Centro Of Biologia Molecular Severo Ochoa

Madrid, Spain

Centro Of Biologia Molecular Severo Ochoa

Madrid, Spain
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PubMed | Institute Of Biologia Molecular Of Barcelona, University of Missouri and Centro Of Biologia Molecular Severo Ochoa
Type: Journal Article | Journal: Journal of virology | Year: 2015

The N-terminal region of the foot-and-mouth disease virus (FMDV) 3D polymerase contains the sequence MRKTKLAPT (residues 16 to 24) that acts as a nuclear localization signal. A previous study showed that substitutions K18E and K20E diminished the transport to the nucleus of 3D and 3CD and severely impaired virus infectivity. These residues have also been implicated in template binding, as seen in the crystal structures of different 3D-RNA elongation complexes. Here, we report the biochemical and structural characterization of different mutant polymerases harboring substitutions at residues 18 and 20, in particular, K18E, K18A, K20E, K20A, and the double mutant K18A K20A (KAKA). All mutant enzymes exhibit low RNA binding activity, low processivity, and alterations in nucleotide recognition, including increased incorporation of ribavirin monophosphate (RMP) relative to the incorporation of cognate nucleotides compared with the wild-type enzyme. The structural analysis shows an unprecedented flexibility of the 3D mutant polymerases, including both global rearrangements of the closed-hand architecture and local conformational changes at loop 9-11 (within the polymerase motif B) and at the template-binding channel. Specifically, in 3D bound to RNA, both K18E and K20E induced the opening of new pockets in the template channel where the downstream templating nucleotide at position +2 binds. The comparisons of free and RNA-bound enzymes suggest that the structural rearrangements may occur in a concerted mode to regulate RNA replication, processivity, and fidelity. Thus, the N-terminal region of FMDV 3D that acts as a nuclear localization signal (NLS) and in template binding is also involved in nucleotide recognition and can affect the incorporation of nucleotide analogues.The study documents multifunctionality of a nuclear localization signal (NLS) located at the N-terminal region of the foot-and-mouth disease viral polymerase (3D). Amino acid substitutions at this polymerase region can impair the transport of 3D to the nucleus, reduce 3D binding to RNA, and alter the relative incorporation of standard nucleoside monophosphate versus ribavirin monophosphate. Structural data reveal that the conformational changes in this region, forming part of the template channel entry, would be involved in nucleotide discrimination. The results have implications for the understanding of viral polymerase function and for lethal mutagenesis mechanisms.


Buonvicino D.,University of Florence | Formentini L.,Centro Of Biologia Molecular Severo Ochoa | Cipriani G.,University of Florence | Chiarugi A.,University of Florence
Journal of Biological Chemistry | Year: 2013

Background: Excessive activation of enzyme poly(ADP-ribose) polymerase-1 (PARP-1) causes ATP depletion and kills cells. Results: We found that in the absence of glucose PARP-1 triggers an adenylate kinase-dependent increase of ATP. Conclusion: PARP-1 hyperactivation is not invariantly related to ATP loss. Significance: This study adds to the complexity of PARP-1 hyperactivity and energy derangement. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.


Gallego-Villar L.,Centro Of Biologia Molecular Severo Ochoa | Perez B.,Centro Of Biologia Molecular Severo Ochoa | Ugarte M.,Centro Of Biologia Molecular Severo Ochoa | Desviat L.R.,Centro Of Biologia Molecular Severo Ochoa | Richard E.,Centro Of Biologia Molecular Severo Ochoa
Biochemical and Biophysical Research Communications | Year: 2014

Propionic acidemia (PA), caused by a deficiency of the mitochondrial biotin dependent enzyme propionyl-CoA carboxylase (PCC) is one of the most frequent organic acidurias in humans. Most PA patients present in the neonatal period with metabolic acidosis and hyperammonemia, developing different neurological symptoms, movement disorders and cardiac complications. There is strong evidence indicating that oxidative damage could be a pathogenic factor in neurodegenerative, mitochondrial and metabolic diseases. Recently, we identified an increase in ROS levels in PA patients-derived fibroblasts. Here, we analyze the capability of seven antioxidants to scavenge ROS production in PA patients' cells. Tiron, trolox, resveratrol and MitoQ significantly reduced ROS content in patients and controls' fibroblasts. In addition, changes in the expression of two antioxidant enzymes, superoxide dismutase and glutathione peroxidase, were observed in PA patients-derived fibroblasts after tiron and resveratrol treatment. Our results in PA cellular models establish the proof of concept of the potential of antioxidants as an adjuvant therapy for PA and pave the way for future assessment of antioxidant strategies in the murine model of PA. © 2014 Elsevier Inc. All rights reserved.


PubMed | Centro Of Biologia Molecular Severo Ochoa
Type: Journal Article | Journal: Biochemical and biophysical research communications | Year: 2014

Propionic acidemia (PA), caused by a deficiency of the mitochondrial biotin dependent enzyme propionyl-CoA carboxylase (PCC) is one of the most frequent organic acidurias in humans. Most PA patients present in the neonatal period with metabolic acidosis and hyperammonemia, developing different neurological symptoms, movement disorders and cardiac complications. There is strong evidence indicating that oxidative damage could be a pathogenic factor in neurodegenerative, mitochondrial and metabolic diseases. Recently, we identified an increase in ROS levels in PA patients-derived fibroblasts. Here, we analyze the capability of seven antioxidants to scavenge ROS production in PA patients cells. Tiron, trolox, resveratrol and MitoQ significantly reduced ROS content in patients and controls fibroblasts. In addition, changes in the expression of two antioxidant enzymes, superoxide dismutase and glutathione peroxidase, were observed in PA patients-derived fibroblasts after tiron and resveratrol treatment. Our results in PA cellular models establish the proof of concept of the potential of antioxidants as an adjuvant therapy for PA and pave the way for future assessment of antioxidant strategies in the murine model of PA.

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