Manuel Pérez-Alonso

Molecular Characterization and Evolution of the Protein Phosphatase 2A B′ Regulatory Subunit Family in Plants

Type 2A serine/threonine protein phosphatases (PP2A) are important components in the reversible protein phosphorylation events in plants and other organisms. PP2A proteins are oligomeric complexes constituted by a catalytic subunit and several regulatory subunits that modulate the activity of these phosphatases. The analysis of the complete genome of Arabidopsis allowed us to characterize four novel genes, AtB′ε, AtB′ζ,AtB′η, and AtB′θ, belonging to the PP2A B′ regulatory subunit family. Because four genes of this type had been described previously, this family is composed of eight members. Reverse transcriptase-polymerase chain reaction experiments showed thatAtB′ε mRNAs are present in all Arabidopsis tissues analyzed, and their levels do not respond significantly to heat stress. Expressed sequence tags corresponding to AtB′ζ,AtB′η, and AtB′θ have been identified, indicating that the new genes are actively transcribed. The genomic organization of this family of PP2A regulatory subunits is reported, as well as its chromosomal location. An extensive survey of the family has been carried out in plants, characterizing B′ subunits in a number of different species, and performing a phylogenetic study that included several B′ regulatory proteins from animals. Our results indicate that the animal and plant proteins have evolved independently, that there is a relationship between the number of B′ isoforms and the complexity of the organism, and that there are at least three main subfamilies of regulatory subunits in plants, which we have named α, η, and κ.

Myotonic dystrophy: candidate small molecule therapeutics

Myotonic dystrophy type 1 (DM1) is a rare multisystemic neuromuscular disorder caused by expansion of CTG trinucleotide repeats in the noncoding region of the DMPK gene. Mutant DMPK transcripts are toxic and alter gene expression at several levels. Chiefly, the secondary structure formed by CUGs has a strong propensity to capture and retain proteins, like those of the muscleblind-like (MBNL) family. Sequestered MBNL proteins cannot then fulfill their normal functions. Many therapeutic approaches have been explored to reverse these pathological consequences. Here, we review the myriad of small molecules that have been proposed for DM1, including examples obtained from computational rational design, HTS, drug repurposing, and therapeutic gene modulation.

Enforcing Conceptual Modeling to improve the understanding of human genome

It is widely accepted that the use of Conceptual Modeling techniques in modern Software Engineering leads to a more accurate description of the problem domain. The application of these techniques in the context of challenging domains as the human genome is a fascinating task. The relevant biological concepts should be properly addressed through the creation of the corresponding conceptual schema. This schema will improve the description of the global process followed from a DNA sequence to a fully functional protein. Once the conceptual model is established, the corresponding database is created. The database is intended to act as a unified repository of integrated information that will allow biologists to perform efficient recovery tasks.

Pentamidine rescues contractility and rhythmicity in a Drosophila model of myotonic dystrophy heart dysfunction

ABSTRACT Up to 80% of individuals with myotonic dystrophy type 1 (DM1) will develop cardiac abnormalities at some point during the progression of their disease, the most common of which is heart blockage of varying degrees. Such blockage is characterized by conduction defects and supraventricular and ventricular tachycardia, and carries a high risk of sudden cardiac death. Despite its importance, very few animal model studies have focused on the heart dysfunction in DM1. Here, we describe the characterization of the heart phenotype in a Drosophila model expressing pure expanded CUG repeats under the control of the cardiomyocyte-specific driver GMH5-Gal4. Morphologically, expression of 250 CUG repeats caused abnormalities in the parallel alignment of the spiral myofibrils in dissected fly hearts, as revealed by phalloidin staining. Moreover, combined immunofluorescence and in situ hybridization of Muscleblind and CUG repeats, respectively, confirmed detectable ribonuclear foci and Muscleblind sequestration, characteristic features of DM1, exclusively in flies expressing the expanded CTG repeats. Similarly to what has been reported in humans with DM1, heart-specific expression of toxic RNA resulted in reduced survival, increased arrhythmia, altered diastolic and systolic function, reduced heart tube diameters and reduced contractility in the model flies. As a proof of concept that the fly heart model can be used for in vivo testing of promising therapeutic compounds, we fed flies with pentamidine, a compound previously described to improve DM1 phenotypes. Pentamidine not only released Muscleblind from the CUG RNA repeats and reduced ribonuclear formation in the Drosophila heart, but also rescued heart arrhythmicity and contractility, and improved fly survival in animals expressing 250 CUG repeats. Summary: The authors characterize a cardiac-dysfunction model of DM1 in Drosophila, and show that pentamidine rescues both contractility and rhythmicity.

Case report: first successful application of preimplantation genetic diagnosis for hereditary angiooedema

Hereditary angiooedema is an autosomal dominant disease caused by mutations in the SERPING1 gene. It is characterized by oedemas in different parts of the body, being particularly dangerous when swelling involves the upper airway. Preimplantation genetic diagnosis (PGD) was performed in a couple where the woman carries a deletion of 2.9Kb that includes exon 4 of the SERPING1 gene. Four polymorphic short tandem repeat markers were tested in order to establish the disease-bearing haplotype and three of them were fully informative. Amplification efficiency at the preclinical work up ranged from 71% to 100% for each locus and allele drop out rates were between 0% and 20% for the polymorphic markers. The couple underwent PGD using fluorescent multiplex heminested polymerase chain reaction. Six embryos were biopsied and five of them were diagnosed as healthy. Two embryos were transferred and a singleton pregnancy was achieved, resulting in the birth of a healthy boy.

Arabidopsis thaliana sequence analysis confirms the presence of cyt b-561 in plants: evidence for a novel protein family.

Recent advances in the Arabidopsis sequencing project has elucidated the presence of two genes Atb561-A and Atb561-B that show limited homology to the DNA sequence encoding for the mammalian chromaffin granule cytochrome b-561 (cyt b-561). Detailed analysis of the structural features and conserved residues reveals, however, that the structural homology between the presumptive Arabidopsis proteins and the animal proteins is very high. All proteins are hydrophobic and show highly conserved transmembrane helices. The presumably heme-binding histidine residues in the plant and animal sequences as well as the suggested binding site for the electron acceptor, monodehydroascorbate, are strictly conserved. In contrast, the suggested electron donor (ascorbate) binding site is not very well conserved between the plant and animal sequences questioning the function of this motif. Sequence analysis of the Atb561-B gene demonstrates a different splicing than that initially predicted in silico resulting in a protein with nine extra amino acids and a significantly higher homology to the other cyt b-561 sequences. The homology between the plant and animal sequences is further supported by the strong similarity between a number of biochemical properties of the chromaffin cyt b-561 and the cyt b-561 isolated from bean hook plasma membranes. Since the mammalian cyt b-561 is considered specific to neuroadrenergic tissues, the identification of a closely related homologue in an aneural organism demonstrates that these proteins constitute a new class of widely occurring membrane proteins. Both the plant and animal cyt b-561 are involved in transmembrane electron transport using ascorbate as an electron donor. The similarity between these proteins therefore suggests, for the first time, that this transport supports a number of different cell physiological processes. An evolutionary relationship between the plant and animal proteins is presented.

Quality management system in PGD/PGS: now is the time

PurposeGovernments and international authorities require an accreditation of the PGD/PGS laboratories in order to ensure the safety and reproducibility of these analytical procedures. The implementation of a Quality Management System is the first mandatory step prior to accreditation. Our aim is to offer a detailed guidance to the PGD/PGS community that would like to implement this system in the future.MethodsThe certification was based on the norm ISO 9001:2000 and requires the identification of procedures, definition of the flowchart, documentation of the processes, recognition of the critical control points, establishment of quality controls, performance of validation and audit system.ResultsThe achievement of ISO certification with the specific scope of “preimplantation genetic diagnosis”.ConclusionCertification of PGD/PGS allows to achieve evaluation of the efficiency to ensure the sensitivity and a continuous improvement of the genetic diagnosis of embryonic single cells.

Preimplantation genetic diagnosis of P450 oxidoreductase deficiency and Huntington Disease using three different molecular approaches simultaneously

PurposeDescription of the confluence of different molecular techniques to detect three different mutations in one cell. The man carries a 20 base pair insertion in exon 12 of the POR gene (c.1551_1552ins20), and the woman carries a point mutation in exon 8 of the POR gene (c.859G>C) plus a triplet repeat expansion in the HTT gene.MethodsHuntington Disease (HD) had to be diagnosed using short tandem repeat (STR) markers linked to the HTT gene. The mutation c.1551_1552ins20 was analyzed by fragment size and c.859G>C was minisequenced. Furthermore, STR markers linked to the POR gene were included to support the diagnosis of P450 oxidoreductase (POR) deficiency.ResultsNine embryos were diagnosed in total: three as POR deficiency affected, two as HD affected, one as POR deficiency and HD affected, and two as carriers of the paternal POR deficiency mutation and healthy for HD. These two last embryos were transferred but no pregnancy was achieved.ConclusionsA successful procedure combining direct and indirect methods for the detection of three different mutations in a single cell has been achieved for the first time.

ZFWD: a novel subfamily of plant proteins containing a C3H zinc finger and seven WD40 repeats

We describe a new subfamily of WD repeat proteins characterised by the presence of a C3H zinc finger at the N-terminal part of the protein associated with seven WD40 repeats. We have identified four members of this subfamily in Arabidopsis thaliana, one of them with associated expressed sequence tags (ESTs). We have also identified homologous ESTs in rice, cotton, maize, poplar, pine tree and the ice plant. We do not observe animal homologues, suggesting that this subfamily could be specific for plants. Our data suggest an important role for these proteins. Based on the high sequence conservation within the conserved domains, we suggest that these proteins could have a regulatory function.

Development of a Drosophila melanogaster spliceosensor system for in vivo high-throughput screening in myotonic dystrophy type 1

Alternative splicing of pre-mRNAs is an important mechanism that regulates cellular function in higher eukaryotes. A growing number of human genetic diseases involve splicing defects that are directly connected to their pathology. In myotonic dystrophy type 1 (DM1), several clinical manifestations have been proposed to be the consequence of tissue-specific missplicing of numerous genes. These events are triggered by an RNA gain-of-function and resultant deregulation of specific RNA-binding factors, such as the nuclear sequestration of muscleblind-like family factors (MBNL1–MBNL3). Thus, the identification of chemical modulators of splicing events could lead to the development of the first valid therapy for DM1 patients. To this end, we have generated and validated transgenic flies that contain a luciferase-reporter-based system that is coupled to the expression of MBNL1-reliant splicing (spliceosensor flies), to assess events that are deregulated in DM1 patients in a relevant disease tissue. We then developed an innovative 96-well plate screening platform to carry out in vivo high-throughput pharmacological screening (HTS) with the spliceosensor model. After a large-scale evaluation (>16,000 chemical entities), several reliable splicing modulators (hits) were identified. Hit validation steps recognized separate DM1-linked therapeutic traits for some of the hits, which corroborated the feasibility of the approach described herein to reveal promising drug candidates to correct missplicing in DM1. This powerful Drosophila-based screening tool might also be applied in other disease models displaying abnormal alternative splicing, thus offering myriad uses in drug discovery.