Assisted protein folding at low temperature: evolutionaryadaptation of the Antarctic fish chaperonin CCT and its clientproteins

Eukaryotic ectotherms of the Southern Ocean face energetic challenges to protein folding assisted by the cytosolic chaperonin CCT. We hypothesize that CCT and its client proteins (CPs) have co-evolved molecular adaptations that facilitate CCT–CP interaction and the ATP-driven folding cycle at low temperature. To test this hypothesis, we compared the functional and structural properties of CCT–CP systems from testis tissues of an Antarctic fish, Gobionotothen gibberifrons (Lo¨nnberg) (habitat/body T=-1.9 to +2˚C), and of the cow (body T=37˚C). We examined the temperature dependence of the binding of denatured CPs (bactin, b-tubulin) by fish and bovine CCTs, both in homologous and heterologous combinations and at temperatures between 24˚C and 20˚C, in a buffer conducive to binding of the denatured CP to the open conformation of CCT. In homologous combination, the percentage of G. gibberifrons CCT bound to CP declined linearly with increasing temperature, whereas the converse was true for bovine CCT. Binding of CCT to heterologous CPs was low, irrespective of temperature. When reactions were supplemented with ATP, G. gibberifrons CCT catalyzed the folding and release of actin at 2˚C. The ATPase activity of apo-CCT from G. gibberifrons at 4˚C was, 2.5-fold greater than that of apo-bovine CCT, whereas equivalent activities were observed at 20˚C. Based on these results, we conclude that the catalytic folding cycle of CCT from Antarctic fishes is partially compensated at their habitat temperature, probably by means of enhanced CP-binding affinity and increased flexibility of the CCT subunits

Obtención de plantas compuestas de olivo mediante transformación con Agrobacterium rhizogenes

La transformación con Agrobacterium rhizogenes ha sido utilizada como herramienta para estudios de genómica funcional en raíces (Collier et al. 2005 Plant Journal 43:449-457; Baranski et al. 2006 Plant Cell Reports 25:190-197). La obtención de plantas compuestas de olivo (sistema radicular transgénico y parte aérea no transgénica), mediante esta técnica, sería de gran ayuda para estudiar la interacción de esta especie con los patógenos de suelo Verticillium dahliae y Rosellinia necatrix. En este trabajo, se presentan las primeras aproximaciones para la transformación de brotes micropropagados de olivo mediante A. rhizogenes. Se han utilizado 2 genotipos procedentes de semilla del cv. Picual, uno con baja capacidad de enraizamiento, P1, y otro, con alta capacidad, P138, y dos cepas de A. rhizogenes: A4, que contiene el plásmido silvestre Ri, y K599, con el plásmido binario pKGWFS7.0-35SP, que incluye el gen marcador gfp. En el caso del genotipo P1, en la fase de co-cultivo con la bacteria, se añadieron al medio 3 mg/l AIB, para facilitar la formación de raíces. En el genotipo P138, se obtuvo un 100% de enraizamiento tanto en el tratamiento control como en el de brotes infectados con la cepa A4; sin embargo, aquéllos inoculados con la cepa K599 sólo alcanzaron un 70% de enraizamiento. Asimismo, se observó que el 61% de las raíces obtenidas tras la infección con K599 mostraron fluorescencia verde bajo el microscopio confocal. En el genotipo P1, el 60% de las plantas control formaron raíces, frente al 10% de plantas infectadas con A4 y ninguna con la cepa K599. La naturaleza transgénica de las raíces obtenidas tras la infección con A4, en ambos genotipos, se evaluará mediante amplificación por PCR del gen RolB.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

Perspectiva de género en la asignatura de farmacología

En este proyecto de innovación docente hemos trabajo para comenzar a poner las bases de posibles diferencias que pueden existir en la farmacología de los medicamentos, atendiendo a si la persona que los toma es un hombre o una mujer. Con alumnos del grado de Nutrición Humana y Dietética y de Odontología hemos buscado bibliografía que nos demuestre si efectivamente se dan estas diferencias en la farmacocinética, en la farmacodinamia y en los efectos adversos. Los resultados encontrados nos han permitido presentar 2 comunicaciones en congresos y realizar un TFGDepto. de Farmacología y ToxicologíaFac. de MedicinaFALSEsubmitte

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Notch1 and IL-7 Receptor Interplay Maintains Proliferation of Human Thymic Progenitors while Suppressing Non-T Cell Fates

Article available at http://www.jimmunol.org/cgi/content/abstract/177/6/3711Notch signaling is critical for T cell development of multipotent hemopoietic progenitors. Yet, how Notch regulates T cell fate specification during early thymopoiesis remains unclear. In this study, we have identified an early subset of CD34highc-kit+flt3+IL-7R+ cells in the human postnatal thymus, which includes primitive progenitors with combined lymphomyeloid potential. To assess the impact of Notch signaling in early T cell development, we expressed constitutively active Notch1 in such thymic lymphomyeloid precursors (TLMPs), or triggered their endogenous Notch pathway in the OP9-Delta-like1 stroma coculture. Our results show that proliferation vs differentiation is a critical decision influenced by Notch at the TLMP stage. We found that Notch signaling plays a prominent role in inhibiting non-T cell differentiation (i.e., macrophages, dendritic cells, and NK cells) of TLMPs, while sustaining the proliferation of undifferentiated thymocytes with T cell potential in response to unique IL-7 signals. However, Notch activation is not sufficient for inducing T-lineage progression of proliferating progenitors. Rather, stroma-derived signals are concurrently required. Moreover, while ectopic IL-7R expression cannot replace Notch for the maintenance and expansion of undifferentiated thymocytes, Notch signals sustain IL-7R expression in proliferating thymocytes and induce IL-7R up-regulation in a T cell line. Thus, IL-7R and Notch pathways cooperate to synchronize cell proliferation and suppression of non-T lineage choices in primitive intrathymic progenitors, which will be allowed to progress along the T cell pathway only upon interaction with an inductive stromal microenvironment. These data provide insight into a mechanism of Notch-regulated amplification of the intrathymic pool of early human T cell progenitorsThis work was supported by grants from Plan Nacional de Biomedicina (SAF2004-01122 and GEN2003-20649-C06-02), Comunidad de Madrid (GR/SAL/0143/ 2004), Fundación La Caixa (ON03/109-00), and Fundación Eugenio Rodríguez Pascual. We thank the Fundación Ramón Areces for an institutional grant to the Centro de Biología Molecular Severo OchoaPeer reviewe

Progressive supranuclear palsy and tau hyperphosphorylation in a patient with a C212Y parkin mutation

Autosomal recessive-juvenile parkinsonism (AR-JP) is one of the most common forms of familial Parkinson's disease (PD) and is related to mutations in the Park-2 gene, encoding for a protein ligase of ubiquitin, parkin. Different mutations located along the parkin gene have been observed in different AR-JP affected families, possibly interfering with the normal function of parkin and the proteasome system. Two cases of patients with AR-JP have been recently described presenting different homo- and heterozygous parkin mutations and limited tau pathology. We report here the case of a patient with clinical and pathological findings compatible with progressive supranuclear palsy (PSP), carrier of a single, heterozygous mutation of the parkin gene, and homozygous for the H1/H1 haplotype in the tau gene. Abnormal tau hyperphosphorylation has been observed in our patient brain samples, suggesting that a partial deficit of parkin, a protein with ubiquitin-ligase function, may trigger tau pathology in individuals with molecular genetic risk factors.Peer reviewe

Bacterial Tubulin Distinct Loop Sequences and Primitive Assembly Properties Support its Origin from a Eukaryotic Tubulin Ancestor

25 p.- 9 fig.-7 fig. supl.-4 tab. supl.The structure of the unique bacterial tubulin BtubA/B from Prosthecobacter is very similar to eukaryotic alphabeta-tubulin, but strikingly, BtubA/B fold without eukaryotic chaperones. Our sequence comparisons indicate that BtubA and BtubB do not really correspond to either alpha or beta-tubulin but have mosaic sequences with intertwining features from both. Their nucleotide binding loops are more conserved and their more divergent sequences correspond to discrete surface zones of tubulin involved in microtubule assembly and binding to eukaryotic chaperonin CCT, which is absent from the P. dejongeii draft genome. BtubA/B cooperatively assembles over a wider range of conditions than alphabeta-tubulin, forming pairs of protofilaments which coalesce into bundles instead of microtubules, and it lacks the abilities to differentially interact with divalent cations and bind typical tubulin drugs. Assembled BtubA/B contain close to one bound GTP and GDP. Both BtubA and BtubB subunits hydrolyze GTP, leading to disassembly. The mutant BtubA/B-S144G in the tubulin signature motif GGG(T/S)G(S/T)G has strongly inhibited GTPase, but BtubA-T147G/B does not, suggesting that BtubB is a more active GTPase, like beta-tubulin. BtubA/B chimera bearing the beta-tubulin loops M, H1-S2 and S9-S10 in BtubB fold, assemble and have reduced GTPase. However, introduction of the alpha-tubulin loop S9-S10 with its unique 8-residue insertion impaired folding. From the sequence analyses, its primitive assembly features and the properties of the chimeras, we propose that BtubA/B were acquired shortly after duplication of a spontaneously folding alpha and beta-tubulin ancestor, possibly by horizontal gene transfer from a primitive eukaryotic cell, followed by divergent evolutionThis work was supported in part by Ministerio de Ciencia e Innovación Grants BFU2008-00013 (to J. M. A.) and BFU2010-15703 (to J. M. V.) and contracts Juan de la Cierva (to A. J. M.-G. and M. A. O.), a contract from the Consejo Superior de Investigaciones Científicas Junta de Ampliación de Estudios-Doctores (to A. J. M.-G.), a Formacion de Personal Universitario fellowship (to L. S.), and a fellowship from the Madrid Regional Government (to M. S.)Peer reviewe