Mapping of quantitative trait loci for cholesterol, LDL, HDL and triglyceride serum concentrations in pigs

The fine mapping of polymorphisms influencing cholesterol (CT), triglyceride (TG), and lipoprotein serum levels in human and mouse has provided a wealth of knowledge about the complex genetic architecture of these traits. The extension of these genetic analyses to pigs would be of utmost importance since they constitute a valuable biological and clinical model for the study of coronary artery disease and myocardial infarction. In the present work, we performed a whole genome scan for serum lipid traits in a half-sib Duroc pig population of 350 individuals. Phenotypic registers included total CT, TG, and low (LDL)- and high (HDL)-density lipoprotein serum concentrations at 45 and 190 days of age. This approach allowed us to identify two genomewide significant quantitative trait loci (QTL) for HDL-to-LDL ratio at 45 days (SSC6, 84 cM) and for TG at 190 days (SSC4, 23 cM) as well as a number of chromosomewide significant QTL. The comparison of QTL locations at 45 and 190 days revealed a notable lack of concordance at these two time points, suggesting that the effects of these QTL are age specific. Moreover, we have observed a considerable level of correspondence among the locations of the most significant porcine lipid QTL and those identified in humans. This finding might suggest that, in mammals, diverse polymorphisms located in a common set of genes are involved in the genetic variation of serum lipid levels.The work was funded by Grants AGL2002-04271-C03 and AGL2007-66707-C02 (Ministerio de Educacion y Ciencia, Spain). D. Gallardo was funded with a fellowship from Universitat Autonoma de Barcelona. R. N. Pena received a contractual grant from Instituto Nacional de Investigacion Agropecuaria (Spain).Peer reviewe

Iodine Atoms: A New Molecular Feature for the Design of Potent Transthyretin Fibrillogenesis Inhibitors

The thyroid hormone and retinol transporter protein known as transthyretin (TTR) is in the origin of one of the 20 or so known amyloid diseases. TTR self assembles as a homotetramer leaving a central hydrophobic channel with two symmetrical binding sites. The aggregation pathway of TTR into amiloid fibrils is not yet well characterized but in vitro binding of thyroid hormones and other small organic molecules to TTR binding channel results in tetramer stabilization which prevents amyloid formation in an extent which is proportional to the binding constant. Up to now, TTR aggregation inhibitors have been designed looking at various structural features of this binding channel others than its ability to host iodine atoms. In the present work, greatly improved inhibitors have been designed and tested by taking into account that thyroid hormones are unique in human biochemistry owing to the presence of multiple iodine atoms in their molecules which are probed to interact with specific halogen binding domains sitting at the TTR binding channel. The new TTR fibrillogenesis inhibitors are based on the diflunisal core structure because diflunisal is a registered salicylate drug with NSAID activity now undergoing clinical trials for TTR amyloid diseases. Biochemical and biophysical evidence confirms that iodine atoms can be an important design feature in the search for candidate drugs for TTR related amyloidosis

Cholera Toxin B Subunits Assemble into Pentamers - Proposition of a Fly-Casting Mechanism

The cholera toxin B pentamer (CtxB5), which belongs to the AB5 toxin family, is used as a model study for protein assembly. The effect of the pH on the reassembly of the toxin was investigated using immunochemical, electrophoretic and spectroscopic methods. Three pH-dependent steps were identified during the toxin reassembly: (i) acquisition of a fully assembly-competent fold by the CtxB monomer, (ii) association of CtxB monomer into oligomers, (iii) acquisition of the native fold by the CtxB pentamer. The results show that CtxB5 and the related heat labile enterotoxin LTB5 have distinct mechanisms of assembly despite sharing high sequence identity (84%) and almost identical atomic structures. The difference can be pinpointed to four histidines which are spread along the protein sequence and may act together. Thus, most of the toxin B amino acids appear negligible for the assembly, raising the possibility that assembly is driven by a small network of amino acids instead of involving all of them

Uptake of Aggregating Transthyretin by Fat Body in a Drosophila Model for TTR-Associated Amyloidosis

Background: A functional link has been established between the severe neurodegenerative disorder Familial amyloidotic polyneuropathy and the enhanced propensity of the plasma protein transthyretin (TTR) to form aggregates in patients with single point mutations in the TTR gene. Previous work has led to the establishment of an experimental model based on transgenic expression of normal or mutant forms of human TTR in Drosophila flies. Remarkably, the severity of the phenotype was greater in flies that expressed a single copy than with two copies of the mutated gene. Methodology/Principal Findings: In this study, we analyze the distribution of normal and mutant TTR in transgenic flies, and the ultrastructure of TTR-positive tissues to clarify if aggregates and/or amyloid filaments are formed. We report the formation of intracellular aggregates of 20 nm spherules and amyloid filaments in thoracic adipose tissue and in brain glia, two tissues that do not express the transgene. The formation of aggregates of nanospherules increased with age and was more considerable in flies with two copies of mutated TTR. Treatment of human neuronal cells with protein extracts prepared from TTR flies of different age showed that the extracts from older flies were less toxic than those from younger flies. Conclusions/Significance: These findings suggest that the uptake of TTR from the circulation and its subsequent segregation into cytoplasmic quasi-crystalline arrays of nanospherules is part of a mechanism that neutralizes the toxic effect of TTR.Original Publication:Malgorzata Pokrzywa, Ingrid Dacklin, Monika Vestling, Dan Hultmark, Erik Lundgren and Rafael Cantera, Uptake of Aggregating Transthyretin by Fat Body in a Drosophila Model for TTR-Associated Amyloidosis, 2010, PLOS ONE, (5), 12.http://dx.doi.org/10.1371/journal.pone.0014343Licensee: Public Library of Science (PLoS)http://www.plos.org

Transthyretin Aggregation Pathway toward the Formation of Distinct Cytotoxic Oligomers

Characterization of small oligomers formed at an early stage of amyloid formation is critical to understanding molecular mechanism of pathogenic aggregation process. Here we identifed and characterized cytotoxic oligomeric intermediates populated during transthyretin (TTR) aggregation process. Under the amyloid-forming conditions, TTR initially forms a dimer through interactions between outer strands. The dimers are then associated to form a hexamer with a spherical shape, which serves as a building block to self-assemble into cytotoxic oligomers. Notably, wild-type (WT) TTR tends to form linear oligomers, while aTTR variant(G53A) prefers forming annular oligomers with pore-like structures. Structural analyses of the amyloidogenic intermediates using circular dichroism (CD) and solid-state NMR revealthatthe dimer and oligomers have a signifcant degree of native-like β-sheet structures (35–38%), but with more disordered regions (~60%)than those of nativeTTR.TheTTR variant oligomers are also less structured than WT oligomers. The partially folded nature of the oligomeric intermediates might be a common structural property of cytotoxic oligomers.The higher fexibility of the dimer and oligomers may also compensate for the entropic loss due to the oligomerization of the monomers

Amitriptyline-Mediated Cognitive Enhancement in Aged 3×Tg Alzheimer's Disease Mice Is Associated with Neurogenesis and Neurotrophic Activity

Approximately 35 million people worldwide suffer from Alzheimer's disease (AD). Existing therapeutics, while moderately effective, are currently unable to stem the widespread rise in AD prevalence. AD is associated with an increase in amyloid beta (Aβ) oligomers and hyperphosphorylated tau, along with cognitive impairment and neurodegeneration. Several antidepressants have shown promise in improving cognition and alleviating oxidative stress in AD but have failed as long-term therapeutics. In this study, amitriptyline, an FDA-approved tricyclic antidepressant, was administered orally to aged and cognitively impaired transgenic AD mice (3×TgAD). After amitriptyline treatment, cognitive behavior testing demonstrated that there was a significant improvement in both long- and short-term memory retention. Amitriptyline treatment also caused a significant potentiation of non-toxic Aβ monomer with a concomitant decrease in cytotoxic dimer Aβ load, compared to vehicle-treated 3×TgAD controls. In addition, amitriptyline administration caused a significant increase in dentate gyrus neurogenesis as well as increases in expression of neurosynaptic marker proteins. Amitriptyline treatment resulted in increases in hippocampal brain-derived neurotrophic factor protein as well as increased tyrosine phosphorylation of its cognate receptor (TrkB). These results indicate that amitriptyline has significant beneficial actions in aged and damaged AD brains and that it shows promise as a tolerable novel therapeutic for the treatment of AD

Amyloid Oligomer Conformation in a Group of Natively Folded Proteins

Recent in vitro and in vivo studies suggest that destabilized proteins with defective folding induce aggregation and toxicity in protein-misfolding diseases. One such unstable protein state is called amyloid oligomer, a precursor of fully aggregated forms of amyloid. Detection of various amyloid oligomers with A11, an anti-amyloid oligomer conformation-specific antibody, revealed that the amyloid oligomer represents a generic conformation and suggested that toxic β-aggregation processes possess a common mechanism. By using A11 antibody as a probe in combination with mass spectrometric analysis, we identified GroEL in bacterial lysates as a protein that may potentially have an amyloid oligomer conformation. Surprisingly, A11 reacted not only with purified GroEL but also with several purified heat shock proteins, including human Hsp27, 40, 70, 90; yeast Hsp104; and bovine Hsc70. The native folds of A11-reactive proteins in purified samples were characterized by their anti-β-aggregation activity in terms of both functionality and in contrast to the β-aggregation promoting activity of misfolded pathogenic amyloid oligomers. The conformation-dependent binding of A11 with natively folded Hsp27 was supported by the concurrent loss of A11 reactivity and anti-β-aggregation activity of heat-treated Hsp27 samples. Moreover, we observed consistent anti-β-aggregation activity not only by chaperones containing an amyloid oligomer conformation but also by several A11-immunoreactive non-chaperone proteins. From these results, we suggest that the amyloid oligomer conformation is present in a group of natively folded proteins. The inhibitory effects of A11 antibody on both GroEL/ES-assisted luciferase refolding and Hsp70-mediated decelerated nucleation of Aβ aggregation suggested that the A11-binding sites on these chaperones might be functionally important. Finally, we employed a computational approach to uncover possible A11-binding sites on these targets. Since the β-sheet edge was a common structural motif having the most similar physicochemical properties in the A11-reactive proteins we analyzed, we propose that the β-sheet edge in some natively folded amyloid oligomers is designed positively to prevent β aggregation

A new era for understanding amyloid structures and disease

The aggregation of proteins into amyloid fibrils and their deposition into plaques and intracellular inclusions is the hallmark of amyloid disease. The accumulation and deposition of amyloid fibrils, collectively known as amyloidosis, is associated with many pathological conditions that can be associated with ageing, such as Alzheimer disease, Parkinson disease, type II diabetes and dialysis-related amyloidosis. However, elucidation of the atomic structure of amyloid fibrils formed from their intact protein precursors and how fibril formation relates to disease has remained elusive. Recent advances in structural biology techniques, including cryo-electron microscopy and solid-state NMR spectroscopy, have finally broken this impasse. The first near-atomic-resolution structures of amyloid fibrils formed in vitro, seeded from plaque material and analysed directly ex vivo are now available. The results reveal cross-β structures that are far more intricate than anticipated. Here, we describe these structures, highlighting their similarities and differences, and the basis for their toxicity. We discuss how amyloid structure may affect the ability of fibrils to spread to different sites in the cell and between organisms in a prion-like manner, along with their roles in disease. These molecular insights will aid in understanding the development and spread of amyloid diseases and are inspiring new strategies for therapeutic intervention

Genetic evaluation combining purebred and crossbred data in a pig breeding scheme

[EN] Genetic evaluations using purebred data alone and combined purebred and crossbred information were performed for lean meat percentage in a pig breeding scheme. One purebred (PB) model and 2 crossbred models (CCPS1 and CCPS2) were used in the analyses. Data were obtained from the Seleccion Batalle S.A. Company (Riudarenes, Spain) and spanned a period of 4 yr (2006 to 2009). The data corresponded to 3 nuclei of purebred populations, Landrace (LD), Duroc (DU), and Pietrain (PI); 1 multiplying farm with animals from a 2-way cross (TB1; DU x LD); and commercial farms with animals from a 3-way cross (TB2; TB1 x PI). Genetic parameters were similar across the models, with the exception of purebred PI. The DU and LD purebreds presented large heritabilities (0.5 to 0.6) for lean meat percentage, whereas the PI purebred showed a lower heritability (approximately 0.1) for the PB model and moderate heritability for the CCPS1 and CCPS2 models (0.2 to 0.3). The mean reliability of the predicted purebred breeding values was clearly increased when the CCPS1 and CCPS2 models were used. Moreover, a reranking of the animals with important changes in the selection decisions was observed in the PI purebred. In a simulation study, the CCPS1 model achieved a greater response to selection than the PB model for the PI purebred. On another hand, between the CCPS1 and CCPS2 models, CCPS1 was slightly superior in terms of predictive ability, exhibiting a greater robustness. These results illustrate the usefulness of using crossbred models to evaluate lean meat percentage in this pig breeding scheme.Financial support was provided by the IRTA, Lleida, Spain (grant 0502-21191).Ibáñez-Escriche, N.; Reixach, J.; Lleonart, N.; Noguera, JL. (2011). Genetic evaluation combining purebred and crossbred data in a pig breeding scheme. Journal of Animal Science. 89(12):3881-3889. https://doi.org/10.2527/jas.2011-3959S388138898912Bijma, P., & van Arendonk, J. A. M. (1998). Maximizing genetic gain for the sire line of a crossbreeding scheme utilizing both purebred and crossbred information. Animal Science, 66(2), 529-542. doi:10.1017/s135772980000970xBusk, H., Olsen, E. V., & Brøndum, J. (1999). Determination of lean meat in pig carcasses with the Autofom classification system. Meat Science, 52(3), 307-314. doi:10.1016/s0309-1740(99)00007-8Dekkers, J. C. M. (2007). Marker-assisted selection for commercial crossbred performance1. Journal of Animal Science, 85(9), 2104-2114. doi:10.2527/jas.2006-683Ducos, A., Bidanel, J., Ducrocq, V., Boichard, D., & Groeneveld, E. (1993). Multivariate restricted maximum likelihood estimation of genetic parameters for growth, carcass and meat quality traits in French Large White and French Landrace pigs. Genetics Selection Evolution, 25(5), 475. doi:10.1186/1297-9686-25-5-475Elzo, M. A. (1994). Restricted maximum likelihood procedures for the estimation of additive and nonadditive genetic variances and covariances in multibreed populations1. Journal of Animal Science, 72(12), 3055-3065. doi:10.2527/1994.72123055xGilbert, H., Bidanel, J.-P., Gruand, J., Caritez, J.-C., Billon, Y., Guillouet, P., … Sellier, P. (2007). Genetic parameters for residual feed intake in growing pigs, with emphasis on genetic relationships with carcass and meat quality traits. Journal of Animal Science, 85(12), 3182-3188. doi:10.2527/jas.2006-590Knapp, P., Willam, A., & Sölkner, J. (1997). Genetic parameters for lean meat content and meat quality traits in different pig breeds. Livestock Production Science, 52(1), 69-73. doi:10.1016/s0301-6226(97)00120-6Lo, L. L., Fernando, R. L., & Grossman, M. (1993). Covariance between relatives in multibreed populations: additive model. Theoretical and Applied Genetics, 87(4), 423-430. doi:10.1007/bf00215087Lutaaya, E., Misztal, I., Mabry, J. W., Short, T., Timm, H. H., & Holzbauer, R. (2001). Genetic parameter estimates from joint evaluation of purebreds and crossbreds in swine using the crossbred model. Journal of Animal Science, 79(12), 3002. doi:10.2527/2001.79123002xLutaaya, E., Misztal, I., Mabry, J. W., Short, T., Timm, H. H., & Holzbauer, R. (2002). Joint evaluation of purebreds and crossbreds in swine. Journal of Animal Science, 80(9), 2263. doi:10.2527/2002.8092263xMunilla Leguizamon, S., & Cantet, R. J. (2010). Equivalence of multibreed animal models and hierarchical Bayes analysis for maternally influenced traits. Genetics Selection Evolution, 42(1), 20. doi:10.1186/1297-9686-42-20Tholen, E., Baulain, U., Henning, M. D., & Schellander, K. (2003). Comparison of different methods to assess the composition of pig bellies in progeny testing. Journal of Animal Science, 81(5), 1177-1184. doi:10.2527/2003.8151177xWei, M. 1992. Combined crossbred and purebred selection in animal breeding. PhD Thesis.Department of Animal Breeding, Wageningen Agricultural University, Wageningen, the Netherlands

Age-Related Oxidative Modifications of Transthyretin Modulate Its Amyloidogenicity

The transthyretin amyloidoses are diseases of protein misfolding characterized by the extracellular deposition of fibrils and other aggregates of the homotetrameric protein transthyretin (TTR) in peripheral nerves, heart, and other tissues. Age is the major risk factor for the development of these diseases. We hypothesized that an age-associated increase in the level of protein oxidation could be involved in the onset of the senile forms of the TTR amyloidoses. To test this hypothesis, we have produced and characterized relevant age-related oxidative modifications of the wild type (WT) and the Val122Ile (V122I) TTR variant, both involved in cardiac TTR deposition in the elderly. Our studies show that methionine/cysteine-oxidized TTR and carbonylated TTR from either the WT or the V122I variant are thermodynamically less stable than their nonoxidized counterparts. Moreover, carbonylated WT and carbonylated V122I TTR have a stronger propensity to form aggregates and fibrils than WT and V122I TTR, respectively, at physiologically attainable pH values. It is well-known that TTR tetramer dissociation, the limiting step for aggregation and amyloid fibril formation, can be prevented by small molecules that bind the TTR tetramer interface. Here, we report that carbonylated WT TTR is less amenable to resveratrol-mediated tetramer stabilization than WT TTR. All the oxidized forms of TTR tested are cytotoxic to a human cardiomyocyte cell line known to be a target for cardiac-specific TTR variants. Overall, these studies demonstrate that age-related oxidative modifications of TTR can contribute to the onset of the senile forms of the TTR amyloidoses