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Ramos M.,University of Zaragoza | Menao S.,University of Zaragoza | Arnedo M.,University of Zaragoza | Puisac B.,University of Zaragoza | And 12 more authors.
European Journal of Medical Genetics | Year: 2013

Mitochondrial HMG-CoA synthase deficiency is a rare inherited metabolic disorder that affects ketone-body synthesis. Acute episodes include vomiting, lethargy, hepatomegaly, hypoglycaemia, dicarboxylic aciduria, and in severe cases, coma. This deficiency may have been under-diagnosed owing to the absence of specific clinical and biochemical markers, limitations in liver biopsy and the lack of an effective method of expression and enzyme assay for verifying the mutations found. To date, eight patients have been reported with nine allelic variants of the HMGCS2 gene. We present a new method of enzyme expression and a modification of the activity assay that allows, for first time, the functional study of missense mutations found in patients with this deficiency. Four of the missense mutations (p.V54M, p.R188H, p.G212R and p.G388R) did not produce proteins that could have been detected in soluble form by western blot; three produced a total loss of activity (p.Y167C, p.M307T and p.R500H) and one, variant p.F174L, gave an enzyme with a catalytic efficiency of 11.5%. This indicates that the deficiency may occur with partial loss of activity of enzyme. In addition, we describe a new patient with this deficiency, in which we detected the missense allelic variant, c.1162G>A (p.G388R) and the nonsense variant c.1270C>T (p.R424X). © 2013 Elsevier Masson SAS. Source


Puisac B.,University of Zaragoza | Teresa-Rodrigo M.E.,University of Zaragoza | Arnedo M.,University of Zaragoza | Gil-Rodriguez M.C.,University of Zaragoza | And 6 more authors.
Molecular Genetics and Metabolism | Year: 2013

Eukaryotic cells can be protected against mutations that generate stop codons by nonsense-mediated mRNA decay (NMD) and/or nonsense-associated altered splicing (NAS). However, the processes are only partially understood and do not always occur. In this work, we study these phenomena in the stop codon mutations c.109G>T (p.Glu37*) and c.504_505delCT; the second and third most frequent mutations in HMG-CoA lyase deficiency (MIM #246450). The deficiency affects the synthesis of ketone bodies and produces severe disorders during early childhood. We used a minigene approach, real-time quantitative PCR and the inhibition of NMD by puromycin treatment, to study the effect of stop codons on splicing (NAS) and NMD in seven patients. Surprisingly, none of the stop codons studied appears to be the direct cause of aberrant splicing. In the mutation c.109G>T, the splicing is due to the base change G>T at position 109, which is critical and cannot be explained by disruption of exonic splicing enhancer (ESE) elements, by the appearance of exonic splicing silencer (ESS) elements which were predicted by bioinformatic tools or by the stop codons. Moreover, the mutation c.504_505delCT produces two mRNA transcripts both with stop codons that generate simultaneous NMD phenomena. The effects of the mutations studied on splicing seemed to be similar in all the patients. Furthermore, we report a Spanish patient with 3-hydroxy-3-methylglutaric aciduria and a novel missense mutation: c.825C>G (p.Asn275Lys). © 2013 Elsevier Inc. Source


Puisac B.,University of Zaragoza | Ramos M.,University of Zaragoza | Arnedo M.,University of Zaragoza | Menao S.,University of Zaragoza | And 12 more authors.
Molecular Biology Reports | Year: 2012

The genes HMGCS2 and HMGCL encode the two main enzymes for ketone-body synthesis, mitochondrial HMG-CoA synthase and HMG-CoA lyase. Here, we identify and describe possible splice variants of these genes in human tissues. We detected an alternative transcript of HMGCS2 carrying a deletion of exon 4, and two alternative transcripts of HMGCL with deletions of exons 5 and 6, and exons 5, 6 and 7, respectively. All splice variants maintained the reading frame. However, Western blot studies and overexpression measurements in eukaryotic or prokaryotic cell models did not reveal HL or mHS protein variants. Both genes showed a similar distribution of the inactive variants in different tissues. Surprisingly, the highest percentages were found in tissues where almost no ketone bodies are synthesized: Heart, skeletal muscle and brain. Our results suggest that alternative splicing might coordinately block the two main enzymes of ketogenesis in specific human tissues. © Springer Science+Business Media B.V. 2011. Source


Martin-Garcia F.,Molecular Modelling Group | Martin-Garcia F.,Biomol Informatics SL | Papaleo E.,Copenhagen University | Gomez-Puertas P.,Molecular Modelling Group | And 2 more authors.
PLoS ONE | Year: 2015

The continued development and utility of molecular dynamics simulations requires improvements in both the physical models used (force fields) and in our ability to sample the Boltzmann distribution of these models. Recent developments in both areas have made available multi-microsecond simulations of two proteins, ubiquitin and Protein G, using a number of different force fields. Although these force fields mostly share a common mathematical form, they differ in their parameters and in the philosophy by which these were derived, and previous analyses showed varying levels of agreement with experimental NMR data. To complement the comparison to experiments, we have performed a structural analysis of and comparison between these simulations, thereby providing insight into the relationship between force-field parameterization, the resulting ensemble of conformations and the agreement with experiments. In particular, our results show that, at a coarse level, many of the motional properties are preserved across several, though not all, force fields. At a finer level of detail, however, there are distinct differences in both the structure and dynamics of the two proteins, which can, together with comparison with experimental data, help to select force fields for simulations of proteins. A noteworthy observation is that force fields that have been reparameterized and improved to provide a more accurate energetic description of the balance between helical and coil structures are difficult to distinguish from their "unbalanced" counterparts in these simulations. This observation implies that simulations of stable, folded proteins, even those reaching 10 microseconds in length, may provide relatively little information that can be used to modify torsion parameters to achieve an accurate balance between different secondary structural elements. © 2015 Martín-García et al. Source


Marcos-Alcalde I.,Molecular Modelling Group | Setoain J.,Complutense University of Madrid | Mendieta-Moreno J.I.,Molecular Modelling Group | Mendieta-Moreno J.I.,Condensed Matter Physics Center | And 3 more authors.
Bioinformatics | Year: 2015

From conformational studies to atomistic descriptions of enzymatic reactions, potential and free energy landscapes can be used to describe biomolecular systems in detail. However, extracting the relevant data of complex 3D energy surfaces can sometimes be laborious. In this article, we present MEPSA (Minimum Energy Path Surface Analysis), a cross-platform user friendly tool for the analysis of energy landscapes from a transition state theory perspective. Some of its most relevant features are: identification of all the barriers and minima of the landscape at once, description of maxima edge profiles, detection of the lowest energy path connecting two minima and generation of transition state theory diagrams along these paths. In addition to a built-in plotting system, MEPSA can save most of the generated data into easily parseable text files, allowing more versatile uses of MEPSA's output such as the generation of molecular dynamics restraints from a calculated path. © The Author 2015. Published by Oxford University Press. All rights reserved. Source

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