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Reudelhuber T. L. - One of the best experts on this subject based on the ideXlab platform.
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The molecular biology of human renin and its gene.
Yale Journal of Biology and Medicine, 2026Co-Authors: Baxter J D, James M. N., Chu W. N., Duncan K., Haidar M. A., Carilli C. T., Reudelhuber T. L.Abstract:The molecular biology of renin, prorenin, and the renin gene have been studied. A tissue-specific pattern of expression was found in rat and human tissues. In the human placenta, the transfected and endogenous renin promoters are active, and renin mRNA levels and transfected promoter activity are increased by a calcium ionophore plus cAMP. Cultured pituitary AtT-20 cells transfected with a Preprorenin expression vector mimick renal renin release by converting prorenin to renin and releasing renin in response to 8Br-cAMP. Studies with mutant renin genes suggest that the body of renin directs renin to the regulated secretory pathway, and renin glycosylation affects its trafficking. Chinese hamster ovary cells were used to produce recombinant prorenin. Infused prorenin was not converted to renin in monkeys. Renin crystals were used to determine its three-dimensional structure. Renin resembles other aspartyl proteases in the active site and core, but it differs in other regions that probably explain renin's unique substrate specificity. Based on structural and mutational analysis, a model for human prorenin was built that suggests lysine -2 of the prosegment interacts with active site aspartate residues, and that the prosegment inactivation of renin is stabilized by binding of an amino terminal beta strand into a groove on renin
Mendes, Dulcyane Neiva - One of the best experts on this subject based on the ideXlab platform.
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Construção de baculovírus recombinantes contendo o gene pré-pró-renina humana
2009Co-Authors: Mendes, Dulcyane NeivaAbstract:Baculovírus são vírus de insetos que têm sido utilizados como bioinseticidas para controle de pragas agrícolas, bem como importante ferramenta em Biotecnologia como vetor de expressão gênica. O sistema de expressão utilizando baculovírus em células de insetos fornece um ambiente apropriado para a síntese de proteínas eucarióticas. A renina humana, uma proteína produzida principalmente pelos rins e responsável pelo controle da pressão sangüínea e do balanço salino, é a chave principal para o desencadeamento do sistema renina-angiotensina-aldosterona que culmina no aumento da pressão sangüínea. Neste trabalho é proposta a produção da renina, com base na construção de baculovírus recombinantes, visando o estudo mais aprofundado tanto de suas propriedades quanto de inibidores que possam ser empregados na indústria farmacêutica. Inicialmente dois baculovírus recombinantes contendo o gene da pré-pró-renina humana foram construídos. No sistema vSyn VI-gal o fragmento da pré-pró-renina (~1,4 Kb) foi inserido no vetor de expressão pSynXIV VI+X3 e por recombinação homóloga com DNA viral levou a formação de vírus recombinante ocluso positivo. No sistema Bac-to-Bac (Invitrogen) o fragmento da pré-pró-renina foi inserido em bacmídeo, por transposição, e levou a formação de vírus recombinante ocluso negativo. A presença do gene da pré-pró-renina nos vírus recombinantes foi confirmada após amplificação do gene por reação em cadeia da polimerase (PCR) com os primers específicos da pró-renina. Para identificação das proteínas virais produzidas durante a infecção foi feita cinética da síntese de proteínas por marcação radioativa (35S-metionina), em células de inseto High five e Sf21, em diferentes tempos pós-infecção (0, 24, 48 e 72 h). Na fase muito tardia da infecção foi observada a síntese de duas proteínas de cerca de 40 kDa e 47 kDa, que correspondem ao peso molecular esperado para a pró-renina e a renina, ou formas glicosiladas da mesma. Como esperado, a análise das proteínas por ensaio imunológico, utilizando anticorpo policlonal contra a renina, mostrou sinal de hibridização nas amostras das células infectadas com os vírus recombinantes contendo o gene da pré-pró-renina (Sf21 e High five). Embora nas células High five não infectadas (mock infected) não tenha havido marcação com o anticorpo, foi, entretanto, detectado sinal de hibridização não esperado em células Sf21 não infectadas (mock infected), o que deve ser devido à hibridização inespecífica. A análise ultraestrutural das células infectadas com vírus recombinante revelou que houve indução de efeitos citopáticos típicos por infecção com baculovírus. Entretanto, no vírus recombinante ocluso positivo parece não ter havido a correta inclusão dos vírions na matriz protéica resultando na formação de poliedros com pouco ou nenhum vírion em seu interior. Finalmente, larvas de Anticarsia gemmatalis e de Spodoptera frugiperda foram infectadas para investigar as alterações morfológicas causadas pelos vírus. Sinais claros de que houve mudanças na fisiologia larval foram observados quando, além dos sintomas da doença, a lagarta infectada apresentou mudança de coloração do tegumento para cor rosa. Essa coloração não é observada na lagarta infectada com o vírus selvagem AcMNPV. As alterações ultraestruturais apresentadas pelas células e as mudanças na coloração da cutícula das larvas infectadas mostram que está havendo diferença na expressão protéica entre o vírus selvagem e os vírus recombinantes. Os resultados obtidos até o momento indicam ter havido a expressão de uma proteína com peso molecular esperado para a renina e que estaria interferindo, de alguma forma, no processo de oclusão da partícula viral bem como na fisiologia do inseto. __________________________________________________________________________________________ ABSTRACTBaculovirus are insect viruses being used as bioinsecticides for agricultural pest control as well as an important tool in Biotechnology as gene expression vector. The baculovirus expression system in insect cells gives an appropriate environment for eukaryotic proteins synthesis. The human renin is a protein produced mainly in the kidney and responsible for blood pressure and salt balance controls and is the principal key to the role of reninangiotensin- aldosteron system that culminates in the blood pressure rise. In this work the renin production is proposed, based on the construction of recombinant baculoviruses, aiming the studies development of the protein properties as well as the inhibitors that may be used in the pharmaceutical industry. Initially, two recombinant baculoviruses containing the Preprorenin gene were constructed. In the vSyn VI-gal system the Preprorenin fragment (~1.4 Kb) was inserted in the pSynXIV VI+X3 vector, homologous recombined with the viral DNA, leading to an occluded virus assembly. In the Bac-to-Bac system (Invitrogen) the Preprorenin fragment was inserted in the bacmid, by transposition, leading to a negative occluded virus assembly. The presence of the Preprorenin gene in the recombinant viruses was confirmed by its amplification by Polymerase Chain Reaction (PCR) technique with specific primers for prorenin. For identification of the viral proteins produced during infection, the kinetics of protein synthesis was done by radioactive labeling (35S-metionin), in High five and Sf21 insect cells, at different times post infection (0, 24, 48 and 72h). In the very late phase of infection the synthesis of two proteins about 40 kDa and 47 kDa was observed. These polypeptides correspond to the molecular weight expected for prorenin and renin, or any of their corresponding glycosilated forms. Protein analysis by immunological assay, using polyclonal antibodies against renin, showed a hybridization signal in infected cells samples with the recombinant viruses containing the Preprorenin gene (Sf21 and High five). Although there was no hybridization signal in mock infected High five cells, there was an unexpected signal detected in mock infected Sf21 cells, which could be somehow due to unspecific labeling of the antibody. Ultrastructural analyses of infected cells showed that there was induction of typical cytophatic effects with baculovirus infection. However, with the occluded recombinant virus it seemed that there was no correct virion inclusion in the protein matrix resulting in polyhedra formation with few or none virions inside. Finally, Anticarsia gemmatalis and Spodoptera frugiperda larva were infected to further investigate the morphological alterations caused by the viruses. Clear signs of larval physiology change were observed when in addition to the common symptoms of the disease the infected larvae presented a tegument change to a pinkish color. This color is not observed in larvae infected with the wild type AcMNPV. The ultrastructural alterations in infected cells and the change in the color cuticle of infected larvae show that there is a protein expression difference between the wild type and the Preprorenin recombinant viruses. The data obtained until now indicate that there is a protein being expressed with the expected molecular weight for renin and that it is somehow interfering with the occlusion process of the viral particle as well as with the insect physiology
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Construção de baculovírus recombinantes contendo o gene pré-pró-renina humana
2006Co-Authors: Mendes, Dulcyane NeivaAbstract:Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, 2006.Baculovírus são vírus de insetos que têm sido utilizados como bioinseticidas para controle de pragas agrícolas, bem como importante ferramenta em Biotecnologia como vetor de expressão gênica. O sistema de expressão utilizando baculovírus em células de insetos fornece um ambiente apropriado para a síntese de proteínas eucarióticas. A renina humana, uma proteína produzida principalmente pelos rins e responsável pelo controle da pressão sangüínea e do balanço salino, é a chave principal para o desencadeamento do sistema renina-angiotensina-aldosterona que culmina no aumento da pressão sangüínea. Neste trabalho é proposta a produção da renina, com base na construção de baculovírus recombinantes, visando o estudo mais aprofundado tanto de suas propriedades quanto de inibidores que possam ser empregados na indústria farmacêutica. Inicialmente dois baculovírus recombinantes contendo o gene da pré-pró-renina humana foram construídos. No sistema vSyn VI-gal o fragmento da pré-pró-renina (~1,4 Kb) foi inserido no vetor de expressão pSynXIV VI+X3 e por recombinação homóloga com DNA viral levou a formação de vírus recombinante ocluso positivo. No sistema Bac-to-Bac (Invitrogen) o fragmento da pré-pró-renina foi inserido em bacmídeo, por transposição, e levou a formação de vírus recombinante ocluso negativo. A presença do gene da pré-pró-renina nos vírus recombinantes foi confirmada após amplificação do gene por reação em cadeia da polimerase (PCR) com os primers específicos da pró-renina. Para identificação das proteínas virais produzidas durante a infecção foi feita cinética da síntese de proteínas por marcação radioativa (35S-metionina), em células de inseto High five e Sf21, em diferentes tempos pós-infecção (0, 24, 48 e 72 h). Na fase muito tardia da infecção foi observada a síntese de duas proteínas de cerca de 40 kDa e 47 kDa, que correspondem ao peso molecular esperado para a pró-renina e a renina, ou formas glicosiladas da mesma. Como esperado, a análise das proteínas por ensaio imunológico, utilizando anticorpo policlonal contra a renina, mostrou sinal de hibridização nas amostras das células infectadas com os vírus recombinantes contendo o gene da pré-pró-renina (Sf21 e High five). Embora nas células High five não infectadas (mock infected) não tenha havido marcação com o anticorpo, foi, entretanto, detectado sinal de hibridização não esperado em células Sf21 não infectadas (mock infected), o que deve ser devido à hibridização inespecífica. A análise ultraestrutural das células infectadas com vírus recombinante revelou que houve indução de efeitos citopáticos típicos por infecção com baculovírus. Entretanto, no vírus recombinante ocluso positivo parece não ter havido a correta inclusão dos vírions na matriz protéica resultando na formação de poliedros com pouco ou nenhum vírion em seu interior. Finalmente, larvas de Anticarsia gemmatalis e de Spodoptera frugiperda foram infectadas para investigar as alterações morfológicas causadas pelos vírus. Sinais claros de que houve mudanças na fisiologia larval foram observados quando, além dos sintomas da doença, a lagarta infectada apresentou mudança de coloração do tegumento para cor rosa. Essa coloração não é observada na lagarta infectada com o vírus selvagem AcMNPV. As alterações ultraestruturais apresentadas pelas células e as mudanças na coloração da cutícula das larvas infectadas mostram que está havendo diferença na expressão protéica entre o vírus selvagem e os vírus recombinantes. Os resultados obtidos até o momento indicam ter havido a expressão de uma proteína com peso molecular esperado para a renina e que estaria interferindo, de alguma forma, no processo de oclusão da partícula viral bem como na fisiologia do inseto. __________________________________________________________________________________________ ABSTRACTBaculovirus are insect viruses being used as bioinsecticides for agricultural pest control as well as an important tool in Biotechnology as gene expression vector. The baculovirus expression system in insect cells gives an appropriate environment for eukaryotic proteins synthesis. The human renin is a protein produced mainly in the kidney and responsible for blood pressure and salt balance controls and is the principal key to the role of reninangiotensin- aldosteron system that culminates in the blood pressure rise. In this work the renin production is proposed, based on the construction of recombinant baculoviruses, aiming the studies development of the protein properties as well as the inhibitors that may be used in the pharmaceutical industry. Initially, two recombinant baculoviruses containing the Preprorenin gene were constructed. In the vSyn VI-gal system the Preprorenin fragment (~1.4 Kb) was inserted in the pSynXIV VI+X3 vector, homologous recombined with the viral DNA, leading to an occluded virus assembly. In the Bac-to-Bac system (Invitrogen) the Preprorenin fragment was inserted in the bacmid, by transposition, leading to a negative occluded virus assembly. The presence of the Preprorenin gene in the recombinant viruses was confirmed by its amplification by Polymerase Chain Reaction (PCR) technique with specific primers for prorenin. For identification of the viral proteins produced during infection, the kinetics of protein synthesis was done by radioactive labeling (35S-metionin), in High five and Sf21 insect cells, at different times post infection (0, 24, 48 and 72h). In the very late phase of infection the synthesis of two proteins about 40 kDa and 47 kDa was observed. These polypeptides correspond to the molecular weight expected for prorenin and renin, or any of their corresponding glycosilated forms. Protein analysis by immunological assay, using polyclonal antibodies against renin, showed a hybridization signal in infected cells samples with the recombinant viruses containing the Preprorenin gene (Sf21 and High five). Although there was no hybridization signal in mock infected High five cells, there was an unexpected signal detected in mock infected Sf21 cells, which could be somehow due to unspecific labeling of the antibody. Ultrastructural analyses of infected cells showed that there was induction of typical cytophatic effects with baculovirus infection. However, with the occluded recombinant virus it seemed that there was no correct virion inclusion in the protein matrix resulting in polyhedra formation with few or none virions inside. Finally, Anticarsia gemmatalis and Spodoptera frugiperda larva were infected to further investigate the morphological alterations caused by the viruses. Clear signs of larval physiology change were observed when in addition to the common symptoms of the disease the infected larvae presented a tegument change to a pinkish color. This color is not observed in larvae infected with the wild type AcMNPV. The ultrastructural alterations in infected cells and the change in the color cuticle of infected larvae show that there is a protein expression difference between the wild type and the Preprorenin recombinant viruses. The data obtained until now indicate that there is a protein being expressed with the expected molecular weight for renin and that it is somehow interfering with the occlusion process of the viral particle as well as with the insect physiology
Baxter J D - One of the best experts on this subject based on the ideXlab platform.
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The molecular biology of human renin and its gene.
Yale Journal of Biology and Medicine, 2026Co-Authors: Baxter J D, James M. N., Chu W. N., Duncan K., Haidar M. A., Carilli C. T., Reudelhuber T. L.Abstract:The molecular biology of renin, prorenin, and the renin gene have been studied. A tissue-specific pattern of expression was found in rat and human tissues. In the human placenta, the transfected and endogenous renin promoters are active, and renin mRNA levels and transfected promoter activity are increased by a calcium ionophore plus cAMP. Cultured pituitary AtT-20 cells transfected with a Preprorenin expression vector mimick renal renin release by converting prorenin to renin and releasing renin in response to 8Br-cAMP. Studies with mutant renin genes suggest that the body of renin directs renin to the regulated secretory pathway, and renin glycosylation affects its trafficking. Chinese hamster ovary cells were used to produce recombinant prorenin. Infused prorenin was not converted to renin in monkeys. Renin crystals were used to determine its three-dimensional structure. Renin resembles other aspartyl proteases in the active site and core, but it differs in other regions that probably explain renin's unique substrate specificity. Based on structural and mutational analysis, a model for human prorenin was built that suggests lysine -2 of the prosegment interacts with active site aspartate residues, and that the prosegment inactivation of renin is stabilized by binding of an amino terminal beta strand into a groove on renin
Jorg Peters - One of the best experts on this subject based on the ideXlab platform.
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Printed in U.S.A. Copyright © 2000 by The Endocrine Society Tissue-Specific Expression of a Rat Renin Transcript Lacking the Coding Sequence for the Prefragment and Its Stimulation by Myocardial Infarction*
2013Co-Authors: Susanne Clausmeyer, Raphaela Farrenkopf, Alexander Reinecke, Thomas Unger, Jorg PetersAbstract:An alternative transcript of the rat renin gene was recently characterized in the adrenal gland, in addition to the known messenger RNA (mRNA) coding for Preprorenin. In the alternative transcript, exon 1 is replaced by exon 1A, a domain originating in intron 1. The reading frame of this mRNA, termed exon 1A-renin transcript, codes for a truncated prorenin that presumably remains intracellular, in contrast to Preprorenin, which is targeted to the secretory pathway by its prefragment. We here demonstrate the tissue-specific regulation of expression of both transcripts by RT and PCR. In many tissues both transcripts are present, for example in the adrenal gland, spleen, liver, and hypothalamus. In some organs, however, only one of the RENIN, THE KEY enzyme of the renin-angiotensin system (RAS), is generally known as a secretory protein, which is synthesized and secreted predominantly by th
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functional significance of prorenin internalization in the rat heart
Circulation Research, 2002Co-Authors: Jorg Peters, Raphaela Farrenkopf, Susanne Clausmeyer, Jutta Zimmer, Surasak Kantachuvesiri, Matthew G F Sharp, John J MullinsAbstract:Intracardiac renin is considered to be involved in the pathogenesis of cardiac hypertrophy, fibrosis, and myocardial infarction. Cardiac renin is predominantly derived from the circulation, because Preprorenin is not expressed locally and uptake of renin has been demonstrated. One mechanism of internalization recently described involves the mannose-6-phosphate receptor and requires glycosylation of renin. Based on previous observations, we considered the existence of another pathway of uptake, not requiring glycosylation and predominantly involving prorenin. This hypothesis and its functional consequences were investigated in vitro and in vivo. We demonstrate that isolated adult cardiomyocytes internalize unglycosylated prorenin, which is followed by the generation of angiotensins. We further show that transgenic rats, expressing the ren-2(d) renin gene in an inducible manner, exhibit markedly enhanced levels of unglycosylated renin within intracellular compartments in the heart as a consequence of the induction of hepatic transgene expression and the rise of circulating unglycosylated prorenin levels. Because in this model severe cardiac damage occurs as a consequence of the rise of circulating prorenin levels, internalization of prorenin into cardiac cells is likely to play a key role in this process.
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an alternative transcript of the rat renin gene can result in a truncated prorenin that is transported into adrenal mitochondria
Circulation Research, 1999Co-Authors: Susanne Clausmeyer, Raphaela Sturzebecher, Jorg PetersAbstract:Characterization of the local renin-angiotensin system in the rat adrenal zona glomerulosa indicated a dual targeting of renin both to the secretory pathway and mitochondria. To investigate the transport of renin into mitochondria, we constructed a series of amino-terminal deletion variants of Preprorenin. One of these variants, lacking the complete signal sequence for the endoplasmic reticulum and 10 amino acids of the profragment, was transported efficiently into isolated mitochondria. The transport was further shown to be dependent on mitochondrial membrane potential and ATP synthesis. Analysis of adrenal RNA revealed the existence of 2 renin transcripts. While one of the transcripts corresponds to the known full-length transcript, the other one lacks exon 1; instead, exon 2 is preceded by a domain of 80 nucleotides originating from intron 1. This domain, as well as the following region of intron 1 being excised, shows all essential sequence elements defining an additional, so-far-unknown exon. The second mRNA possibly derives from an additional transcription start in intron 1 and an alternative splicing process. Translation of this mRNA could result in a truncated prorenin representing a cytosolic form of renin, which is required for transport into mitochondria. This truncated prorenin corresponds exactly to the deletion variant being imported into mitochondria in vitro.
James M. N. - One of the best experts on this subject based on the ideXlab platform.
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The molecular biology of human renin and its gene.
Yale Journal of Biology and Medicine, 2026Co-Authors: Baxter J D, James M. N., Chu W. N., Duncan K., Haidar M. A., Carilli C. T., Reudelhuber T. L.Abstract:The molecular biology of renin, prorenin, and the renin gene have been studied. A tissue-specific pattern of expression was found in rat and human tissues. In the human placenta, the transfected and endogenous renin promoters are active, and renin mRNA levels and transfected promoter activity are increased by a calcium ionophore plus cAMP. Cultured pituitary AtT-20 cells transfected with a Preprorenin expression vector mimick renal renin release by converting prorenin to renin and releasing renin in response to 8Br-cAMP. Studies with mutant renin genes suggest that the body of renin directs renin to the regulated secretory pathway, and renin glycosylation affects its trafficking. Chinese hamster ovary cells were used to produce recombinant prorenin. Infused prorenin was not converted to renin in monkeys. Renin crystals were used to determine its three-dimensional structure. Renin resembles other aspartyl proteases in the active site and core, but it differs in other regions that probably explain renin's unique substrate specificity. Based on structural and mutational analysis, a model for human prorenin was built that suggests lysine -2 of the prosegment interacts with active site aspartate residues, and that the prosegment inactivation of renin is stabilized by binding of an amino terminal beta strand into a groove on renin