Edel Reytor - Academia.edu (original) (raw)

Papers by Edel Reytor

The methyl donor S-adenosylmethionine is synthesized in mammalian cytosol by three isoenzymes. Me... more The methyl donor S-adenosylmethionine
is synthesized in mammalian cytosol by three isoenzymes.
Methionine adenosyltransferase II is ubiquitously
expressed, whereas isoenzymes I (homotetramer)
and III (homodimer) are considered the hepatic
enzymes. In this work, we identified methionine adenosyltransferase
I/III in most rat tissues, both in the
cytoplasm and the nucleus. Nuclear localization was the
preferred distribution observed in extrahepatic tissues,
where the protein colocalizes with nuclear matrix markers.
A battery of mutants used in several cell lines to
decipher the determinants involved in methionine adenosyltransferase
subcellular localization demonstrated,
by confocal microscopy and subcellular fractionation,
the presence of two partially overlapping
areas at the C-terminal end of the protein involved both
in cytoplasmic retention and nuclear localization. Immunoprecipitation
of coexpressed FLAG and EGFP
fusions and gel-filtration chromatography allowed detection
of tetramers and monomers in nuclear fractions
that also exhibited S-adenosylmethionine synthesis.
Neither nuclear localization nor matrix binding required
activity, as demonstrated with the inactive
F251D mutant. Nuclear accumulation of the active
enzyme only correlated with histone H3K27 trimethylation
among the epigenetic modifications evaluated,
therefore pointing to the necessity of methionine adenosyltransferase
I/III to guarantee the supply of Sadenosylmethionine
for specific methylations. However,
nuclear monomers may exhibit additional roles.

Human Molecular Genetics, Jan 1, 2013

Hedgehog (Hh) signaling is involved in patterning and morphogenesis of most organs in the develop... more Hedgehog (Hh) signaling is involved in patterning and morphogenesis of most organs in the developing mammalian embryo. Despite many advances in understanding core components of the pathway, little is known about how the activity of the Hh pathway is adjusted in organ- and tissue-specific developmental processes. Mutations in EVC or EVC2 disrupt Hh signaling in tooth and bone development. Using mouse models, we show here that Evc and Evc2 are mutually required for localizing to primary cilia and also for maintaining their normal protein levels. Consistent with Evc and Evc2 functioning as a complex, the skeletal phenotypes in either single or double homozygous mutant mice are virtually indistinguishable. Smo translocation to the cilium was normal in Evc2-deficient chondrocytes following Hh activation with the Smo-agonist SAG. However, Gli3 recruitment to cilia tips was reduced and Sufu/Gli3 dissociation was impaired. Interestingly, we found Smo to co-precipitate with Evc/Evc2, indicating that in some cells Hh signaling requires direct interaction of Smo with the Evc/Evc2 complex. Expression of a dominantly acting Evc2 mutation previously identified in Weyer's acrodental dysostosis (Evc2Δ43) caused mislocalization of Evc/Evc2Δ43 within the cilium and also reproduced the Gli3-related molecular defects observed in Evc2(-/-) chondrocytes. Moreover, Evc silencing in Sufu(-/-) cells attenuated the output of the Hh pathway, suggesting that Evc/Evc2 also promote Hh signaling in the absence of Sufu. Together our data reveal that the Hh pathway involves Evc/Evc2-dependent modulations that are necessary for normal endochondral bone formation.

Faseb Journal, 2009

The methyl donor S-adenosylmethionine is synthesized in mammalian cytosol by three isoenzymes. Me... more The methyl donor S-adenosylmethionine is synthesized in mammalian cytosol by three isoenzymes. Methionine adenosyltransferase II is ubiquitously expressed, whereas isoenzymes I (homotetramer) and III (homodimer) are considered the hepatic enzymes. In this work, we identified methionine adenosyltransferase I/III in most rat tissues both in the cytoplasm and the nucleus. Nuclear localization was the preferred distribution observed in extrahepatic tissues, where the protein colocalizes with nuclear matrix markers. A battery of mutants used in several cell lines to decipher the determinants involved in methionine adenosyltransferase subcellular localization demonstrated, by confocal microscopy and subcellular fractionation, the presence of two partially overlapping areas at the C-terminal end of the protein involved both in cytoplasmic retention and nuclear localization. Immunoprecipitation of coexpressed FLAG and EGFP fusions and gel filtration chromatography allowed detection of tetramers and monomers in nuclear fractions that also exhibited S-adenosylmethionine synthesis. Neither nuclear localization nor matrix binding required activity, as demonstrated with the inactive F251D mutant. Nuclear accumulation of the active enzyme only correlated with histone H3K27 trimethylation among the epigenetic modifications evaluated, therefore pointing to the necessity of methionine adenosyltransferase I/III to guarantee the supply of S-adenosylmethionine for specific methylations. However, nuclear monomers may exhibit additional roles. Key words: S-adenosylmethionine synthetase, epigenetic modifications, structural determinants of localization, tissular expression, nuclear methylations. Control of gene expression and synthesis of adrenaline or phospholipids such as phosphatidylcholine are some examples of processes involving S-adenosylmethionine (AdoMet)dependent methylations . AdoMet also participates in other important reactions such as those catalyzed by SAM radical proteins (i.e. biotin synthesis) or, upon decarboxylation, in spermidine and spermine synthesis (2, 3). In fact, all the radicals surrounding the AdoMet sulfur atom can be donated, as well as the carboxyl and amino groups of the methionine and the ribosyl moiety (4, 5). Thus, the number of processes involving AdoMet has been calculated to be as large as that of reactions using ATP. This small positively charged compound is synthesized from methionine and ATP in a singular reaction catalyzed by methionine adenosyltransferases (MATs)(6). These enzymes are present in the cytoplasm of eukaryotic cells , though reactions using this compound exhibit a wider subcellular distribution. Transfer of AdoMet to other cell compartments is therefore needed and carried out by specific transporters (7). Changes in AdoMet levels have been detected in several diseases, ranging from Parkinson (8) to alcohol liver cirrhosis (9). However, a direct correlation between AdoMet levels and pathology was only obtained by generating MAT1A knockout mice, which spontaneously develop hepatocellular carcinoma (HCC)(10).

Biotechnology Techniques, 1997

Two single-step purification methods were used to isolate the recombinant protein, rBm86, produce... more Two single-step purification methods were used to isolate the recombinant protein, rBm86, produced in Pichia pastoris. Salting-out results in a compromise between final purity and recovery of rBm86. At 15, 25 and 35% of ammonium sulphate saturation (pH 7), rBm86 concentration in the supernatant phase was proportional to the initial amount of protein. Acid precipitation of contaminants resulted in 98% purity and 98% recovery of rBm86. High aggregation of rBm86, forming particles of 28 nm, changed the isoelectric point of monomers (5.5), considering only the aminoacid sequence, to 4.5 for particles.

Journal of Biotechnology, 2007

Previous literature addressing the production of recombinant proteins in heterologous systems has... more Previous literature addressing the production of recombinant proteins in heterologous systems has consistently shown that proteins capable of forming complex structures tend to accumulate within host cells at relatively higher levels than monomeric forms. In this report, we translationally fused a 21-aminoacids long highly immunogenic peptide (2L21), derived from canine parvovirus (CPV) VP2 protein to a 41-aminoacid long tetramerization domain (TD) from the transcriptional factor p53. The chimerical DNA construction 2L21-TD was cloned in a binary plant transformation vector and used to transform Arabidopsis thaliana plants. Fifteen of the 25 transgenic lines obtained in the experiment showed detectable 2L21-TD RNA accumulation and from these we chose 4 to study 2L21-TD protein accumulation. Non-denaturing immunoblotting assays revealed that 2L21-TD chimeras effectively formed tetrameric complexes with yields reaching up to 12 g/mg of soluble protein. Mice immunized by oral or intraperitoneal routes with crude protein extracts containing 2L21-TD protein were able to detect both 2L21-synthetic peptide and CPV VP2 proteins, with titers similar to those elicited by a previously reported fusion between 2L21 and the ␤-glucuronidase protein. These results demonstrate that multimerization directed by the small TD domain contributed to the stabilization and consequently to the accumulation of the 2L21 peptide in transgenic plants, without altering its native antigenicity and immunogenicity.

Biotechnology Techniques, 1998

A competitive enzyme linked immonosorbent assay (ELISA) was developed for quantification of recom... more A competitive enzyme linked immonosorbent assay (ELISA) was developed for quantification of recombinant Bm86 protein (rBm86), antigen of the vaccine GavacTM against the cattle tick Boophilus microplus. Monoclonal antibody (Mab) SSBm1 showed identical recognition to folded and denatured antigen and was used in the competition assay. This ELISA was sensitive enough to detect 60 ng/ml of rBm86. Within-assay coefficient of variation was 5.7 to 6.2 % and between-assay variation was 7.7 to 11.6 %. The level of expression of recombinant Bm86 in Pichia pastoris reached about 2.7 g per liter of culture after 80 hour of fermentation. © Rapid Science Ltd. 1998

Biochemical and Biophysical Research Communications, 2003

In this study, 13 samples of liver biopsies from patients with chronic hepatitis C were studied b... more In this study, 13 samples of liver biopsies from patients with chronic hepatitis C were studied by transmission electron microscopy (EM) and immunoelectron microscopy (IEM). The 13 biopsies showed ultrastructural cell damage typical of acute viral hepatitis. In four of the 13 liver biopsies enveloped virus-like particles (VLPs) inside cytoplasmic vesicles and in the cytoplasm of hepatocytes were observed. We also detected the presence of unenveloped VLPs mainly in the cytoplasm and in the endoplasmic reticulum. IEM using anti-core, E1 and E2 monoclonal antibodies (mAbs) confirmed the specific localization of these proteins, in vivo, inside cytoplasm and endoplasmic reticulum. Thus, this work provided evidence for hepatocellular injury related to HCV infection. It also suggested the presence of HCV-related replicating structures in the cytoplasm of hepatocytes and raised the possibility of hepatitis C virion morphogenesis in intracellular vesicles.

The methyl donor S-adenosylmethionine is synthesized in mammalian cytosol by three isoenzymes. Me... more The methyl donor S-adenosylmethionine
is synthesized in mammalian cytosol by three isoenzymes.
Methionine adenosyltransferase II is ubiquitously
expressed, whereas isoenzymes I (homotetramer)
and III (homodimer) are considered the hepatic
enzymes. In this work, we identified methionine adenosyltransferase
I/III in most rat tissues, both in the
cytoplasm and the nucleus. Nuclear localization was the
preferred distribution observed in extrahepatic tissues,
where the protein colocalizes with nuclear matrix markers.
A battery of mutants used in several cell lines to
decipher the determinants involved in methionine adenosyltransferase
subcellular localization demonstrated,
by confocal microscopy and subcellular fractionation,
the presence of two partially overlapping
areas at the C-terminal end of the protein involved both
in cytoplasmic retention and nuclear localization. Immunoprecipitation
of coexpressed FLAG and EGFP
fusions and gel-filtration chromatography allowed detection
of tetramers and monomers in nuclear fractions
that also exhibited S-adenosylmethionine synthesis.
Neither nuclear localization nor matrix binding required
activity, as demonstrated with the inactive
F251D mutant. Nuclear accumulation of the active
enzyme only correlated with histone H3K27 trimethylation
among the epigenetic modifications evaluated,
therefore pointing to the necessity of methionine adenosyltransferase
I/III to guarantee the supply of Sadenosylmethionine
for specific methylations. However,
nuclear monomers may exhibit additional roles.

Human Molecular Genetics, Jan 1, 2013

Hedgehog (Hh) signaling is involved in patterning and morphogenesis of most organs in the develop... more Hedgehog (Hh) signaling is involved in patterning and morphogenesis of most organs in the developing mammalian embryo. Despite many advances in understanding core components of the pathway, little is known about how the activity of the Hh pathway is adjusted in organ- and tissue-specific developmental processes. Mutations in EVC or EVC2 disrupt Hh signaling in tooth and bone development. Using mouse models, we show here that Evc and Evc2 are mutually required for localizing to primary cilia and also for maintaining their normal protein levels. Consistent with Evc and Evc2 functioning as a complex, the skeletal phenotypes in either single or double homozygous mutant mice are virtually indistinguishable. Smo translocation to the cilium was normal in Evc2-deficient chondrocytes following Hh activation with the Smo-agonist SAG. However, Gli3 recruitment to cilia tips was reduced and Sufu/Gli3 dissociation was impaired. Interestingly, we found Smo to co-precipitate with Evc/Evc2, indicating that in some cells Hh signaling requires direct interaction of Smo with the Evc/Evc2 complex. Expression of a dominantly acting Evc2 mutation previously identified in Weyer's acrodental dysostosis (Evc2Δ43) caused mislocalization of Evc/Evc2Δ43 within the cilium and also reproduced the Gli3-related molecular defects observed in Evc2(-/-) chondrocytes. Moreover, Evc silencing in Sufu(-/-) cells attenuated the output of the Hh pathway, suggesting that Evc/Evc2 also promote Hh signaling in the absence of Sufu. Together our data reveal that the Hh pathway involves Evc/Evc2-dependent modulations that are necessary for normal endochondral bone formation.

Faseb Journal, 2009

The methyl donor S-adenosylmethionine is synthesized in mammalian cytosol by three isoenzymes. Me... more The methyl donor S-adenosylmethionine is synthesized in mammalian cytosol by three isoenzymes. Methionine adenosyltransferase II is ubiquitously expressed, whereas isoenzymes I (homotetramer) and III (homodimer) are considered the hepatic enzymes. In this work, we identified methionine adenosyltransferase I/III in most rat tissues both in the cytoplasm and the nucleus. Nuclear localization was the preferred distribution observed in extrahepatic tissues, where the protein colocalizes with nuclear matrix markers. A battery of mutants used in several cell lines to decipher the determinants involved in methionine adenosyltransferase subcellular localization demonstrated, by confocal microscopy and subcellular fractionation, the presence of two partially overlapping areas at the C-terminal end of the protein involved both in cytoplasmic retention and nuclear localization. Immunoprecipitation of coexpressed FLAG and EGFP fusions and gel filtration chromatography allowed detection of tetramers and monomers in nuclear fractions that also exhibited S-adenosylmethionine synthesis. Neither nuclear localization nor matrix binding required activity, as demonstrated with the inactive F251D mutant. Nuclear accumulation of the active enzyme only correlated with histone H3K27 trimethylation among the epigenetic modifications evaluated, therefore pointing to the necessity of methionine adenosyltransferase I/III to guarantee the supply of S-adenosylmethionine for specific methylations. However, nuclear monomers may exhibit additional roles. Key words: S-adenosylmethionine synthetase, epigenetic modifications, structural determinants of localization, tissular expression, nuclear methylations. Control of gene expression and synthesis of adrenaline or phospholipids such as phosphatidylcholine are some examples of processes involving S-adenosylmethionine (AdoMet)dependent methylations . AdoMet also participates in other important reactions such as those catalyzed by SAM radical proteins (i.e. biotin synthesis) or, upon decarboxylation, in spermidine and spermine synthesis (2, 3). In fact, all the radicals surrounding the AdoMet sulfur atom can be donated, as well as the carboxyl and amino groups of the methionine and the ribosyl moiety (4, 5). Thus, the number of processes involving AdoMet has been calculated to be as large as that of reactions using ATP. This small positively charged compound is synthesized from methionine and ATP in a singular reaction catalyzed by methionine adenosyltransferases (MATs)(6). These enzymes are present in the cytoplasm of eukaryotic cells , though reactions using this compound exhibit a wider subcellular distribution. Transfer of AdoMet to other cell compartments is therefore needed and carried out by specific transporters (7). Changes in AdoMet levels have been detected in several diseases, ranging from Parkinson (8) to alcohol liver cirrhosis (9). However, a direct correlation between AdoMet levels and pathology was only obtained by generating MAT1A knockout mice, which spontaneously develop hepatocellular carcinoma (HCC)(10).

Biotechnology Techniques, 1997

Two single-step purification methods were used to isolate the recombinant protein, rBm86, produce... more Two single-step purification methods were used to isolate the recombinant protein, rBm86, produced in Pichia pastoris. Salting-out results in a compromise between final purity and recovery of rBm86. At 15, 25 and 35% of ammonium sulphate saturation (pH 7), rBm86 concentration in the supernatant phase was proportional to the initial amount of protein. Acid precipitation of contaminants resulted in 98% purity and 98% recovery of rBm86. High aggregation of rBm86, forming particles of 28 nm, changed the isoelectric point of monomers (5.5), considering only the aminoacid sequence, to 4.5 for particles.

Journal of Biotechnology, 2007

Previous literature addressing the production of recombinant proteins in heterologous systems has... more Previous literature addressing the production of recombinant proteins in heterologous systems has consistently shown that proteins capable of forming complex structures tend to accumulate within host cells at relatively higher levels than monomeric forms. In this report, we translationally fused a 21-aminoacids long highly immunogenic peptide (2L21), derived from canine parvovirus (CPV) VP2 protein to a 41-aminoacid long tetramerization domain (TD) from the transcriptional factor p53. The chimerical DNA construction 2L21-TD was cloned in a binary plant transformation vector and used to transform Arabidopsis thaliana plants. Fifteen of the 25 transgenic lines obtained in the experiment showed detectable 2L21-TD RNA accumulation and from these we chose 4 to study 2L21-TD protein accumulation. Non-denaturing immunoblotting assays revealed that 2L21-TD chimeras effectively formed tetrameric complexes with yields reaching up to 12 g/mg of soluble protein. Mice immunized by oral or intraperitoneal routes with crude protein extracts containing 2L21-TD protein were able to detect both 2L21-synthetic peptide and CPV VP2 proteins, with titers similar to those elicited by a previously reported fusion between 2L21 and the ␤-glucuronidase protein. These results demonstrate that multimerization directed by the small TD domain contributed to the stabilization and consequently to the accumulation of the 2L21 peptide in transgenic plants, without altering its native antigenicity and immunogenicity.

Biotechnology Techniques, 1998

A competitive enzyme linked immonosorbent assay (ELISA) was developed for quantification of recom... more A competitive enzyme linked immonosorbent assay (ELISA) was developed for quantification of recombinant Bm86 protein (rBm86), antigen of the vaccine GavacTM against the cattle tick Boophilus microplus. Monoclonal antibody (Mab) SSBm1 showed identical recognition to folded and denatured antigen and was used in the competition assay. This ELISA was sensitive enough to detect 60 ng/ml of rBm86. Within-assay coefficient of variation was 5.7 to 6.2 % and between-assay variation was 7.7 to 11.6 %. The level of expression of recombinant Bm86 in Pichia pastoris reached about 2.7 g per liter of culture after 80 hour of fermentation. © Rapid Science Ltd. 1998

Biochemical and Biophysical Research Communications, 2003

In this study, 13 samples of liver biopsies from patients with chronic hepatitis C were studied b... more In this study, 13 samples of liver biopsies from patients with chronic hepatitis C were studied by transmission electron microscopy (EM) and immunoelectron microscopy (IEM). The 13 biopsies showed ultrastructural cell damage typical of acute viral hepatitis. In four of the 13 liver biopsies enveloped virus-like particles (VLPs) inside cytoplasmic vesicles and in the cytoplasm of hepatocytes were observed. We also detected the presence of unenveloped VLPs mainly in the cytoplasm and in the endoplasmic reticulum. IEM using anti-core, E1 and E2 monoclonal antibodies (mAbs) confirmed the specific localization of these proteins, in vivo, inside cytoplasm and endoplasmic reticulum. Thus, this work provided evidence for hepatocellular injury related to HCV infection. It also suggested the presence of HCV-related replicating structures in the cytoplasm of hepatocytes and raised the possibility of hepatitis C virion morphogenesis in intracellular vesicles.