Increased Serum Hepcidin Levels in Subjects with the Metabolic Syndrome: A Population Study

The recent discovery of hepcidin, the key iron regulatory hormone, has changed our view of iron metabolism, which in turn is long known to be linked with insulin resistant states, including type 2 diabetes mellitus and the Metabolic Syndrome (MetS). Serum ferritin levels are often elevated in MetS (Dysmetabolic hyperferritinemia - DHF), and are sometimes associated with a true mild-to-moderate hepatic iron overload (dysmetabolic iron overload syndrome - DIOS). However, the pathophysiological link between iron and MetS remains unclear. This study was aimed to investigate, for the first time, the relationship between MetS and hepcidin at population level. We measured serum hepcidin levels by Mass Spectrometry in 1,391 subjects from the Val Borbera population, and evaluated their relationship with classical MetS features. Hepcidin levels increased significantly and linearly with increasing number of MetS features, paralleling the trend of serum ferritin. In multivariate models adjusted for relevant variables including age, C-Reactive Protein, and the HFE C282Y mutation, ferritin was the only significant independent predictor of hepcidin in males, while in females MetS was also independently associated with hepcidin. Overall, these data indicate that the fundamental iron regulatory feedback is preserved in MetS, i.e. that hepcidin tends to progressively increase in response to the increase of iron stores. Due to recently discovered pleiotropic effects of hepcidin, this may worsen insulin resistance and contribute to the cardiovascular complications of MetS.     

Evolutionary Reconstructions of the Transferrin Receptor of Caniforms Supports Canine Parvovirus Being a Re-emerged and Not a Novel Pathogen in Dogs

Parvoviruses exploit transferrin receptor type-1 (TfR) for cellular entry in carnivores, and specific interactions are key to control of host range. We show that several key mutations acquired by TfR during the evolution of Caniforms (dogs and related species) modified the interactions with parvovirus capsids by reducing the level of binding. These data, along with signatures of positive selection in the TFRC gene, are consistent with an evolutionary arms race between the TfR of the Caniform clade and parvoviruses. As well as the modifications of amino acid sequence which modify binding, we found that a glycosylation site mutation in the TfR of dogs which provided resistance to the carnivore parvoviruses which were in circulation prior to about 1975 predates the speciation of coyotes and dogs. Because the closely-related black-backed jackal has a TfR similar to their common ancestor and lacks the glycosylation site, reconstructing this mutation into the jackal TfR shows the potency of that site in blocking binding and infection and explains the resistance of dogs until recent times. This alters our understanding of this well-known example of viral emergence by indicating that canine parvovirus emergence likely resulted from the re-adaptation of a parvovirus to the resistant receptor of a former host.     

Scale‐dependent post‐establishment spread and genetic diversity in an invading mollusc in South America

Aims Our study aimed to characterize the dispersal dynamics and population genetic structure of the introduced golden mussel Limnoperna fortunei throughout its invaded range in South America and to determine how different dispersal methods, that is, human‐mediated dispersal and downstream natural dispersal, contribute to genetic variation among populations. Location Paraná–Uruguay–Río de la Plata watershed in Argentina, Brazil, Paraguay and Uruguay. Methods We performed genetic analyses based on a comprehensive sampling strategy encompassing 22 populations (N = 712) throughout the invaded range in South America, using the mitochondrial cytochrome c oxidase subunit I (COI) gene and eight polymorphic nuclear microsatellites. We employed both population genetics and phylogenetic analyses to clarify the dispersal dynamics and population genetic structure. Results We detected relatively high genetic differentiation between populations (F_ST = ?0.041 to 0.111 for COI, ?0.060 to 0.108 for microsatellites) at both fine and large geographical scales. Bayesian clustering and three‐dimensional factorial correspondence analyses consistently revealed two genetically distinct clusters, highlighting genetic discontinuities in the invaded range. Results of all genetic analyses suggest ship‐mediated ‘jump’ dispersal as the dominant mode of spread of golden mussels in South America, while downstream natural dispersal has had limited effects on contemporary genetic patterns. Main conclusions Our study provides new evidence that post‐establishment dispersal dynamics and genetic patterns vary across geographical scales. While ship‐mediated ‘jump’ dispersal dominates post‐establishment spread of golden mussels in South America, once colonies become established in upstream locations, larvae produced may be advected downstream to infill patchy distributions. Moreover, genetic structuring at fine geographical scales, especially within the same drainages, suggests a further detailed understanding of dynamics of larval dispersal and settlement in different water systems. Knowledge of the mechanisms by which post‐establishment spread occurs can, in some cases, be used to limit dispersal of golden mussels and other introduced species.     

Identification of collagen-induced arthritis loci in aged multiparous female mice

Collagen-induced arthritis in mice is one of the most commonly used autoimmune experimental models, with many similarities to rheumatoid arthritis. Since collagen-induced arthritis is a complex polygenic disease there is a need for identification of several major disease-controlling genes. Because rheumatoid arthritis particularly affects aged women, we have in the present study identified new genetic regions critical for collagen-induced arthritis by studying aged female mice of a cross between NFR/N and B10.Q (H-2^q haplotype). The mice in the present study had different reproductive histories, which did not significantly affect the onset, incidence or severity of the disease. A total of 200 female mice were used in a total genome-wide screening with 125 microsatellite markers. We found one new significant quantitative trait locus affecting the arthritis incidence, severity and day of onset on chromosome 11 (denoted Cia40), which colocalizes with a locus controlling pregnancy failure. Furthermore, a quantitative trait locus of suggestive significance associated with the incidence, severity and day of onset was identified on chromosome 1. Finally, a suggestively significant quantitative trait locus associated with collagen type II antibody titers was identified on chromosome 13. This study indicates that several gene loci control arthritis in aged multiparous females, and that at least one of these loci coincides with pregnancy failure.     

The NatA Acetyltransferase Couples Sup35 Prion Complexes to the [PSI+] Phenotype

Protein-only (prion) epigenetic elements confer unique phenotypes by adopting alternate conformations that specify new traits. Given the conformational flexibility of prion proteins, protein-only inheritance requires efficient self-replication of the underlying conformation. To explore the cellular regulation of conformational self-replication and its phenotypic effects, we analyzed genetic interactions between [PSI⁺], a prion form of the S. cerevisiae Sup35 protein (Sup35^[PSI+]), and the three N^α-acetyltransferases, NatA, NatB, and NatC, which collectively modify ~50% of yeast proteins. Although prion propagation proceeds normally in the absence of NatB or NatC, the [PSI⁺] phenotype is reversed in strains lacking NatA. Despite this change in phenotype, [PSI⁺] NatA mutants continue to propagate heritable Sup35^[PSI+]. This uncoupling of protein state and phenotype does not arise through a decrease in the number or activity of prion templates (propagons) or through an increase in soluble Sup35. Rather, NatA null strains are specifically impaired in establishing the translation termination defect that normally accompanies Sup35 incorporation into prion complexes. The NatA effect cannot be explained by the modification of known components of the [PSI⁺] prion cycle including Sup35; thus, novel acetylated cellular factors must act to establish and maintain the tight link between Sup35^[PSI+] complexes and their phenotypic effects.     

Differential interactions of fluorescent agonists and antagonists with the yeast G protein coupled receptor Ste2p

We describe a rapid method to probe for mutations in cell surface ligand-binding proteins that affect the environment of bound ligand. The method uses fluorescence-activated cell sorting to screen randomly mutated receptors for substitutions that alter the fluorescence emission spectrum of environmentally sensitive fluorescent ligands. When applied to the yeast α-factor receptor Ste2p, a G protein-coupled receptor, the procedure identified 22 substitutions that red shift the emission of a fluorescent agonist, including substitutions at residues previously implicated in ligand binding and at additional sites. A separate set of substitutions, identified in a screen for mutations that alter the emission of a fluorescent α-factor antagonist, occurs at sites that are unlikely to contact the ligand directly. Instead, these mutations alter receptor conformation to increase ligand-binding affinity and provide signaling in response to antagonists of normal receptors. These results suggest that receptor-agonist interactions involve at least two sites, of which only one is specific for the activated conformation of the receptor.     

Functional mimicry of the LDL receptor-associated protein through multimerization of a minimized third domain

The LDL receptor-associated protein (RAP) is a ligand for the LDL receptor-related protein (LRP1). The first and third domains of RAP can each bind to one of many sequence-related pairs of complement-type repeats (CR) found within the LRP1 ectodomain. Multiple sites of interaction between the multivalent RAP ligand and the multivalent LRP1 receptor yield strong binding avidity for the complex. The third domain of RAP can be significantly truncated, with material retention of monovalent CR pair-binding affinity, provided that the minimized sequence is stabilized with an intramolecular disulfide bond. We demonstrate that the avidity of full-length RAP for LRP1 in vitro can be partially reconstituted by assembly of truncated, disulfide-linked RAP peptides on tetravalent streptavidin or bivalent immunoglobulin scaffolds. The peptide complex with streptavidin shows pronounced hepatotropism in vivo, replicating the biodistribution of full-length RAP.     

Catalytic activity, stability, unfolding, and degradation pathways of engineered and reconstituted myoglobins

The structural and functional consequences of engineering a positively charged Lys residue and replacing the natural heme with a heme-L-His derivative in the active site of sperm whale myoglobin (Mb) have been investigated. The main structural change caused by the distal T67K mutation appears to be mobilization of the propionate-7 group. Reconstitution of wild-type and T67K Mb with heme-L-His relaxes the protein fragment around the heme because it involves the loss of the interaction of one of the propionate groups which stabilize heme binding to the protein. This modification increases the accessibility of exogenous ligands or substrates to the active site. The catalytic activity of the reconstituted proteins in peroxidase-type reactions is thus significantly increased, particularly with T67K Mb. The T67K mutation slightly reduces the thermodynamic stability and the chemical stability of Mb during catalysis, but somewhat more marked effects are observed by cofactor reconstitution. Hydrogen peroxide, in fact, induces pseudo-peroxidase activity but also promotes oxidative damage of the protein. The mechanism of protein degradation involves two pathways, which depend on the evolution of radical species generated on protein residues by the Mb active species and on the reactivity of phenoxy radicals produced during turnover. Both protein oligomers and heme-protein cross-links have been detected upon inactivation.     

Cytosolic prion protein (PrP) is not toxic in N2a cells and primary neurons expressing pathogenic PrP mutations

Inherited prion diseases are linked to mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. It has been proposed that neuronal death can be triggered by accumulation of PrP in the cytosol because of impairment of proteasomal degradation of misfolded PrP molecules retrotranslocated from the endoplasmic reticulum (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). To test whether this neurotoxic mechanism is operative in inherited prion diseases, we evaluated the effect of proteasome inhibitors on the viability of transfected N2a cells and primary neurons expressing mouse PrP homologues of the D178N and nine octapeptide mutations. We found that the inhibitors caused accumulation of an unglycosylated, aggregated form of PrP exclusively in transfected N2a expressing PrP from the cytomegalovirus promoter. This form contained an uncleaved signal peptide, indicating that it represented polypeptide chains that had failed to translocate into the ER lumen during synthesis, rather than retrogradely translocated PrP. Quantification of N2a viability in the presence of proteasome inhibitors demonstrated that accumulation of this form was not toxic. No evidence of cytosolic PrP was found in cerebellar granule neurons from transgenic mice expressing wild-type or mutant PrPs from the endogenous promoter, nor were these neurons more susceptible to proteasome inhibitor toxicity than neurons from PrP knock-out mice. Our analysis fails to confirm the previous observation that mislocation of PrP in the cytosol is neurotoxic, and argues against the hypothesis that perturbation of PrP metabolism through the proteasomal pathway plays a pathogenic role in prion diseases.