Translation elongation factor eEF1A2 is essential for post-weaning survival in mice

Translation elongation factor eEF1A, formerly known as EF-1 alpha, exists as two variant forms; eEF1A1, which is almost ubiquitously expressed, and eEF1A2, whose expression is restricted to muscle and brain at the level of whole tissues. Expression analysis of these genes has been complicated by a general lack of availability of antibodies that specifically recognize each variant form. Wasted mice (wst/wst) have a 15.8-kilobase deletion that abolishes activity of eEF1A2, but before this study it was unknown whether the deletion also affected neighboring genes. We have generated a panel of anti-peptide antibodies and used them to show that eEF1A2 is expressed at high levels in specific cell types in tissues previously thought not to express this variant, such as pancreatic islet cells and enteroendocrine cells in colon crypts. Expression of eEF1A1 and eEF1A2 is shown to be generally mutually exclusive, and we relate the expression pattern of eEF1A2 to the phenotype seen in wasted mice. We then carried out a series of transgenic experiments to establish whether the expression of other genes is affected by the deletion in wasted mice. We show that aspects of the phenotype such as motor neuron degeneration relate precisely to the relative expression of eEF1A1 and eEF1A2, whereas the immune system abnormalities are likely to result from a stress response. We conclude that loss of eEF1A2 function is solely responsible for the abnormalities seen in these mice.     

Interaction of cartilage oligomeric matrix protein/thrombospondin 5 with aggrecan

Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP5) is a major component of the extracellular matrix (ECM) of the musculoskeletal system. Its importance is underscored by its association with several growth disorders. In this report, we investigated its interaction with aggrecan, a major component of cartilage ECM. We also tested a COMP/TSP5 mutant, designated MUT3 that accounts for 30% of human pseudoachondroplasia cases, to determine if the mutation affects function. Using a solid-phase binding assay, we have shown that COMP/TSP5 can bind aggrecan. This binding was decreased with MUT3, or when COMP/TSP5 was treated with EDTA, indicating the presence of a conformation-dependent aggrecan binding site. Soluble glycosaminoglycans (GAGs) partially inhibited binding, suggesting that the interaction was mediated in part through aggrecan GAG side chains. Using affinity co-electrophoresis, we showed that COMP/TSP5, in its calcium-replete conformation, bound to heparin, chondroitin sulfates, and heparan sulfate; this binding was reduced with EDTA treatment of COMP/TSP5. MUT3 showed weaker binding than calcium-repleted COMP/TSP5. Using recombinant COMP/TSP5 fragments, we found that the "signature domain" could bind to aggrecan, suggesting that this domain can mediate the interaction of COMP/TSP5 and aggrecan. In summary, our data indicate that COMP/TSP5 is an aggrecan-binding protein, and this interaction is regulated by the calcium-sensitive conformation of COMP/TSP5; interaction of COMP with aggrecan can be mediated through the GAG side chains on aggrecan and the "signature domain" of COMP/TSP5. Our results suggest that COMP/TSP5 may function to support matrix interactions in cartilage ECM.     

Rat liver carnitine palmitoyltransferase 1 forms an oligomeric complex within the outer mitochondrial membrane

Carnitine palmitoyltransferase (CPT) 1A catalyzes the rate-limiting step in the transport of long chain acyl-CoAs from cytoplasm to the mitochondrial matrix by converting them to acylcarnitines. Located within the outer mitochondrial membrane, CPT1A activity is inhibited by malonyl-CoA, its allosteric inhibitor. In this study, we investigate for the first time the quaternary structure of rat CPT1A. Chemical cross-linking studies using intact mitochondria isolated from fed rat liver or from Saccharomyces cerevisiae expressing CPT1A show that CPT1A self-assembles into an oligomeric complex. Size exclusion chromatography experiments using solubilized mitochondrial extracts suggest that the fundamental unit of its quaternary structure is a trimer. When studied in blue native-PAGE, the CPT1A hexamer could be observed, however, suggesting that under these native conditions CPT1A trimers might be arranged as dimers. Moreover, the oligomeric state of CPT1A was found unchanged by starvation and by streptozotocin-induced diabetes, conditions characterized by changes in malonyl-CoA sensitivity of CPT1A. Finally, gel filtration analysis of several yeast-expressed chimeric CPTs demonstrates that the first 147 N-terminal residues of CPT1A, encompassing its two transmembrane segments, trigger trimerization independently of its catalytic C-terminal domain. Deletion of residues 1-82, including transmembrane 1, did not abrogate oligomerization, but the latter is limited to a trimer by the presence of the large catalytic C-terminal domain on the cytosolic face of mitochondria. Based on these findings, we proposed that the oligomeric structure of CPT1A would allow the newly formed acylcarnitines to gain direct access into the intermembrane space, hence facilitating substrate channeling.     

Identification of the Propionicin F Bacteriocin Immunity Gene (pcfI) and Development of a Food-Grade Cloning System for Propionibacterium freudenreichii

This report describes the first functional analysis of a bacteriocin immunity gene from Propionibacterium freudenreichii and its use as a selection marker for food-grade cloning. Cloning of the pcfI gene (previously orf5 [located as part of the pcfABC propionicin F operon]) rendered the sensitive host 1,000-fold more tolerant to the propionicin F bacteriocin. The physiochemical properties of the 127-residue large PcfI protein resemble those of membrane-bound immunity proteins from bacteriocin systems found in lactic acid bacteria. The high level of immunity conferred by pcfI allowed its use as a selection marker for plasmid transformation in P. freudenreichii. Electroporation of P. freudenreichii IFO12426 by use of the pcfI expression plasmid pSL102 and propionicin F selection (200 bacteriocin units/ml) yielded 10⁷ transformants/μg DNA. The 2.7-kb P. freudenreichii food-grade cloning vector pSL104 consists of the pLME108 replicon, a multiple cloning site, and pcfI expressed from the constitutive P_pampS promoter for selection. The pSL104 vector efficiently facilitated cloning of the propionicin T1 bacteriocin in P. freudenreichii. High-level propionicin T1 production (640 BU/ml) was obtained with the IFO12426 strain, and the food-grade propionicin T1 expression plasmid pSL106 was maintained by ~91% of the cells over 25 generations in the absence of selection. To the best of our knowledge this is the first report of an efficient cloning system that facilitates the generation of food-grade recombinant P. freudenreichii strains.     

11beta-Hydroxysteroid Dehydrogenase Type 1 Regulation by Intracellular Glucose 6-Phosphate Provides Evidence for a Novel Link between Glucose Metabolism and Hypothalamo-Pituitary-Adrenal Axis Function

Microsomal glucose-6-phosphatase-alpha (G6Pase-alpha) and glucose 6-phosphate transporter (G6PT) work together to increase blood glucose concentrations by performing the terminal step in both glycogenolysis and gluconeogenesis. Deficiency of the G6PT in liver gives rise to glycogen storage disease type 1b (GSD1b), whereas deficiency of G6Pase-alpha leads to GSD1a. G6Pase-alpha shares its substrate (glucose 6-phosphate; G6P) with hexose-6-phosphate-dehydrogenase (H6PDH), a microsomal enzyme that regenerates NADPH within the endoplasmic reticulum lumen, thereby conferring reductase activity upon 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1). 11beta-HSD1 interconverts hormonally active C11beta-hydroxy steroids (cortisol in humans and corticosterone in rodents) to inactive C11-oxo steroids (cortisone and 11-dehydrocorticosterone, respectively). In vivo reductase activity predominates, generating active glucocorticoid. We hypothesized that substrate (G6P) availability to H6PDH in patients with GSD1b and GSD1a will decrease or increase 11beta-HSD1 reductase activity, respectively. We investigated 11beta-HSD1 activity in GSD1b and GSD1a mice and in two patients with GSD1b and five patients diagnosed with GSD1a. We confirmed our hypothesis by assessing 11beta-HSD1 in vivo and in vitro, revealing a significant decrease in reductase activity in GSD1b animals and patients, whereas GSD1a patients showed a marked increase in activity. The cellular trafficking of G6P therefore directly regulates 11beta-HSD1 reductase activity and provides a novel link between glucose metabolism and function of the hypothalamo-pituitary-adrenal axis.     

Discovery of novel 2,3-diarylfuro[2,3-b]pyridin-4-amines as potent and selective inhibitors of Lck: synthesis, SAR, and pharmacokinetic properties

2,3-Diarylfuro[2,3-b]pyridine-4-amines are a novel class of potent and selective inhibitors of Lck. The discovery, synthesis, and structure activity relationships of this series of inhibitors are reported. The most promising compounds were also profiled to deduce their pharmacokinetic properties.     

Multiple common susceptibility variants near BMP pathway loci GREM1, BMP4, and BMP2 explain part of the missing heritability of colorectal cancer

Genome-wide association studies (GWAS) have identified 14 tagging single nucleotide polymorphisms (tagSNPs) that are associated with the risk of colorectal cancer (CRC), and several of these tagSNPs are near bone morphogenetic protein (BMP) pathway loci. The penalty of multiple testing implicit in GWAS increases the attraction of complementary approaches for disease gene discovery, including candidate gene- or pathway-based analyses. The strongest candidate loci for additional predisposition SNPs are arguably those already known both to have functional relevance and to be involved in disease risk. To investigate this proposition, we searched for novel CRC susceptibility variants close to the BMP pathway genes GREM1 (15q13.3), BMP4 (14q22.2), and BMP2 (20p12.3) using sample sets totalling 24,910 CRC cases and 26,275 controls. We identified new, independent CRC predisposition SNPs close to BMP4 (rs1957636, P = 3.93×10(-10)) and BMP2 (rs4813802, P = 4.65×10(-11)). Near GREM1, we found using fine-mapping that the previously-identified association between tagSNP rs4779584 and CRC actually resulted from two independent signals represented by rs16969681 (P = 5.33×10(-8)) and rs11632715 (P = 2.30×10(-10)). As low-penetrance predisposition variants become harder to identify-owing to small effect sizes and/or low risk allele frequencies-approaches based on informed candidate gene selection may become increasingly attractive. Our data emphasise that genetic fine-mapping studies can deconvolute associations that have arisen owing to independent correlation of a tagSNP with more than one functional SNP, thus explaining some of the apparently missing heritability of common diseases.     

New inflammation-related biomarkers during malaria infection

Malaria is one of the most prevalent infectious diseases worldwide with more than 250 million cases and one million deaths each year. One of the well-characterized malarial-related molecules is hemozoin (HZ), which is a dark-brown crystal formed by the parasite and released into the host during the burst of infected red blood cells. HZ has a stimulatory effect on the host immune system such as its ability to induce pro-inflammatory mediators responsible for some of the malaria related clinical symptoms such as fever. However, the host serum proteins interacting with malarial HZ as well as how this interaction modifies its recognition by phagocytes remained elusive. In the actual study, using proteomic liquid chromatographic mass spectrometry (LC-MS/MS) and immunochemical approaches, we compared the serum protein profiles of malaria patients and healthy individuals. Particularly, we utilized the malarial HZ itself to capture serum proteins capable to bind to HZ, enabling us to identify several proteins such as apolipoprotein E (ApoE), serum amyloid A (SAA), gelsolin, complement factor H and fibrinogen that were found to differ among healthy and malaria individual. Of particular interest is LPS binding protein (LBP), which is reported herein for the first time in the context of malaria. LBP is usually produced during innate inflammatory response to gram-negative bacterial infections. The exact role of these biomarkers and acute phase responses in malaria in general and HZ in particular remains to be investigated. The identification of these inflammation-related biomarkers in malaria paves the way to potentially utilize them as diagnostic and therapeutic targets.     

Impact of changing drug treatment and malaria endemicity on the heritability of malaria phenotypes in a longitudinal family-based cohort study

Despite considerable success of genome wide association (GWA) studies in identifying causal variants for many human diseases, their success in unraveling the genetic basis to complex diseases has been more mitigated. Pathogen population structure may impact upon the infectious phenotype, especially with the intense short-term selective pressure that drug treatment exerts on pathogens. Rigorous analysis that accounts for repeated measures and disentangles the influence of genetic and environmental factors must be performed. Attempts should be made to consider whether pathogen diversity will impact upon host genetic responses to infection.We analyzed the heritability of two Plasmodium falciparum phenotypes, the number of clinical malaria episodes (PFA) and the proportion of these episodes positive for gametocytes (Pfgam), in a family-based cohort followed for 19 years, during which time there were four successive drug treatment regimes, with documented appearance of drug resistance. Repeated measures and variance components analyses were performed with fixed environmental, additive genetic, intra-individual and maternal effects for each drug period. Whilst there was a significant additive genetic effect underlying PFA during the first drug period of study, this was lost in subsequent periods. There was no additive genetic effect for Pfgam. The intra-individual effect increased significantly in the chloroquine period.The loss of an additive genetic effect following novel drug treatment may result in significant loss of power to detect genes in a GWA study. Prior genetic analysis must be a pre-requisite for more detailed GWA studies. The temporal changes in the individual genetic and the intra-individual estimates are consistent with those expected if there were specific host-parasite interactions. The complex basis to the human response to malaria parasite infection likely includes dominance/epistatic genetic effects encompassed within the intra-individual variance component. Evaluating their role in influencing the outcome of infection through host genotype by parasite genotype interactions warrants research effort.