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Robert M Dores - One of the best experts on this subject based on the ideXlab platform.
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Tracking the evolution of the Proenkephalin gene in tetrapods.
General and Comparative Endocrinology, 2007Co-Authors: Erin Roberts, Stephanie Lecaude, Pezhman Shoureshi, Katarzyna Kozak, Laura Szynskie, Andrea Baron, Robert M DoresAbstract:In gnathostomes there is remarkable consistency in the organization of the Proenkephalin gene. This opioid precursor encodes seven opioid (YGGF) sequences: five pentapeptide sequences, a met-enkephalin-7 sequence and a met-enkephalin-8 sequence. Yet, within vertebrate lineages there can be distinct sets of pentapeptide opioids (YGGFM or YGGFL). In the Sarcopterygii, the sixth opioid position in lungfishes and anuran amphibian Proenkephalin genes encodes a met-enkephalin (YGGFM) sequence. However, in mammalian Proenkephalin there is a leu-enkephalin (YGGFL) sequence at this position. This study was done to test the hypothesis that the presence of the leu-enkephalin sequence in mammals is a feature common to amniote vertebrates, but not present in anamniote vertebrates. To resolve this issue, Proenkephalin cDNAs were cloned from the urodele amphibians, Amphiuma means and Necturus maculosus, and two amniote vertebrates, the turtle, Chrysemys scripta, and the brown snake, Storeria dekayi. As predicted, a met-enkephalin sequence is present at the sixth opioid position in urodele amphibians; whereas, a leu-enkephalin sequence is present at this opioid site in the reptile Proenkephalin sequences. These data are consistent with the conclusion that the transition from a met-enkephalin sequence to a leu-enkephalin sequence at the sixth opioid position in tetrapod Proenkephalins occurred in the ancestral proto-reptiles. Phylogenetic analyses, using the Maximum Parsimony and Neighbor-Joining algorithms, of the amphibian Proenkephalin sequences supported the position that anuran and urodele amphibians are a monophyletic assemblage. The same analysis of reptile-related Proenkephalin sequences, including the deduced amino acid sequence of a partially characterized alligator Proenkephalin cDNA, could not conclusively resolve the phylogeny of the major reptilian orders.
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Trends in the evolution of the Proenkephalin and prodynorphin genes in gnathostomes.
Annals of the New York Academy of Sciences, 2005Co-Authors: Ashley Khalap, Phillip B. Danielson, Stephanie Lecaude, Brian Bagrosky, John H. Youson, Robert M DoresAbstract:The opioid/orphanin gene family provides a model system for analyzing the outcomes of genome duplication events. Recent studies on the Proenkephalin gene provide additional evidence that the organizational plan for this gene has been conserved throughout the extensive radiation of the gnathostome vertebrates. However, an analysis of the amino acid sequence of Proenkephalin from the zebrafish, Danio rerio, suggests that novel forms of this opioid precursor may be evolving in teleosts. Analyses of sarcopterygian prodynorphin sequences revealed a Proenkephalin signature in prodynorphin. Current studies on the opioid/orphanin gene family point to the duplication events that shaped this family occurring prior to the radiation of the gnathostomes.
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Analyzing the radiation of the Proenkephalin gene in tetrapods: cloning of a Bombina orientalis Proenkephalin cDNA.
Peptides, 2001Co-Authors: Robert M Dores, David Costantino, Jeannine Walnutt, Phillip B. Danielson, Stephanie Lecaude, Stephanie LecaudeAbstract:Analyzing the Radiation of the Proenkephalin Gene in Tetrapods: Cloning of a Bombina orientalis Proenkephalin cDNA: A Proenkephalin cDNA was cloned from the brain of the anuran amphibian, Bombina orientalis (Family: Discoglossidae). This cDNA is 1358 nucleotides in length, and contains an open reading frame that codes for 251 amino acids. Within the open reading frame there are seven opioid (YGGF) sequences. There were five Met-enkephalin (YGGFM) sequences that are flanked by sets of paired basic amino acid proteolytic cleavage sites and two C-terminally extended Met-enkephalin sequences: YGGFMRGY and YGGFMRF. No Leu-enkephalin sequences were found in B. orientalis Proenkephalin. It was possible to align the amino acid sequences of Proenkephalin from several vertebrate taxa (human, Australian lungfish, B. orientalis, Xenopus laevis, Spea multiplicatus) by inserting a minimum of nine gaps. This alignment was then used to analyze the corresponding nucleotides for each Proenkephalin sequence using maximum likelihood. This analysis yielded a single tree. In this tree, the Australian lungfish sequence was the outgroup or the tetrapod ingroup. The amphibian sequences form a clade separate from the human sequence. The bootstrap value for the amphibian clade was 100%. Within the amphibian clade the Bombina sequence was the sister group to a clade composed of the X. laevis and S. multiplicatus sequences. The bootstrap value for the X. laevis/S. multiplicatus clade was 94%. Collectively, these data indicate that the sequence of Bombina Proenkephalin may be more similar to the proposed ancestral anuran Proenkephalin sequence, than either X. laevis or S. multiplicatus Proenkephalin.
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In the African Lungfish Met-Enkephalin and Leu-Enkephalin Are Derived from Separate Genes: Cloning of a Proenkephalin cDNA
Neuroendocrinology, 2000Co-Authors: Robert M Dores, Isabelle Lihrmann, Mauro Vallarino, Jenny Lee, Cristina Sollars, Phillip Danielson, Hubert VaudryAbstract:A full-length Proenkephalin cDNA (accession number: AF232670) was cloned from an African lungfish (Protopterus annectens) brain cDNA library. The 1,351-bp African lungfish Proenkephalin contains an open reading frame that codes 266 amino acids and a stop codon. Within the sequence of lungfish Proenkephalin there are 5 pentapeptide opioid sequences (all YGGFM), 1 octapeptide opioid sequence (YGGFMRSL) and 1 heptapeptide opioid sequence (YGGFMGY). A Leu-enkephalin sequence was conspicuously absent in lungfish Proenkephalin. These results, coupled with observations on the organization of amphibian Proenkephalin and mammalian Proenkephalin, indicate that among the Sarcopterygii (lobed finned fish and tetrapods), the appearance of a Leu-enkephalin sequence in Proenkephalin may have evolved in either the ancestral amniotes or the ancestral mammals, but not earlier in sarcopterygian evolution. Furthermore, the detection of neurons in the lungfish CNS that are only immunopositive for Met-enkephalin, coupled with earlier anatomical studies on the presence of neurons in the lungfish CNS that are only immunopositive for Leu-enkephalin, indicates that a Leu-enkephalin-coding opioid gene must be present in the CNS of the lungfish. This gene may be the lungfish form of prodynorphin. Given the phylogenetic position of the lungfish in vertebrate evolution, the putative Leu-enkephalin-coding gene must have evolved in the ancestral sarcopterygian vertebrates, or in the ancestral gnathostomes. The apparent slow rate of lungfish evolution makes these organisms interesting models for investigating the evolution of the opioid/orphanin gene family.
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Deciphering the origin of Met-enkephalin and Leu-enkephalin in Lobe-finned fish: cloning of australian lungfish Proenkephalin.
Brain research, 2000Co-Authors: Cristina Sollars, Phillip B. Danielson, Jean M.p. Joss, Robert M DoresAbstract:The previous detection of Met-enkephalin and Leu-enkephalin in the CNS of the Australian lungfish, Neoceratodus forsteri, in a molar ratio comparable to mammals suggested that the lungfish Proenkephalin precursor should contain the sequences of both Met-enkephalin and Leu-enkephalin as seen for mammalian Proenkephalin. However, the cloning of a full-length Proenkephalin cDNA from the CNS of the Australian lungfish indicates that the organization of this precursor is more similar to amphibian Proenkephalin than mammalian Proenkephalin. The Australian lungfish cDNA is 1284 nucleotides in length and the open reading frame (267 amino acids) contains seven opioid sequences (GenBank #AF232671). There are five copies of the Met-enkephalin sequence flanked by sets of paired basic amino acid proteolytic cleavage sites and two C-terminally extended forms of Met-enkephalin: YGGFMRSL and YGGFMGY. As seen for amphibians, no Leu-enkephalin sequence was detected in the Australian lungfish Proenkephalin cDNA. The fact that Leu-enkephalin has been identified by radioimmunoassay and HPLC analysis in the CNS of the Australian lungfish indicates that a Leu-enkephalin-coding gene, distinct from Proenkephalin, must be expressed in lungfish. Potential candidates may include a prodynorphin- or other opioid-like gene. Furthermore, the absence of a Leu-enkephalin sequence in lungfish and amphibian Proenkephalin would suggest that the mutations that yielded this opioid sequence in tetrapod Proenkephalin occurred at some point in the radiation of the amniote vertebrates.
Stephanie Lecaude - One of the best experts on this subject based on the ideXlab platform.
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Tracking the evolution of the Proenkephalin gene in tetrapods.
General and Comparative Endocrinology, 2007Co-Authors: Erin Roberts, Stephanie Lecaude, Pezhman Shoureshi, Katarzyna Kozak, Laura Szynskie, Andrea Baron, Robert M DoresAbstract:In gnathostomes there is remarkable consistency in the organization of the Proenkephalin gene. This opioid precursor encodes seven opioid (YGGF) sequences: five pentapeptide sequences, a met-enkephalin-7 sequence and a met-enkephalin-8 sequence. Yet, within vertebrate lineages there can be distinct sets of pentapeptide opioids (YGGFM or YGGFL). In the Sarcopterygii, the sixth opioid position in lungfishes and anuran amphibian Proenkephalin genes encodes a met-enkephalin (YGGFM) sequence. However, in mammalian Proenkephalin there is a leu-enkephalin (YGGFL) sequence at this position. This study was done to test the hypothesis that the presence of the leu-enkephalin sequence in mammals is a feature common to amniote vertebrates, but not present in anamniote vertebrates. To resolve this issue, Proenkephalin cDNAs were cloned from the urodele amphibians, Amphiuma means and Necturus maculosus, and two amniote vertebrates, the turtle, Chrysemys scripta, and the brown snake, Storeria dekayi. As predicted, a met-enkephalin sequence is present at the sixth opioid position in urodele amphibians; whereas, a leu-enkephalin sequence is present at this opioid site in the reptile Proenkephalin sequences. These data are consistent with the conclusion that the transition from a met-enkephalin sequence to a leu-enkephalin sequence at the sixth opioid position in tetrapod Proenkephalins occurred in the ancestral proto-reptiles. Phylogenetic analyses, using the Maximum Parsimony and Neighbor-Joining algorithms, of the amphibian Proenkephalin sequences supported the position that anuran and urodele amphibians are a monophyletic assemblage. The same analysis of reptile-related Proenkephalin sequences, including the deduced amino acid sequence of a partially characterized alligator Proenkephalin cDNA, could not conclusively resolve the phylogeny of the major reptilian orders.
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Trends in the evolution of the Proenkephalin and prodynorphin genes in gnathostomes.
Annals of the New York Academy of Sciences, 2005Co-Authors: Ashley Khalap, Phillip B. Danielson, Stephanie Lecaude, Brian Bagrosky, John H. Youson, Robert M DoresAbstract:The opioid/orphanin gene family provides a model system for analyzing the outcomes of genome duplication events. Recent studies on the Proenkephalin gene provide additional evidence that the organizational plan for this gene has been conserved throughout the extensive radiation of the gnathostome vertebrates. However, an analysis of the amino acid sequence of Proenkephalin from the zebrafish, Danio rerio, suggests that novel forms of this opioid precursor may be evolving in teleosts. Analyses of sarcopterygian prodynorphin sequences revealed a Proenkephalin signature in prodynorphin. Current studies on the opioid/orphanin gene family point to the duplication events that shaped this family occurring prior to the radiation of the gnathostomes.
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Analyzing the radiation of the Proenkephalin gene in tetrapods: cloning of a Bombina orientalis Proenkephalin cDNA.
Peptides, 2001Co-Authors: Robert M Dores, David Costantino, Jeannine Walnutt, Phillip B. Danielson, Stephanie Lecaude, Stephanie LecaudeAbstract:Analyzing the Radiation of the Proenkephalin Gene in Tetrapods: Cloning of a Bombina orientalis Proenkephalin cDNA: A Proenkephalin cDNA was cloned from the brain of the anuran amphibian, Bombina orientalis (Family: Discoglossidae). This cDNA is 1358 nucleotides in length, and contains an open reading frame that codes for 251 amino acids. Within the open reading frame there are seven opioid (YGGF) sequences. There were five Met-enkephalin (YGGFM) sequences that are flanked by sets of paired basic amino acid proteolytic cleavage sites and two C-terminally extended Met-enkephalin sequences: YGGFMRGY and YGGFMRF. No Leu-enkephalin sequences were found in B. orientalis Proenkephalin. It was possible to align the amino acid sequences of Proenkephalin from several vertebrate taxa (human, Australian lungfish, B. orientalis, Xenopus laevis, Spea multiplicatus) by inserting a minimum of nine gaps. This alignment was then used to analyze the corresponding nucleotides for each Proenkephalin sequence using maximum likelihood. This analysis yielded a single tree. In this tree, the Australian lungfish sequence was the outgroup or the tetrapod ingroup. The amphibian sequences form a clade separate from the human sequence. The bootstrap value for the amphibian clade was 100%. Within the amphibian clade the Bombina sequence was the sister group to a clade composed of the X. laevis and S. multiplicatus sequences. The bootstrap value for the X. laevis/S. multiplicatus clade was 94%. Collectively, these data indicate that the sequence of Bombina Proenkephalin may be more similar to the proposed ancestral anuran Proenkephalin sequence, than either X. laevis or S. multiplicatus Proenkephalin.
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Organization of Proenkephalin in amphibians: cloning of a Proenkephalin cDNA from the brain of the anuran amphibian, Spea multiplicatus.
Peptides, 2000Co-Authors: Stephanie Lecaude, Cristina Sollars, Phillip B. Danielson, Jasem Alrubaian, Catherine R. Propper, Robert M DoresAbstract:Cloning of a Proenkephalin cDNA from the pelobatid anuran amphibian, Spea multiplicatus, provides additional evidence that Leu-enkephalin, although present in the brain of anuran amphibians, is not encoded by the Proenkephalin gene. The S. multiplicatus Proenkephalin cDNA is 1375 nucleotides in length, and the open reading frame contains the sequences of seven opioid sequences. There are five copies of the Met-enkephalin sequence, as well as an octapeptide opioid sequence (YGGFMRNY) and a heptapeptide opioid sequence (YGGFMRF). In the Proenkephalin sequence of S. multiplicatus the penultimate opioid is a Met-enkephalin sequence rather than the Leu-enkephalin present in mammalian sequences. The same order of opioid sequences also is observed for the Proenkephalin sequence of the pipid anuran amphibian, Xenopus laevis. Hence, from a phylogenetic standpoint the organization of tetrapod Proenkephalin has been remarkably conserved. What remains to be resolved is whether the Leu-enkephalin sequence found in mammalian Proenkephalin is an ancestral trait or a derived trait for the tetrapods. Unlike the Proenkephalin precursor of X. laevis, all of the opioid sequences in the S. multiplicatus Proenkephalin cDNA are flanked by paired-basic amino acid proteolytic cleavage sites. In this regard the Proenkephalin sequence for S. multiplicatus is more similar to mammalian Proenkephalins than the Proenkephalin sequence of X. laevis. However, a comparison of the Proenkephalin sequences in human, X. laevis, and S. multiplicatus revealed several conserved features in the evolution of the tetrapod Proenkephalin gene. By contrast, a comparison of tetrapod Proenkephalin sequences with the partial sequence of a sturgeon Proenkephalin cDNA indicates that the position occupied by the penultimate opioid sequence in vertebrate Proenkephalins may be a highly variable locus in this gene.
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Cloning of Proopiomelanocortin from the Brain of the African Lungfish, Protopterus annectens, and the Brain of the Western Spadefoot Toad, Spea multiplicatus
Neuroendocrinology, 1999Co-Authors: Jenny Lee, Isabelle Lihrmann, Hubert Vaudry, Cristina Sollars, Phillip Danielson, Stephanie Lecaude, Jasem Alrubaian, Catherine Propper, Robert M DoresAbstract:A degenerate primer, specific for the opioid core sequence YGGFM, was used to clone and sequence proopiomelanocortin (POMC) cDNAs from the brain of the African lungfish, Protopterus annectens, and from the brain of the western spadefoot toad, Spea multiplicatus. In addition, the opioid-specific primer was used to clone and sequence a 3'RACE product corresponding to a portion of the open reading frame of S. multiplicatus Proenkephalin. For both species, cDNA was made from a single brain and a degenerate opioid-specific primer provided a reliable probe for detecting opioid-related cDNAs. The African lungfish POMC cDNA was 1,168 nucleotides in length, and contained regions that are similar to tetrapod POMCs and fish POMCs. The African lungfish POMC encodes a tetrapod-like gamma-MSH sequence that is flanked by sets of paired basic amino acid proteolytic cleavage sites. The gamma-MSH region in ray-finned fish POMCs either has degenerate cleavage sites or is totally absent in some species. However, the African lungfish gamma-MSH sequence does contain a deletion which has not been observed in tetrapod gamma-MSH sequences. The beta-endorphin region of lungfish POMC has the di-amino acid sequence tryptophan-aspartic acid in the N-terminal region and an additional glutamic acid residue in the C-terminal region of beta-endorphin - features found in fish beta-endorphin, but not tetrapod beta-endorphins. The western spadefoot toad POMC was 1,186 nucleotides in length, and exhibited an organizational scheme typical for tetrapod POMCs. However, the toad POMC did lack a paired basic amino acid proteolytic cleavage site N-terminal to the beta-MSH sequence. Thus, like rat POMC, it is doubtful that beta-MSH is an end product in either the toad brain or intermediate pituitary. At the amino acid level, the toad POMC had 76% sequence identity with Xenopus laevis POMC and 68% sequence identity with Rana ribidunda POMC. The use of these POMC sequences to assess phylogenetic relationships within anuran amphibians will be discussed. With respect to the fragment of S. multiplicatus Proenkephalin cDNA, two metenkephalin sequences and the metenkephalin-RF sequence were found encoded in this fragment. As seen for X. laevis and R. ridibunda Proenkephalin, a leuenkephalin sequence was not detected in the C-terminal region of the S. multiplicatus Proenkephalin. The absence of a leuenkephalin sequence may be a common feature of anuran amphibian Proenkephalins.
Cristina Sollars - One of the best experts on this subject based on the ideXlab platform.
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In the African Lungfish Met-Enkephalin and Leu-Enkephalin Are Derived from Separate Genes: Cloning of a Proenkephalin cDNA
Neuroendocrinology, 2000Co-Authors: Robert M Dores, Isabelle Lihrmann, Mauro Vallarino, Jenny Lee, Cristina Sollars, Phillip Danielson, Hubert VaudryAbstract:A full-length Proenkephalin cDNA (accession number: AF232670) was cloned from an African lungfish (Protopterus annectens) brain cDNA library. The 1,351-bp African lungfish Proenkephalin contains an open reading frame that codes 266 amino acids and a stop codon. Within the sequence of lungfish Proenkephalin there are 5 pentapeptide opioid sequences (all YGGFM), 1 octapeptide opioid sequence (YGGFMRSL) and 1 heptapeptide opioid sequence (YGGFMGY). A Leu-enkephalin sequence was conspicuously absent in lungfish Proenkephalin. These results, coupled with observations on the organization of amphibian Proenkephalin and mammalian Proenkephalin, indicate that among the Sarcopterygii (lobed finned fish and tetrapods), the appearance of a Leu-enkephalin sequence in Proenkephalin may have evolved in either the ancestral amniotes or the ancestral mammals, but not earlier in sarcopterygian evolution. Furthermore, the detection of neurons in the lungfish CNS that are only immunopositive for Met-enkephalin, coupled with earlier anatomical studies on the presence of neurons in the lungfish CNS that are only immunopositive for Leu-enkephalin, indicates that a Leu-enkephalin-coding opioid gene must be present in the CNS of the lungfish. This gene may be the lungfish form of prodynorphin. Given the phylogenetic position of the lungfish in vertebrate evolution, the putative Leu-enkephalin-coding gene must have evolved in the ancestral sarcopterygian vertebrates, or in the ancestral gnathostomes. The apparent slow rate of lungfish evolution makes these organisms interesting models for investigating the evolution of the opioid/orphanin gene family.
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Deciphering the origin of Met-enkephalin and Leu-enkephalin in Lobe-finned fish: cloning of australian lungfish Proenkephalin.
Brain research, 2000Co-Authors: Cristina Sollars, Phillip B. Danielson, Jean M.p. Joss, Robert M DoresAbstract:The previous detection of Met-enkephalin and Leu-enkephalin in the CNS of the Australian lungfish, Neoceratodus forsteri, in a molar ratio comparable to mammals suggested that the lungfish Proenkephalin precursor should contain the sequences of both Met-enkephalin and Leu-enkephalin as seen for mammalian Proenkephalin. However, the cloning of a full-length Proenkephalin cDNA from the CNS of the Australian lungfish indicates that the organization of this precursor is more similar to amphibian Proenkephalin than mammalian Proenkephalin. The Australian lungfish cDNA is 1284 nucleotides in length and the open reading frame (267 amino acids) contains seven opioid sequences (GenBank #AF232671). There are five copies of the Met-enkephalin sequence flanked by sets of paired basic amino acid proteolytic cleavage sites and two C-terminally extended forms of Met-enkephalin: YGGFMRSL and YGGFMGY. As seen for amphibians, no Leu-enkephalin sequence was detected in the Australian lungfish Proenkephalin cDNA. The fact that Leu-enkephalin has been identified by radioimmunoassay and HPLC analysis in the CNS of the Australian lungfish indicates that a Leu-enkephalin-coding gene, distinct from Proenkephalin, must be expressed in lungfish. Potential candidates may include a prodynorphin- or other opioid-like gene. Furthermore, the absence of a Leu-enkephalin sequence in lungfish and amphibian Proenkephalin would suggest that the mutations that yielded this opioid sequence in tetrapod Proenkephalin occurred at some point in the radiation of the amniote vertebrates.
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Organization of Proenkephalin in amphibians: cloning of a Proenkephalin cDNA from the brain of the anuran amphibian, Spea multiplicatus.
Peptides, 2000Co-Authors: Stephanie Lecaude, Cristina Sollars, Phillip B. Danielson, Jasem Alrubaian, Catherine R. Propper, Robert M DoresAbstract:Cloning of a Proenkephalin cDNA from the pelobatid anuran amphibian, Spea multiplicatus, provides additional evidence that Leu-enkephalin, although present in the brain of anuran amphibians, is not encoded by the Proenkephalin gene. The S. multiplicatus Proenkephalin cDNA is 1375 nucleotides in length, and the open reading frame contains the sequences of seven opioid sequences. There are five copies of the Met-enkephalin sequence, as well as an octapeptide opioid sequence (YGGFMRNY) and a heptapeptide opioid sequence (YGGFMRF). In the Proenkephalin sequence of S. multiplicatus the penultimate opioid is a Met-enkephalin sequence rather than the Leu-enkephalin present in mammalian sequences. The same order of opioid sequences also is observed for the Proenkephalin sequence of the pipid anuran amphibian, Xenopus laevis. Hence, from a phylogenetic standpoint the organization of tetrapod Proenkephalin has been remarkably conserved. What remains to be resolved is whether the Leu-enkephalin sequence found in mammalian Proenkephalin is an ancestral trait or a derived trait for the tetrapods. Unlike the Proenkephalin precursor of X. laevis, all of the opioid sequences in the S. multiplicatus Proenkephalin cDNA are flanked by paired-basic amino acid proteolytic cleavage sites. In this regard the Proenkephalin sequence for S. multiplicatus is more similar to mammalian Proenkephalins than the Proenkephalin sequence of X. laevis. However, a comparison of the Proenkephalin sequences in human, X. laevis, and S. multiplicatus revealed several conserved features in the evolution of the tetrapod Proenkephalin gene. By contrast, a comparison of tetrapod Proenkephalin sequences with the partial sequence of a sturgeon Proenkephalin cDNA indicates that the position occupied by the penultimate opioid sequence in vertebrate Proenkephalins may be a highly variable locus in this gene.
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Cloning of Proopiomelanocortin from the Brain of the African Lungfish, Protopterus annectens, and the Brain of the Western Spadefoot Toad, Spea multiplicatus
Neuroendocrinology, 1999Co-Authors: Jenny Lee, Isabelle Lihrmann, Hubert Vaudry, Cristina Sollars, Phillip Danielson, Stephanie Lecaude, Jasem Alrubaian, Catherine Propper, Robert M DoresAbstract:A degenerate primer, specific for the opioid core sequence YGGFM, was used to clone and sequence proopiomelanocortin (POMC) cDNAs from the brain of the African lungfish, Protopterus annectens, and from the brain of the western spadefoot toad, Spea multiplicatus. In addition, the opioid-specific primer was used to clone and sequence a 3'RACE product corresponding to a portion of the open reading frame of S. multiplicatus Proenkephalin. For both species, cDNA was made from a single brain and a degenerate opioid-specific primer provided a reliable probe for detecting opioid-related cDNAs. The African lungfish POMC cDNA was 1,168 nucleotides in length, and contained regions that are similar to tetrapod POMCs and fish POMCs. The African lungfish POMC encodes a tetrapod-like gamma-MSH sequence that is flanked by sets of paired basic amino acid proteolytic cleavage sites. The gamma-MSH region in ray-finned fish POMCs either has degenerate cleavage sites or is totally absent in some species. However, the African lungfish gamma-MSH sequence does contain a deletion which has not been observed in tetrapod gamma-MSH sequences. The beta-endorphin region of lungfish POMC has the di-amino acid sequence tryptophan-aspartic acid in the N-terminal region and an additional glutamic acid residue in the C-terminal region of beta-endorphin - features found in fish beta-endorphin, but not tetrapod beta-endorphins. The western spadefoot toad POMC was 1,186 nucleotides in length, and exhibited an organizational scheme typical for tetrapod POMCs. However, the toad POMC did lack a paired basic amino acid proteolytic cleavage site N-terminal to the beta-MSH sequence. Thus, like rat POMC, it is doubtful that beta-MSH is an end product in either the toad brain or intermediate pituitary. At the amino acid level, the toad POMC had 76% sequence identity with Xenopus laevis POMC and 68% sequence identity with Rana ribidunda POMC. The use of these POMC sequences to assess phylogenetic relationships within anuran amphibians will be discussed. With respect to the fragment of S. multiplicatus Proenkephalin cDNA, two metenkephalin sequences and the metenkephalin-RF sequence were found encoded in this fragment. As seen for X. laevis and R. ridibunda Proenkephalin, a leuenkephalin sequence was not detected in the C-terminal region of the S. multiplicatus Proenkephalin. The absence of a leuenkephalin sequence may be a common feature of anuran amphibian Proenkephalins.
Anahit V Azaryan - One of the best experts on this subject based on the ideXlab platform.
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the processing proteases prohormone thiol protease pc1 3 and pc2 and 70 kda aspartic proteinase show preferences among Proenkephalin proneuropeptide y and proopiomelanocortin substrates
Archives of Biochemistry and Biophysics, 1996Co-Authors: Vivian Hook, Martin R Schiller, Anahit V AzaryanAbstract:Abstract Proteases of cysteine, aspartic, and subtilisin classes have been indicated as candidate prohormone processing enzymes. The chromaffin granule Proenkephalin processing proteases have been characterized as the novel cysteine protease prohormone thiol protease (PTP), a 70-kDa aspartic proteinase, and the subtilisin-like PC1/3 and PC2 enzymes. The goal of this study was to assess whether these processing proteases possess preference(s) for prohormone substrates. The recombinant prohormones Proenkephalin, proneuropeptide Y (pro-NPY), and proopiomelanocortin (POMC) were expressed in Escherichia coli using the T7 expression system and purified for in vitro processing studies. Results indicated that the chromaffin granule processing proteases possess selectivity for particular prohormones. PTP preferred Proenkephalin, with good cleavage of pro-NPY and slow processing of POMC. In contrast, the 70-kDa aspartic proteinase cleaved POMC most readily, with cleavage of Proenkephalin and some processing of pro-NPY. PC1/3 and PC2 preferred POMC among the prohormones tested. Importantly, these results indicate that prohormone selectivity of processing proteases may be an important factor in predicting the primary and rate-limiting protease(s) required for processing a particular prohormone.
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The processing proteases prohormone thiol protease, PC1/3 and PC2, and 70-kDa aspartic proteinase show preferences among Proenkephalin, proneuropeptide Y, and proopiomelanocortin substrates.
Archives of biochemistry and biophysics, 1996Co-Authors: Vivian Hook, Martin R Schiller, Anahit V AzaryanAbstract:Abstract Proteases of cysteine, aspartic, and subtilisin classes have been indicated as candidate prohormone processing enzymes. The chromaffin granule Proenkephalin processing proteases have been characterized as the novel cysteine protease prohormone thiol protease (PTP), a 70-kDa aspartic proteinase, and the subtilisin-like PC1/3 and PC2 enzymes. The goal of this study was to assess whether these processing proteases possess preference(s) for prohormone substrates. The recombinant prohormones Proenkephalin, proneuropeptide Y (pro-NPY), and proopiomelanocortin (POMC) were expressed in Escherichia coli using the T7 expression system and purified for in vitro processing studies. Results indicated that the chromaffin granule processing proteases possess selectivity for particular prohormones. PTP preferred Proenkephalin, with good cleavage of pro-NPY and slow processing of POMC. In contrast, the 70-kDa aspartic proteinase cleaved POMC most readily, with cleavage of Proenkephalin and some processing of pro-NPY. PC1/3 and PC2 preferred POMC among the prohormones tested. Importantly, these results indicate that prohormone selectivity of processing proteases may be an important factor in predicting the primary and rate-limiting protease(s) required for processing a particular prohormone.
Vivian Hook - One of the best experts on this subject based on the ideXlab platform.
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α1 antichymotrypsin like proteins i and ii purified from bovine adrenal medulla are enriched in chromaffin granules and inhibit the Proenkephalin processing enzyme prohormone thiol protease
Journal of Neurochemistry, 2002Co-Authors: Vivian Hook, Nikolaos Tezapsidis, Shinrong Hwang, Catherine Sei, Michael Byrne, Sukkid YasothornsrikulAbstract:Abstract: Proteolytic processing of inactive Proenkephalin and proneuropeptides is essential for the production of biologically active enkephalins and many neuropeptides. The incomplete processing of Proenkephalin in adrenal medulla suggests that endogenous protease inhibitors may inhibit Proenkephalin processing enzymes. This study demonstrates the isolation and characterization of two isoforms of adrenal medullary α1-antichymotrypsin (ACT), referred to as ACT-like proteins I and II, which are colocalized with enkephalin in chromaffin granules and which inhibit the Proenkephalin processing enzyme known as prohormone thiol protease (PTP). Subcellular fractionation demonstrated enrichment of 56- and 60-kDa ACT-like proteins I and II, respectively, to enkephalin-containing chromaffin granules (secretory vesicles). Immunofluorescence cytochemistry of chromaffin cells indicated a discrete, punctate pattern of ACT immunostaining that resembles that of [Met]enkephalin that is stored in secretory vesicles. Chromatography of adrenal medullary extracts through DEAE-Sepharose and chromatofocusing resulted in the separation of ACT-like proteins I and II that possess different isoelectric points of 5.5 and 4.0, respectively. The 56-kDa ACT-like protein I was purified to apparent homogeneity by Sephacryl S200 chromatography; the 60-kDa ACT-like protein II was isolated by butyl-Sepharose, Sephacryl S200, and concanavalin A-Sepharose columns. The Proenkephalin processing enzyme PTP was potently inhibited by ACT-like protein I, with a Ki,app of 35 nM, but ACT-like protein II was less effective. ACT-like proteins I and II had little effect on chymotrypsin. These results demonstrate the biochemical identification of two secretory vesicle ACT-like proteins that differentially inhibit PTP. The colocalization of the ACT-like proteins and PTP within chromaffin granules indicates that they could interact in vivo. Results from this study suggest that these ACT-like proteins may be considered as candidate inhibitors of PTP, which could provide a mechanism for limited Proenkephalin processing in adrenal medulla.
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the processing proteases prohormone thiol protease pc1 3 and pc2 and 70 kda aspartic proteinase show preferences among Proenkephalin proneuropeptide y and proopiomelanocortin substrates
Archives of Biochemistry and Biophysics, 1996Co-Authors: Vivian Hook, Martin R Schiller, Anahit V AzaryanAbstract:Abstract Proteases of cysteine, aspartic, and subtilisin classes have been indicated as candidate prohormone processing enzymes. The chromaffin granule Proenkephalin processing proteases have been characterized as the novel cysteine protease prohormone thiol protease (PTP), a 70-kDa aspartic proteinase, and the subtilisin-like PC1/3 and PC2 enzymes. The goal of this study was to assess whether these processing proteases possess preference(s) for prohormone substrates. The recombinant prohormones Proenkephalin, proneuropeptide Y (pro-NPY), and proopiomelanocortin (POMC) were expressed in Escherichia coli using the T7 expression system and purified for in vitro processing studies. Results indicated that the chromaffin granule processing proteases possess selectivity for particular prohormones. PTP preferred Proenkephalin, with good cleavage of pro-NPY and slow processing of POMC. In contrast, the 70-kDa aspartic proteinase cleaved POMC most readily, with cleavage of Proenkephalin and some processing of pro-NPY. PC1/3 and PC2 preferred POMC among the prohormones tested. Importantly, these results indicate that prohormone selectivity of processing proteases may be an important factor in predicting the primary and rate-limiting protease(s) required for processing a particular prohormone.
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The processing proteases prohormone thiol protease, PC1/3 and PC2, and 70-kDa aspartic proteinase show preferences among Proenkephalin, proneuropeptide Y, and proopiomelanocortin substrates.
Archives of biochemistry and biophysics, 1996Co-Authors: Vivian Hook, Martin R Schiller, Anahit V AzaryanAbstract:Abstract Proteases of cysteine, aspartic, and subtilisin classes have been indicated as candidate prohormone processing enzymes. The chromaffin granule Proenkephalin processing proteases have been characterized as the novel cysteine protease prohormone thiol protease (PTP), a 70-kDa aspartic proteinase, and the subtilisin-like PC1/3 and PC2 enzymes. The goal of this study was to assess whether these processing proteases possess preference(s) for prohormone substrates. The recombinant prohormones Proenkephalin, proneuropeptide Y (pro-NPY), and proopiomelanocortin (POMC) were expressed in Escherichia coli using the T7 expression system and purified for in vitro processing studies. Results indicated that the chromaffin granule processing proteases possess selectivity for particular prohormones. PTP preferred Proenkephalin, with good cleavage of pro-NPY and slow processing of POMC. In contrast, the 70-kDa aspartic proteinase cleaved POMC most readily, with cleavage of Proenkephalin and some processing of pro-NPY. PC1/3 and PC2 preferred POMC among the prohormones tested. Importantly, these results indicate that prohormone selectivity of processing proteases may be an important factor in predicting the primary and rate-limiting protease(s) required for processing a particular prohormone.
