Structural analysis of mevalonate‐3‐kinase provides insight into the mechanisms of isoprenoid pathway decarboxylases

In animals, cholesterol is made from 5‐carbon building blocks produced by the mevalonate pathway. Drugs that inhibit the mevalonate pathway such as atorvastatin (lipitor) have led to successful treatments for high cholesterol in humans. Another potential target for the inhibition of cholesterol synthesis is mevalonate diphosphate decarboxylase (MDD), which catalyzes the phosphorylation of (R)‐mevalonate diphosphate, followed by decarboxylation to yield isopentenyl pyrophosphate. We recently discovered an MDD homolog, mevalonate‐3‐kinase (M3K) from Thermoplasma acidophilum, which catalyzes the identical phosphorylation of (R)‐mevalonate, but without concomitant decarboxylation. Thus, M3K catalyzes half the reaction of the decarboxylase, allowing us to separate features of the active site that are required for decarboxylation from features required for phosphorylation. Here we determine the crystal structure of M3K in the apo form, and with bound substrates, and compare it to MDD structures. Structural and mutagenic analysis reveals modifications that allow M3K to bind mevalonate rather than mevalonate diphosphate. Comparison to homologous MDD structures show that both enzymes employ analogous Arg or Lys residues to catalyze phosphate transfer. However, an invariant active site Asp/Lys pair of MDD previously thought to play a role in phosphorylation is missing in M3K with no functional replacement. Thus, we suggest that the invariant Asp/Lys pair in MDD may be critical for decarboxylation rather than phosphorylation.     

Thyroid receptor ligands. Part 8: Thyromimetics derived from N-acylated-alpha-amino acid derivatives displaying modulated pharmacological selectivity compared with KB-141

Based on the scaffold of the pharmacologically selective thyromimetic 2b, structurally a close analog to KB-141 (2a), a number of novel N-acylated-alpha-amino acid derivatives were synthesized and tested in a TR radioligand binding assay as well as in a reporter cell assay. On the basis of TRbeta(1)-isoform selectivity and affinity, as well as affinity to the reporter cell assay, 3d was selected for further studies in the cholesterol-fed rat model. In this model 3d revealed an improved therapeutic window between cholesterol and TSH lowering but decreased margins versus tachycardia compared with 2a.     

Long‐term caloric restriction ameliorates deleterious effects of aging on white and brown adipose tissue plasticity

Age‐related increased adiposity is an important contributory factor in the development of insulin resistance (IR) and is associated with metabolic defects. Caloric restriction (CR) is known to induce weight loss and to decrease adiposity while preventing metabolic risk factors. Here, we show that moderate 20% CR delays early deleterious effects of aging on white and brown adipose tissue (WAT and BAT, respectively) function and improves peripheral IR. To elucidate the role of CR in delaying early signs of aging, young (3 months), middle‐aged (12 months), and old (20 months) mice fed al libitum and middle‐aged and old mice subjected to early‐onset CR were used. We show that impaired plasticity of subcutaneous WAT (scWAT) contributes to IR, which is already evident in middle‐aged mice. Moreover, alteration of thyroid axis status with age is an important factor contributing to BAT dysfunction in middle‐aged animals. Both defects in WAT and BAT/beige cells are ameliorated by CR. Accordingly, CR attenuated the age‐related decline in scWAT function and decreased the extent of fibro‐inflammation. Furthermore, CR promoted scWAT browning. In brief, our study identifies the contribution of scWAT impairment to age‐associated metabolic dysfunction and identifies browning in response to food restriction, as a potential therapeutic strategy to prevent the adverse metabolic effects in middle‐aged animals.     

Guidelines for a priori grouping of species in hierarchical community models

Recent methodological advances permit the estimation of species richness and occurrences for rare species by linking species‐level occurrence models at the community level. The value of such methods is underscored by the ability to examine the influence of landscape heterogeneity on species assemblages at large spatial scales. A salient advantage of community‐level approaches is that parameter estimates for data‐poor species are more precise as the estimation process “borrows” from data‐rich species. However, this analytical benefit raises a question about the degree to which inferences are dependent on the implicit assumption of relatedness among species. Here, we assess the sensitivity of community/group‐level metrics, and individual‐level species inferences given various classification schemes for grouping species assemblages using multispecies occurrence models. We explore the implications of these groupings on parameter estimates for avian communities in two ecosystems: tropical forests in Puerto Rico and temperate forests in northeastern United States. We report on the classification performance and extent of variability in occurrence probabilities and species richness estimates that can be observed depending on the classification scheme used. We found estimates of species richness to be most precise and to have the best predictive performance when all of the data were grouped at a single community level. Community/group‐level parameters appear to be heavily influenced by the grouping criteria, but were not driven strictly by total number of detections for species. We found different grouping schemes can provide an opportunity to identify unique assemblage responses that would not have been found if all of the species were analyzed together. We suggest three guidelines: (1) classification schemes should be determined based on study objectives; (2) model selection should be used to quantitatively compare different classification approaches; and (3) sensitivity of results to different classification approaches should be assessed. These guidelines should help researchers apply hierarchical community models in the most effective manner.     

Plasma metabolites and migration physiology of semipalmated sandpipers: refueling performance at five latitudes

Long-distance bird migration is fueled by energy gathered at stopover sites along the migration route. The refueling rate at stopover sites is a determinant of time spent at stopovers and impacts the overall speed of migration. Refueling rate during spring migration may influence the fitness of individuals via changes in the probability of successful migration and reproduction during the subsequent breeding season. We evaluated four plasma lipid metabolites (triglycerides, phospholipids, β-OH-butyrate, and glycerol) as measures of refueling rate in free-living semipalmated sandpipers (Calidris pusilla) captured at non-breeding areas. We described the spatial and temporal variation in metabolite concentrations among one winter site in the Dominican Republic and four stopover sites in the South Atlantic and Mid-Atlantic Coastal Plain regions of North America. Triglycerides and β-OH-butyrate clearly identified spatial variation in refueling rate and stopover habitat quality. Metabolite profiles indicated that birds had higher refueling rates at one site in the Mid-Atlantic Coastal Plain than at three sites on the South Atlantic Coastal Plain and one site in the Dominican Republic. Temporal variation in lipid metabolites during the migration season suggested that male semipalmated sandpipers gained more weight at stopovers on the South Atlantic Coastal Plain than did females, evidence of differential migration strategies for the sexes. Plasma lipid metabolites provide information on migration physiology that may help determine stopover habitat quality and reveal how migratory populations use stopover sites to refuel and successfully complete long-distance migrations.     

Poster Sessions CP02: Aging. Combination of NGF treatment and enrichment environment in old rats: biochemical and behavioral consequences

Brain glutathione system (Glut‐Syst) exhibits functional changes with age as well as during neurodegenerative diseases. After NGF treatment, cognitive functions and Glut‐Syst activity are favorably modified in aged rats. also, the environmental enrichment (E‐E) activates molecular mechanisms linked to cognition and sensorimotor coordination. We evaluate the functional repercussion of the combination of both factors. Old cognitively impaired rats received intracerebroventricular infusion of NGF (ICV, 22 mg/mL) or intraparenquimal (IP‐nbM, 5.5 mg/mL) during 14 days using ALZET osmotic minipumps. Simultaneously, these animals received a passive training in an E‐E during 4 weeks. A control group received training + saline infusion. Animals were assessed in the water maze task, avoidance passive test, open field test and transverse bridges test. At the end of the week 4, glutathione content and Choline Acetyltransferase (ChAT) activity were measured in brain areas of interest. E‐E or NGF treatments, particularly the IP route, improve the rat's overall behavioral performance but a synergic effect was observed when NGF and E‐E were applied simultaneously. A trend to hyperactivity was detected in the ICV group. Glutathione content and ChAT activity exhibited significant changes according to the group and brain area. It's well known that activity/levels of antioxidant enzymes and ChAT activity are related to age, brain region and neurotrophins activity. Results point out the possibilities of neurotrophic therapy if an adequate route of delivery is used as well as the benefit of combining a neurorehabilitation program on both, behavioural and protection from oxidative stress.     

Structural basis for the methylation site specificity of SET7/9

Human SET7/9 is a protein lysine methyltransferase (PKMT) that methylates histone H3, the tumor suppressor p53 and the TBP-associated factor TAF10. To elucidate the determinants of its substrate specificity, we have solved the enzyme's structure bound to a TAF10 peptide and examined its ability to methylate histone H3, TAF10 and p53 substrates bearing either mutations or covalent modifications within their respective methylation sites. Collectively, our data reveal that SET7/9 recognizes a conserved K/R-S/T/A motif preceding the lysine substrate and has a propensity to bind aspartates and asparagines on the C-terminal side of the lysine target. We then used a sequence-based approach with this motif to identify novel substrates for this PKMT. Among the putative targets is TAF7, which is methylated at Lys5 by the enzyme in vitro. These results demonstrate the predictive value of the consensus motif in identifying novel substrates for SET7/9.