated in view of our current knowledge of this enzyme, i.e., (1) its being cytochrome-b5 reductase and (2) its presence in two forms: soluble and membrane-bound. We found that red cells from cord blood and newborns exhibited a 50% decrease of soluble cytochrome-b5 reductase activity, whereas membrane-bound activity was in the adult range. Ghosts from these cells possessed diminished ability to solubilize membrane-bound cytochrome-b5 reductase in the course of in vitro auto-incubation. This autosolubilizing ability increased with age and reached adult level concomitantly with soluble cytochrome-b5 reductase activity at 6 mo. We conclude that the relative deficiency of soluble cytochrome-b5 reductase observed at birth is due to diminished post-translational processing of the membrane-bound enzyme during erythropoiesis of fetal cells. This processing is calcium-dependent related to calmodulin.

Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats

1991 ◽  
Vol 69 (8) ◽  
pp. 499-508 ◽  
Author(s):  
Andrea G. Bodnar ◽  
Richard A. Rachubinski
Keyword(s):  
Endoplasmic Reticulum ◽  
Immunoblot Analysis ◽  
In Vitro Translation ◽  
Peroxisome Proliferator ◽  
Peroxisomal Membrane ◽  
Molecular Masses ◽  
Fold Induction ◽  
Membrane Bound ◽  
Liver Peroxisomes

We have characterized the integral membrane polypeptides of liver peroxisomes from untreated rats and rats treated with clofibrate, a peroxisome proliferator. Membranes, prepared by treatment of purified peroxisomes with sodium carbonate, were used to raise an antiserum in rabbits. Immunoblot analysis demonstrated the reaction of this antiserum with six peroxisomal integral membrane polypeptides (molecular masses, 140, 69, 50, 36, 22, and 15 kDa). Treatment of rats with the hypolipidemic drug clofibrate caused a 4- to 10-fold induction in the 69-kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50- and 36-kDa integral membrane polypeptides of the peroxisome. Biochemical and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22-, 36-, and 69-kDa integral membrane polypeptides were synthesized on free polysomes, while the 50-kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50-kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis.Key words: peroxisomes, integral membrane polypeptides, clofibrate, free polysomes, membrane-bound polysomes.

Abstract 15696: Cytochrome B5 Reductase 3 Sensitizes Soluble Guanylate Cyclase to Nitric Oxide

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Anh T Nguyen ◽  
Mizanur M Rahaman ◽  
Stephanie M Mutchler ◽  
Megan Miller ◽  
Josef T Prchal ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Blood Pressure ◽  
Soluble Guanylate Cyclase ◽  
Cytochrome B5 ◽  
Heme Iron ◽  
Proximity Ligation Assay ◽  
Cytochrome B5 Reductase ◽  
Cgmp Production ◽  
Iron Reductase

Impaired soluble guanylyl cyclase (sGC)-dependent nitric oxide (NO) signaling has been linked to numerous cardiovascular diseases (CVD) such as hypertension, myocardial infarction and atherosclerosis. Despite emerging evidence indicating the importance of sGC function within the cardiovascular system, the basic mechanisms that regulate sGC activity remain incompletely understood. Herein, we provide in vitro and in vivo evidence that cytochrome b5 reductase 3 (Cyb5R3) is an sGC heme iron reductase and regulates downstream cGMP signaling. Of major significance, we also demonstrate that a Cyb5R3 T116S polymorphism with allele frequency of 0.23 in African Americans associates with increase blood pressure and is incapable of reducing sGC. Proximity ligation assay (PLA) experiments show that endogenous Cyb5R3 and oxidized sGC associate. Knockdown of Cyb5R3 results in reduced cGMP production and downstream signaling in rat aortic smooth muscle cells (SMC). Overexpression of Cyb5R3 not only rescues cGMP production but also increases baseline cGMP, whereas T116S mutant does not. Finally, inhibition of Cyb5R3 in mice significantly increases systemic blood pressure. Our studies are the first to identify an sGC heme iron reductase, provide evidence for Cyb5R3 as a key biological regulator of sGC activity and vascular tone in SMC, and link a human polymorphism of Cyb5R3 to increased blood pressure; all of which may lead to the development of novel therapeutics targeting Cyb5R3 for the treatment of CVD. Importantly, the co-expression of Cyb5R3 and sGC in multiple cells types suggests that this regulation of sGC activity may have broad applications for multiple physiological and pathophysiological processes. Results: Conclusions:

scholarly journals c-erbA encodes multiple proteins in chicken erythroid cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4155-4161 ◽  
Author(s):  
J Bigler ◽  
R N Eisenman
Keyword(s):  
Expression Vector ◽  
In Vitro Translation ◽  
Vector System ◽  
Mrna Levels ◽  
Erythroid Cells ◽  
Avian Erythroblastosis Virus ◽  
Related Proteins ◽  
Translational Mechanisms

To identify and characterize the proteins encoded by the erbA proto-oncogene, we expressed the C-terminal region of v-erbA in a bacterial trpE expression vector system and used the fusion protein to prepare antiserum. The anti-trp-erbA serum recognized the P75gag-erbA protein encoded by avian erythroblastosis virus and specifically precipitated six highly related proteins ranging in size from 27 to 46 kilodaltons from chicken embryonic erythroid cells. In vitro translation of a chicken erbA cDNA produced essentially the same pattern of proteins. Partial proteolytic maps and antigenicity and kinetic analyses of the in vivo and in vitro proteins indicated that they are related and that the multiple bands are likely to arise from internal initiations within c-erbA to generate a nested set of proteins. All of the c-erbA proteins are predominantly associated with chicken erythroblast nuclei. However, Nonidet P-40 treatment resulted in extraction of the three smaller proteins, whereas the larger proteins were retained. During differentiation of erythroid cells in chicken embryos, we found maximal levels of c-erbA protein synthesis at days 7 to 8 of embryogenesis. By contrast, c-erbA mRNA levels remained essentially constant from days 5 to 12. Together, our results indicate that posttranscriptional or translational mechanisms are involved in regulation of c-erbA expression and in the complexity of its protein products.

scholarly journals Light-regulated translation of chloroplast proteins. I. Transcripts of psaA-psaB, psbA, and rbcL are associated with polysomes in dark-grown and illuminated barley seedlings

1988 ◽  
Vol 106 (2) ◽  
pp. 289-301 ◽  
Author(s):  
RR Klein ◽  
HS Mason ◽  
JE Mullet
Keyword(s):  
Chlorophyll A ◽  
Photosystem I ◽  
In Vitro Translation ◽  
Chain Elongation ◽  
Selective Activation ◽  
Translation Elongation ◽  
In Vitro Synthesis ◽  
Chloroplast Translation ◽  
Membrane Bound

We have previously observed (Klein, R. R., and J. E. Mullet, 1986, J. Biol. Chem. 261:11138-11145) that translation of two 65-70-kD chlorophyll a-apoproteins of Photosystem I (gene products of psaA and psaB) and a 32-kD quinone-binding protein of Photosystem II (gene product of psbA) was not detected in plastids of dark-grown barley seedlings even though transcripts for these proteins were present. In the present study it was found that nearly all of the psaA-psaB transcripts in plastids of dark-grown plants were associated with membrane-bound polysomes. Membrane-associated polysomes from plastids of dark-grown plants synthesized the 65-70-kD chlorophyll a-apoproteins at low levels when added to a homologous in vitro translation extract capable of translation elongation. However, when etioplast membranes were disrupted with detergent, in vitro synthesis of the 65-70-kD chlorophyll a-apoproteins increased to levels observed with polysomes of plastids from illuminated plants. These results suggest that synthesis of the chlorophyll a-apoproteins of Photosystem I is arrested on membrane-bound polysomes at the level of polypeptide chain elongation. In addition to the selective activation of chlorophyll a-apoprotein translation, illumination also caused an increase in chloroplast polysomes (membrane-associated and stromal) and induced a recruitment of psbA and rbcL transcripts into chloroplast polysomes. These results indicate that in conjunction with the selective activation of chlorophyll a-apoprotein elongation, illumination also caused a general stimulation of chloroplast translation initiation.

c-erbA encodes multiple proteins in chicken erythroid cells

1988 ◽  
Vol 8 (10) ◽  
pp. 4155-4161
Author(s):  
J Bigler ◽  
R N Eisenman
Keyword(s):  
Expression Vector ◽  
In Vitro Translation ◽  
Vector System ◽  
Mrna Levels ◽  
Erythroid Cells ◽  
Avian Erythroblastosis Virus ◽  
Related Proteins ◽  
Translational Mechanisms

To identify and characterize the proteins encoded by the erbA proto-oncogene, we expressed the C-terminal region of v-erbA in a bacterial trpE expression vector system and used the fusion protein to prepare antiserum. The anti-trp-erbA serum recognized the P75gag-erbA protein encoded by avian erythroblastosis virus and specifically precipitated six highly related proteins ranging in size from 27 to 46 kilodaltons from chicken embryonic erythroid cells. In vitro translation of a chicken erbA cDNA produced essentially the same pattern of proteins. Partial proteolytic maps and antigenicity and kinetic analyses of the in vivo and in vitro proteins indicated that they are related and that the multiple bands are likely to arise from internal initiations within c-erbA to generate a nested set of proteins. All of the c-erbA proteins are predominantly associated with chicken erythroblast nuclei. However, Nonidet P-40 treatment resulted in extraction of the three smaller proteins, whereas the larger proteins were retained. During differentiation of erythroid cells in chicken embryos, we found maximal levels of c-erbA protein synthesis at days 7 to 8 of embryogenesis. By contrast, c-erbA mRNA levels remained essentially constant from days 5 to 12. Together, our results indicate that posttranscriptional or translational mechanisms are involved in regulation of c-erbA expression and in the complexity of its protein products.

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