M. Monsalve - Academia.edu (original) (raw)

Papers by M. Monsalve

Research paper thumbnail of Failure in mitochondrial biogenesis causes mtDNA depletion in secondary biliary cirrhosis in rats

Free Radical Research, 2007

Research paper thumbnail of PGC-1α regulates TLS activity: Role in oxidative stress gene expression

Free Radical Biology and Medicine, 2012

Research paper thumbnail of Transcription Activation and Repression by Interaction of a Regulator with the α Subunit of RNA Polymerase: The Model of Phage ϕ29 Protein p4

Progress in Nucleic Acid Research and Molecular Biology, 1998

Regulatory protein p4, encoded by Bacillus subtilis phage phi 29, has proved to be a very useful ... more Regulatory protein p4, encoded by Bacillus subtilis phage phi 29, has proved to be a very useful model to analyze the molecular mechanisms of transcription regulation. Protein p4 modulates the transcription of phage phi 29 genome by activating the late A3 promoter (PA3) and simultaneously repressing the two main early promoters, A2b and A2c (or PA2b and PA2c). This review describes in detail the regulatory mechanism leading to activation or repression, and discusses them in the context of the recent findings on the role of the RNA polymerase alpha subunit in transcription regulation. Activation of PA3 implies the p4-mediated stabilization of RNA polymerase at the promoter as a closed complex. Repression of the early A2b promoter occurs by binding of protein p4 to a site that partially overlaps the -35 consensus region of the promoter, therefore preventing the binding of RNA polymerase to the promoter. Repression of the A2c promoter, located 96 bp downstream from PA2b, occurs by a different mechanism that implies the simultaneous binding of protein p4 and RNA polymerase to the promoter in such a way that promoter clearance is inhibited. Interestingly, activation of PA3 and repression of PA2c require an interaction between protein p4 and RNA polymerase, and in both cases this interaction occurs between the same surface of protein p4 and the C-terminal domain of the alpha subunit of RNA polymerase, which provides new insights into how a protein can activate or repress transcription by subtle variations in the protein-DNA complexes formed at promoters.

Research paper thumbnail of Transcription Activation or Repression by Phage Φ29 Protein p4 Depends on the Strength of the RNA Polymerase–Promoter Interactions

Molecular Cell, 1997

1992), or the escape of RNAP from the promoter (Monsalve et al., 1996a). At least in some of thes... more 1992), or the escape of RNAP from the promoter (Monsalve et al., 1996a). At least in some of these cases, repression involves an interaction between the repressor and the RNAP ␣ subunit (Choy et al., 1995; Monsalve et al., 1996b).

Research paper thumbnail of Transcriptional activator of phage ?29 late promoter: mapping of residues involved in interaction with RNA polymerase and in DNA bending

Molecular Microbiology, 1996

Phage @29 regulatory protein p4 activates transcription from the late A3 promoter by stabilizing ... more Phage @29 regulatory protein p4 activates transcription from the late A3 promoter by stabilizing aA-RNA polymerase at the promoter as a closed complex. Activation requires interaction between both proteins. Protein p4 bends the DNA upon binding. We have performed a detailed mutagenesis study of the carboxyl end of the protein, which is involved in both transcription activation and DNA bending. The results indicate that Arg-120 is the most critical residue for activation, probably mediating the interaction with RNA polymerase. Several basic residues have been identified, including Arg-120, that contribute to maintenance of the DNA bending, probably via electrostatic interactions with the DNA backbone. The degree or stability of the induced bend apparently relies on the additive contribution of all basic residues of the carboxyl end of the protein. Therefore, the activation and DNA bending surfaces overlap, and Arg-120 should interact with both DNA and RNA polymerase. As we show that protein p4 is a dimer in solution, and is bound to DNA as a tetramer, the results suggest a model in which two of the p4 subunits interact with the DNA, bending it, while the other two subunits remain accessible to interact with RNA polymerase.

Research paper thumbnail of Functional Analysis of TFIID Components

Cold Spring Harbor Symposia on Quantitative Biology, 1998

... In higher eukaryotes, TBP interacts with hTAFII250 (Takada et al. 1992; Hisatake et al. 1993;... more ... In higher eukaryotes, TBP interacts with hTAFII250 (Takada et al. 1992; Hisatake et al. 1993;Kokubo et al. 1993; Ruppert et al. 1993; Zhou et al. ... Mol. Cell 1: 895. Kokubo T., Gong D.-W., Yamashita S., Horikoshi M., Roeder RG, and Nakatani Y. 1993. ...

Research paper thumbnail of Direct Coupling of Transcription and mRNA Processing through the Thermogenic Coactivator PGC-1

Molecular Cell, 2000

The notion of transcription and processing as coordinated events has led to the proposition that ... more The notion of transcription and processing as coordinated events has led to the proposition that there might also be mechanisms that allow transcription factors to

Research paper thumbnail of Mitochondrial dysfunction in human pathologies

Frontiers in Bioscience, 2007

Research paper thumbnail of Perspective: Mitochondria-ER Contacts in Metabolic Cellular Stress Assessed by Microscopy

Cells, 2018

The interplay of mitochondria with the endoplasmic reticulum and their connections, called mitoch... more The interplay of mitochondria with the endoplasmic reticulum and their connections, called mitochondria-ER contacts (MERCs) or mitochondria-associated ER membranes (MAMs), are crucial hubs in cellular stress. These sites are essential for the passage of calcium ions, reactive oxygen species delivery, the sorting of lipids in whole-body metabolism. In this perspective article, we focus on microscopic evidences of the pivotal role of MERCs/MAMs and their changes in metabolic diseases, like obesity, diabetes, and neurodegeneration.

Research paper thumbnail of European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS)

Redox biology, Oct 18, 2017

The European Cooperation in Science and Technology (COST) provides an ideal framework to establis... more The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associat...

Research paper thumbnail of The non-canonical NOTCH ligand DLK1 exhibits a novel vascular role as a strong inhibitor of angiogenesis

Cardiovascular Research, 2011

The epidermal growth factor-like protein Delta-like 1 (DLK1) regulates multiple differentiation p... more The epidermal growth factor-like protein Delta-like 1 (DLK1) regulates multiple differentiation processes. It resembles NOTCH ligands structurally and is considered a non-canonical ligand. Given the crucial role of the NOTCH pathway in angiogenesis, we hypothesized that DLK1 could regulate angiogenesis by interfering with NOTCH. We therefore investigated the expression and function of DLK1 in the vascular endothelium and its role in the regulation of angiogenesis. Methods and results We report DLK1 expression in the endothelium of different species, including human, cow, pig, and mouse. Angiogenesis was studied by using in vitro and in vivo models of angiotube formation in endothelial cells, retinal phenotypes in Dlk1-null mice, and vessel development in zebrafish. DLK1 overexpression strongly inhibited angiotube formation, whereas lung endothelial cells from Dlk1-null mice were highly angiogenic. In vivo studies demonstrated DLK1mediated inhibition of neovessel formation and revealed an altered pattern of angiogenesis in the retinas of Dlk1null mice. The expression of human DLK1 in zebrafish embryos severely altered the formation of intersegmental vessels, while knockdown of the orthologous gene was associated with ectopic and increased tumour-induced angiogenesis. NOTCH-dependent signalling as determined by gene expression reporters was inhibited by the presence of DLK1 in vascular endothelial cells. In contrast, Dlk1-null mice showed increased levels of NOTCH downstream targets, such as Snail and Slug. Conclusion Our results unveil a novel inhibitory role for DLK1 in the regulation of angiogenesis, mediated by antagonism of the NOTCH pathway, and establish the basis for investigating its action in pathological settings.

Research paper thumbnail of Melatonin Effects on Non-Alcoholic Fatty Liver Disease Are Related to MicroRNA-34a-5p/Sirt1 Axis and Autophagy

Cells, 2019

Melatonin, an indole produced by pineal and extrapineal tissues, but also taken with a vegetarian... more Melatonin, an indole produced by pineal and extrapineal tissues, but also taken with a vegetarian diet, has strong anti-oxidant, anti-inflammatory and anti-obesogenic potentials. Non-alcoholic fatty liver disease (NAFLD) is the hepatic side of the metabolic syndrome. NAFLD is a still reversible phase but may evolve into steatohepatitis (NASH), cirrhosis and carcinoma. Currently, an effective therapy for blocking NAFLD staging is lacking. Silent information regulator 1 (SIRT1), a NAD+ dependent histone deacetylase, modulates the energetic metabolism in the liver. Micro-RNA-34a-5p, a direct inhibitor of SIRT1, is an emerging indicator of NAFLD grading. Thus, here we analyzed the effects of oral melatonin against NAFLD and underlying molecular mechanisms, focusing on steatosis, ER stress, mitochondrial shape and autophagy. Male C57BL/6J (WT) and SIRT1 heterozygous (HET) mice were placed either on a high-fat diet (58.4% energy from lard) (HFD) or on a standard maintenance diet (8.4% ene...

Research paper thumbnail of Activation and repression of transcription at two different phage phi29 promoters are mediated by interaction of the same residues of regulatory protein p4 with RNA polymerase

The EMBO Journal, 1996

Phage 029 regulatory protein p4 activates transcription from the late A3 promoter and represses t... more Phage 029 regulatory protein p4 activates transcription from the late A3 promoter and represses the main early promoters, named A2b and A2c. Activation involves stabilization of RNA polymerase (RNAP) at the A3 promoter as a closed complex and is mediated by interaction between RNAP and a small domain of protein p4 in which residue Argl20 plays an essential role. We show that protein p4 represses the A2c promoter by binding to DNA immediately upstream from RNAP in a way that does not hinder RNAP binding; rather, the two proteins bind cooperatively to DNA. In the presence of protein p4, RNAP can form an initiated complex at the A2c promoter that generates short abortive transcripts, but cannot leave the promoter. Mutation of protein p4 residue Argl20, which relieves the contact between the two proteins, leads to a loss of repression. Therefore, the contact between protein p4 and RNAP through the protein p4 domain containing Argl20 can activate or repress transcription, depending on the promoter. The relative position of protein p4 and RNAP, which is different at each promoter, together with the distinct characteristics of the two promoters, may determine whether protein p4 activates or represses transcription.

Research paper thumbnail of Transcription Regulation in Bacillus subtilis Phage Φ29: Expression of the Viral Promoters throughout the Infection Cycle

Research paper thumbnail of Substitution of the C-terminal domain of the Escherichia coli RNA polymerase α subunit by that from Bacillus subtilis makes the enzyme responsive to a Bacillus subtilis transcriptional activator

Journal of Molecular Biology, 1998

Regulatory protein p4 of Bacillus subtilis phage È29 activates transcription from the viral late ... more Regulatory protein p4 of Bacillus subtilis phage È29 activates transcription from the viral late A3 promoter by interacting with the C-terminal domain (CTD) of the B. subtilis RNA polymerase a subunit, thereby stabilizing the holoenzyme at the promoter. Protein p4 does not interact with the Escherichia coli RNA polymerase and cannot activate transcription with this enzyme. We have constructed a chimerical a subunit containing the N-terminal domain of the E. coli a subunit and the CTD of the B. subtilis a subunit. Reconstitution of RNA polymerases containing this chimerical a subunit, the E. coli b and b H subunits, and the vegetative s factor from either E. coli (s 70) or B. subtilis (s A), generated hybrid enzymes that were responsive to protein p4 and ef®ciently supported activation at the A3 promoter. Protein p4 activated transcription with the chimerical enzymes through the same activation surface used with B. subtilis RNA polymerase. Therefore, the B. subtilis a-CTD allowed activation by p4 even when the rest of the RNA polymerase subunits belonged to E. coli, a distantly related bacterium. These results strongly suggest that protein p4 works essentially by serving as an anchor that stabilizes RNA polymerase at the promoter.

Research paper thumbnail of The switch from early to late transcription in phage GA-1: characterization of the regulatory protein p4G

Journal of Molecular Biology, 1999

Research paper thumbnail of Binding of phage Φ29 protein p4 to the early A2c promoter: recruitment of a repressor by the RNA polymerase

Journal of Molecular Biology, 1998

Regulatory protein p4 from Bacillus subtilis phage È29 represses the early A2c promoter by bindin... more Regulatory protein p4 from Bacillus subtilis phage È29 represses the early A2c promoter by binding upstream from RNA polymerase and interacting with the C-terminal domain of the RNA polymerase a subunit. This interaction stabilizes the RNA polymerase at the promoter in such a way that promoter clearance is prevented. Here, the binding of protein p4 to the A2c promoter has been studied. In the absence of RNA polymerase, protein p4 was found to bind with low af®nity to a site centered at position À39 relative to the transcription start site. When RNA polymerase was present, protein p4 was displaced from this site and bound instead to a different target centered at position À71. Stable binding to this site requires the interaction of protein p4 with the C-terminal domain of the RNA polymerase a-subunit. Both sites contain sequences resembling the well-characterized p4 binding site present at the late A3 promoter, to which p4 binds with high af®nity. A mutational analysis revealed that the site at À71 is critical for a stable interaction between protein p4 and RNA polymerase, and for ef®cient repression, whereas mutation of the site at À39 had only a small effect on repression ef®ciency. Therefore, RNA polymerase plays an active role in the repression mechanism by stabilizing the repressor at the promoter, generating a nucleoprotein complex that is too stable to allow promoter clearance.

Research paper thumbnail of A mutation in the C-terminal domain of the RNA polymerase alpha subunit that destabilizes the open complexes formed at the phage φ29 late A3 promoter

Journal of Molecular Biology, 2001

Regulatory protein p4 from Bacillus subtilis phage f29 activates the viral late A3 promoter mainl... more Regulatory protein p4 from Bacillus subtilis phage f29 activates the viral late A3 promoter mainly by stabilizing the binding of RNA polymerase (RNAP) to it as a closed complex. This requires an interaction between protein p4 residue Arg120 and the C-terminal domain (CTD) of the RNAP a subunit. Several acidic residues of the a-CTD, considered as plausible targets for p4 residue Arg120, were individually changed into alanine. In addition, a truncated a subunit lacking the last four residues, two of which are acidic, was obtained. The modi®ed a subunits were puri®ed and reconstituted into RNAP holoenzyme in vitro. Protein p4 was found to be unable to activate the late A3 promoter when residue Glu297 of the a subunit was changed to Ala, a modi®cation that did not impair transcription from several other promoters. Interestingly, protein p4 could stabilize the modi®ed RNAP at the A3 promoter as a closed complex, although the open complexes formed were unstable and did not proceed to elongation complexes. Our results indicate that the change of the a residue Glu297 into Ala destabilizes the open complexes formed at this promoter, but not at other promoters. Considered in the context of earlier ®ndings indicating that the RNAP a-CTD may participate in the transition from closed to intermediate complexes at some other promoters, the new results expand and clarify our view of its role in transcription initiation.

Research paper thumbnail of PGC-1α regulates the mitochondrial antioxidant defense system in vascular endothelial cells

Cardiovascular Research, 2005

Research paper thumbnail of European Contribution to the study of ROS: A Summary of the Findings and Prospects for the Future from the COST Action BM1203 (EU-ROS)

The European Cooperation in Science and Technology (COST) provides an ideal framework to establis... more The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associat...

Research paper thumbnail of Failure in mitochondrial biogenesis causes mtDNA depletion in secondary biliary cirrhosis in rats

Free Radical Research, 2007

Research paper thumbnail of PGC-1α regulates TLS activity: Role in oxidative stress gene expression

Free Radical Biology and Medicine, 2012

Research paper thumbnail of Transcription Activation and Repression by Interaction of a Regulator with the α Subunit of RNA Polymerase: The Model of Phage ϕ29 Protein p4

Progress in Nucleic Acid Research and Molecular Biology, 1998

Regulatory protein p4, encoded by Bacillus subtilis phage phi 29, has proved to be a very useful ... more Regulatory protein p4, encoded by Bacillus subtilis phage phi 29, has proved to be a very useful model to analyze the molecular mechanisms of transcription regulation. Protein p4 modulates the transcription of phage phi 29 genome by activating the late A3 promoter (PA3) and simultaneously repressing the two main early promoters, A2b and A2c (or PA2b and PA2c). This review describes in detail the regulatory mechanism leading to activation or repression, and discusses them in the context of the recent findings on the role of the RNA polymerase alpha subunit in transcription regulation. Activation of PA3 implies the p4-mediated stabilization of RNA polymerase at the promoter as a closed complex. Repression of the early A2b promoter occurs by binding of protein p4 to a site that partially overlaps the -35 consensus region of the promoter, therefore preventing the binding of RNA polymerase to the promoter. Repression of the A2c promoter, located 96 bp downstream from PA2b, occurs by a different mechanism that implies the simultaneous binding of protein p4 and RNA polymerase to the promoter in such a way that promoter clearance is inhibited. Interestingly, activation of PA3 and repression of PA2c require an interaction between protein p4 and RNA polymerase, and in both cases this interaction occurs between the same surface of protein p4 and the C-terminal domain of the alpha subunit of RNA polymerase, which provides new insights into how a protein can activate or repress transcription by subtle variations in the protein-DNA complexes formed at promoters.

Research paper thumbnail of Transcription Activation or Repression by Phage Φ29 Protein p4 Depends on the Strength of the RNA Polymerase–Promoter Interactions

Molecular Cell, 1997

1992), or the escape of RNAP from the promoter (Monsalve et al., 1996a). At least in some of thes... more 1992), or the escape of RNAP from the promoter (Monsalve et al., 1996a). At least in some of these cases, repression involves an interaction between the repressor and the RNAP ␣ subunit (Choy et al., 1995; Monsalve et al., 1996b).

Research paper thumbnail of Transcriptional activator of phage ?29 late promoter: mapping of residues involved in interaction with RNA polymerase and in DNA bending

Molecular Microbiology, 1996

Phage @29 regulatory protein p4 activates transcription from the late A3 promoter by stabilizing ... more Phage @29 regulatory protein p4 activates transcription from the late A3 promoter by stabilizing aA-RNA polymerase at the promoter as a closed complex. Activation requires interaction between both proteins. Protein p4 bends the DNA upon binding. We have performed a detailed mutagenesis study of the carboxyl end of the protein, which is involved in both transcription activation and DNA bending. The results indicate that Arg-120 is the most critical residue for activation, probably mediating the interaction with RNA polymerase. Several basic residues have been identified, including Arg-120, that contribute to maintenance of the DNA bending, probably via electrostatic interactions with the DNA backbone. The degree or stability of the induced bend apparently relies on the additive contribution of all basic residues of the carboxyl end of the protein. Therefore, the activation and DNA bending surfaces overlap, and Arg-120 should interact with both DNA and RNA polymerase. As we show that protein p4 is a dimer in solution, and is bound to DNA as a tetramer, the results suggest a model in which two of the p4 subunits interact with the DNA, bending it, while the other two subunits remain accessible to interact with RNA polymerase.

Research paper thumbnail of Functional Analysis of TFIID Components

Cold Spring Harbor Symposia on Quantitative Biology, 1998

... In higher eukaryotes, TBP interacts with hTAFII250 (Takada et al. 1992; Hisatake et al. 1993;... more ... In higher eukaryotes, TBP interacts with hTAFII250 (Takada et al. 1992; Hisatake et al. 1993;Kokubo et al. 1993; Ruppert et al. 1993; Zhou et al. ... Mol. Cell 1: 895. Kokubo T., Gong D.-W., Yamashita S., Horikoshi M., Roeder RG, and Nakatani Y. 1993. ...

Research paper thumbnail of Direct Coupling of Transcription and mRNA Processing through the Thermogenic Coactivator PGC-1

Molecular Cell, 2000

The notion of transcription and processing as coordinated events has led to the proposition that ... more The notion of transcription and processing as coordinated events has led to the proposition that there might also be mechanisms that allow transcription factors to

Research paper thumbnail of Mitochondrial dysfunction in human pathologies

Frontiers in Bioscience, 2007

Research paper thumbnail of Perspective: Mitochondria-ER Contacts in Metabolic Cellular Stress Assessed by Microscopy

Cells, 2018

The interplay of mitochondria with the endoplasmic reticulum and their connections, called mitoch... more The interplay of mitochondria with the endoplasmic reticulum and their connections, called mitochondria-ER contacts (MERCs) or mitochondria-associated ER membranes (MAMs), are crucial hubs in cellular stress. These sites are essential for the passage of calcium ions, reactive oxygen species delivery, the sorting of lipids in whole-body metabolism. In this perspective article, we focus on microscopic evidences of the pivotal role of MERCs/MAMs and their changes in metabolic diseases, like obesity, diabetes, and neurodegeneration.

Research paper thumbnail of European contribution to the study of ROS: A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS)

Redox biology, Oct 18, 2017

The European Cooperation in Science and Technology (COST) provides an ideal framework to establis... more The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associat...

Research paper thumbnail of The non-canonical NOTCH ligand DLK1 exhibits a novel vascular role as a strong inhibitor of angiogenesis

Cardiovascular Research, 2011

The epidermal growth factor-like protein Delta-like 1 (DLK1) regulates multiple differentiation p... more The epidermal growth factor-like protein Delta-like 1 (DLK1) regulates multiple differentiation processes. It resembles NOTCH ligands structurally and is considered a non-canonical ligand. Given the crucial role of the NOTCH pathway in angiogenesis, we hypothesized that DLK1 could regulate angiogenesis by interfering with NOTCH. We therefore investigated the expression and function of DLK1 in the vascular endothelium and its role in the regulation of angiogenesis. Methods and results We report DLK1 expression in the endothelium of different species, including human, cow, pig, and mouse. Angiogenesis was studied by using in vitro and in vivo models of angiotube formation in endothelial cells, retinal phenotypes in Dlk1-null mice, and vessel development in zebrafish. DLK1 overexpression strongly inhibited angiotube formation, whereas lung endothelial cells from Dlk1-null mice were highly angiogenic. In vivo studies demonstrated DLK1mediated inhibition of neovessel formation and revealed an altered pattern of angiogenesis in the retinas of Dlk1null mice. The expression of human DLK1 in zebrafish embryos severely altered the formation of intersegmental vessels, while knockdown of the orthologous gene was associated with ectopic and increased tumour-induced angiogenesis. NOTCH-dependent signalling as determined by gene expression reporters was inhibited by the presence of DLK1 in vascular endothelial cells. In contrast, Dlk1-null mice showed increased levels of NOTCH downstream targets, such as Snail and Slug. Conclusion Our results unveil a novel inhibitory role for DLK1 in the regulation of angiogenesis, mediated by antagonism of the NOTCH pathway, and establish the basis for investigating its action in pathological settings.

Research paper thumbnail of Melatonin Effects on Non-Alcoholic Fatty Liver Disease Are Related to MicroRNA-34a-5p/Sirt1 Axis and Autophagy

Cells, 2019

Melatonin, an indole produced by pineal and extrapineal tissues, but also taken with a vegetarian... more Melatonin, an indole produced by pineal and extrapineal tissues, but also taken with a vegetarian diet, has strong anti-oxidant, anti-inflammatory and anti-obesogenic potentials. Non-alcoholic fatty liver disease (NAFLD) is the hepatic side of the metabolic syndrome. NAFLD is a still reversible phase but may evolve into steatohepatitis (NASH), cirrhosis and carcinoma. Currently, an effective therapy for blocking NAFLD staging is lacking. Silent information regulator 1 (SIRT1), a NAD+ dependent histone deacetylase, modulates the energetic metabolism in the liver. Micro-RNA-34a-5p, a direct inhibitor of SIRT1, is an emerging indicator of NAFLD grading. Thus, here we analyzed the effects of oral melatonin against NAFLD and underlying molecular mechanisms, focusing on steatosis, ER stress, mitochondrial shape and autophagy. Male C57BL/6J (WT) and SIRT1 heterozygous (HET) mice were placed either on a high-fat diet (58.4% energy from lard) (HFD) or on a standard maintenance diet (8.4% ene...

Research paper thumbnail of Activation and repression of transcription at two different phage phi29 promoters are mediated by interaction of the same residues of regulatory protein p4 with RNA polymerase

The EMBO Journal, 1996

Phage 029 regulatory protein p4 activates transcription from the late A3 promoter and represses t... more Phage 029 regulatory protein p4 activates transcription from the late A3 promoter and represses the main early promoters, named A2b and A2c. Activation involves stabilization of RNA polymerase (RNAP) at the A3 promoter as a closed complex and is mediated by interaction between RNAP and a small domain of protein p4 in which residue Argl20 plays an essential role. We show that protein p4 represses the A2c promoter by binding to DNA immediately upstream from RNAP in a way that does not hinder RNAP binding; rather, the two proteins bind cooperatively to DNA. In the presence of protein p4, RNAP can form an initiated complex at the A2c promoter that generates short abortive transcripts, but cannot leave the promoter. Mutation of protein p4 residue Argl20, which relieves the contact between the two proteins, leads to a loss of repression. Therefore, the contact between protein p4 and RNAP through the protein p4 domain containing Argl20 can activate or repress transcription, depending on the promoter. The relative position of protein p4 and RNAP, which is different at each promoter, together with the distinct characteristics of the two promoters, may determine whether protein p4 activates or represses transcription.

Research paper thumbnail of Transcription Regulation in Bacillus subtilis Phage Φ29: Expression of the Viral Promoters throughout the Infection Cycle

Research paper thumbnail of Substitution of the C-terminal domain of the Escherichia coli RNA polymerase α subunit by that from Bacillus subtilis makes the enzyme responsive to a Bacillus subtilis transcriptional activator

Journal of Molecular Biology, 1998

Regulatory protein p4 of Bacillus subtilis phage È29 activates transcription from the viral late ... more Regulatory protein p4 of Bacillus subtilis phage È29 activates transcription from the viral late A3 promoter by interacting with the C-terminal domain (CTD) of the B. subtilis RNA polymerase a subunit, thereby stabilizing the holoenzyme at the promoter. Protein p4 does not interact with the Escherichia coli RNA polymerase and cannot activate transcription with this enzyme. We have constructed a chimerical a subunit containing the N-terminal domain of the E. coli a subunit and the CTD of the B. subtilis a subunit. Reconstitution of RNA polymerases containing this chimerical a subunit, the E. coli b and b H subunits, and the vegetative s factor from either E. coli (s 70) or B. subtilis (s A), generated hybrid enzymes that were responsive to protein p4 and ef®ciently supported activation at the A3 promoter. Protein p4 activated transcription with the chimerical enzymes through the same activation surface used with B. subtilis RNA polymerase. Therefore, the B. subtilis a-CTD allowed activation by p4 even when the rest of the RNA polymerase subunits belonged to E. coli, a distantly related bacterium. These results strongly suggest that protein p4 works essentially by serving as an anchor that stabilizes RNA polymerase at the promoter.

Research paper thumbnail of The switch from early to late transcription in phage GA-1: characterization of the regulatory protein p4G

Journal of Molecular Biology, 1999

Research paper thumbnail of Binding of phage Φ29 protein p4 to the early A2c promoter: recruitment of a repressor by the RNA polymerase

Journal of Molecular Biology, 1998

Regulatory protein p4 from Bacillus subtilis phage È29 represses the early A2c promoter by bindin... more Regulatory protein p4 from Bacillus subtilis phage È29 represses the early A2c promoter by binding upstream from RNA polymerase and interacting with the C-terminal domain of the RNA polymerase a subunit. This interaction stabilizes the RNA polymerase at the promoter in such a way that promoter clearance is prevented. Here, the binding of protein p4 to the A2c promoter has been studied. In the absence of RNA polymerase, protein p4 was found to bind with low af®nity to a site centered at position À39 relative to the transcription start site. When RNA polymerase was present, protein p4 was displaced from this site and bound instead to a different target centered at position À71. Stable binding to this site requires the interaction of protein p4 with the C-terminal domain of the RNA polymerase a-subunit. Both sites contain sequences resembling the well-characterized p4 binding site present at the late A3 promoter, to which p4 binds with high af®nity. A mutational analysis revealed that the site at À71 is critical for a stable interaction between protein p4 and RNA polymerase, and for ef®cient repression, whereas mutation of the site at À39 had only a small effect on repression ef®ciency. Therefore, RNA polymerase plays an active role in the repression mechanism by stabilizing the repressor at the promoter, generating a nucleoprotein complex that is too stable to allow promoter clearance.

Research paper thumbnail of A mutation in the C-terminal domain of the RNA polymerase alpha subunit that destabilizes the open complexes formed at the phage φ29 late A3 promoter

Journal of Molecular Biology, 2001

Regulatory protein p4 from Bacillus subtilis phage f29 activates the viral late A3 promoter mainl... more Regulatory protein p4 from Bacillus subtilis phage f29 activates the viral late A3 promoter mainly by stabilizing the binding of RNA polymerase (RNAP) to it as a closed complex. This requires an interaction between protein p4 residue Arg120 and the C-terminal domain (CTD) of the RNAP a subunit. Several acidic residues of the a-CTD, considered as plausible targets for p4 residue Arg120, were individually changed into alanine. In addition, a truncated a subunit lacking the last four residues, two of which are acidic, was obtained. The modi®ed a subunits were puri®ed and reconstituted into RNAP holoenzyme in vitro. Protein p4 was found to be unable to activate the late A3 promoter when residue Glu297 of the a subunit was changed to Ala, a modi®cation that did not impair transcription from several other promoters. Interestingly, protein p4 could stabilize the modi®ed RNAP at the A3 promoter as a closed complex, although the open complexes formed were unstable and did not proceed to elongation complexes. Our results indicate that the change of the a residue Glu297 into Ala destabilizes the open complexes formed at this promoter, but not at other promoters. Considered in the context of earlier ®ndings indicating that the RNAP a-CTD may participate in the transition from closed to intermediate complexes at some other promoters, the new results expand and clarify our view of its role in transcription initiation.

Research paper thumbnail of PGC-1α regulates the mitochondrial antioxidant defense system in vascular endothelial cells

Cardiovascular Research, 2005

Research paper thumbnail of European Contribution to the study of ROS: A Summary of the Findings and Prospects for the Future from the COST Action BM1203 (EU-ROS)

The European Cooperation in Science and Technology (COST) provides an ideal framework to establis... more The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associat...