The Acute Phase Protein Serum Amyloid A Induces Lipolysis and Inflammation in Human Adipocytes through Distinct Pathways

Background: The acute phase response (APR) is characterized by alterations in lipid and glucose metabolism leading to an increased delivery of energy substrates. In adipocytes, there is a coordinated decrease in Free Fatty acids (FFAs) and glucose storage, in addition to an increase in FFAs mobilization. Serum Amyloid A (SAA) is an acute phase protein mainly associated with High Density Lipoproteins (HDL). We hypothesized that enrichment of HDL with SAA, during the APR, could be implicated in the metabolic changes occurring in adipocytes. Methodology/Principal Findings: In vitro differentiated human adipocytes (hMADS) were treated with SAA enriched HDL or recombinant SAA and the metabolic phenotype of the cells analyzed. In hMADS, SAA induces an increased lipolysis through an ERK dependent pathway. At the molecular level, SAA represses PPARc2, C/EBPa and SREBP-1c gene expression, three transcription factors involved in adipocyte differentiation or lipid synthesis. In addition, the activation of the NF-kB pathway by SAA leads to the induction of pro-inflammatory cytokines and chemokines, as in the case of immune cells. These latter findings were replicated in freshly isolated mature human adipocytes. Conclusions/Significance: Besides its well-characterized role in cholesterol metabolism, SAA has direct metabolic effects on human adipocytes. These metabolic changes could be at least partly responsible for alterations of adipocyte metabolism observed during the APR as well as during pathophysiological conditions such as obesity and conditions leading to insuli

Complete Genome Sequence of Mycoplasma suis and Insights into Its Biology and Adaption to an Erythrocyte Niche

Mycoplasma suis, the causative agent of porcine infectious anemia, has never been cultured in vitro and mechanisms by which it causes disease are poorly understood. Thus, the objective herein was to use whole genome sequencing and analysis of M. suis to define pathogenicity mechanisms and biochemical pathways. M. suis was harvested from the blood of an experimentally infected pig. Following DNA extraction and construction of a paired end library, whole-genome sequencing was performed using GS-FLX (454) and Titanium chemistry. Reads on paired-end constructs were assembled using GS De Novo Assembler and gaps closed by primer walking; assembly was validated by PFGE. Glimmer and Manatee Annotation Engine were used to predict and annotate protein-coding sequences (CDS). The M. suis genome consists of a single, 742,431 bp chromosome with low G+C content of 31.1%. A total of 844 CDS, 3 single copies, unlinked rRNA genes and 32 tRNAs were identified. Gene homologies and GC skew graph show that M. suis has a typical Mollicutes oriC. The predicted metabolic pathway is concise, showing evidence of adaptation to blood environment. M. suis is a glycolytic species, obtaining energy through sugars fermentation and ATP-synthase. The pentose-phosphate pathway, metabolism of cofactors and vitamins, pyruvate dehydrogenase and NAD+ kinase are missing. Thus, ribose, NADH, NADPH and coenzyme A are possibly essential for its growth. M. suis can generate purines from hypoxanthine, which is secreted by RBCs, and cytidine nucleotides from uracil. Toxins orthologs were not identified. We suggest that M. suis may cause disease by scavenging and competing for host' nutrients, leading to decreased life-span of RBCs. In summary, genome analysis shows that M. suis is dependent on host cell metabolism and this characteristic is likely to be linked to its pathogenicity. The prediction of essential nutrients will aid the development of in vitro cultivation systems

Anticodon recognition in evolution: switching tRNA specificity of an aminoacyl-tRNA synthetase by site-directed peptide transplantation.

International audienceThe highly conserved aspartyl-, asparaginyl-, and lysyl-tRNA synthetases compose one subclass of aminoacyl-tRNA synthetases, called IIb. The three enzymes possess an OB-folded extension at their N terminus. The function of this extension is to specifically recognize the anticodon triplet of the tRNA. Three-dimensional models of bacterial aspartyl- and lysyl-tRNA synthetases complexed to tRNA indicate that a rigid scaffold of amino acid residues along the five beta-strands of the OB-fold accommodates the base U at the center of the anticodon. The binding of the adjacent anticodon bases occurs through interactions with a flexible loop joining strands 4 and 5 (L45). As a result, a switching of the specificity of lysyl-tRNA synthetase from tRNALys (anticodon UUU) toward tRNAAsp (GUC) could be attempted by transplanting the small loop L45 of aspartyl-tRNA synthetase inside lysyl-tRNA synthetase. Upon this transplantation, lysyl-tRNA synthetase loses its capacity to aminoacylate tRNALys. In exchange, the chimeric enzyme acquires the capacity to charge tRNAAsp with lysine. Upon giving the tRNAAsp substrate the discriminator base of tRNALys, the specificity shift is improved. The change of specificity was also established in vivo. Indeed, the transplanted lysyl-tRNA synthetase succeeds in suppressing a missense Lys --> Asp mutation inserted into the beta-lactamase gene. These results functionally establish that sequence variation in a small peptide region of subclass IIb aminoacyl-tRNA synthetases contributes to specification of nucleic acid recognition. Because this peptide element is not part of the core catalytic structure, it may have evolved independently of the active sites of these synthetases

Copper tolerance of three Crotalaria species from southeastern D.R. Congo at the early development stage

Description of the subject. Microevolutionary processes in metallophytes established on copper enriched soils can lead to a diversity of plant species showing distinct tolerance capacities among genus. Researches about the relationship between these endangered plants and soil copper concentrations are critical in order to improve ex situ conservation methods in southeastern Democratic Republic of Congo (Katangan Copperbelt). Objectives. The aim of the study was to test the effect of copper on the germination and root elongation of three Crotalaria species naturally occurring along a natural copper gradient. The hypothesis is that copper concentrations have different effects on germination and root elongation according to the species of Crotalaria genus. Method. Three species were selected: Crotalaria cobalticola, Crotalaria peschiana and Crotalaria cornetii, occurring on soils with the highest to the lowest copper concentrations respectively. Germination and root elongation tests were performed in vitro (MS vitamin-enriched medium) in six copper mediums ranging from 0 to 125 µM Cu2+. Results. No significant differences in germination percentage were observed according to the copper concentrations. Crotalaria cornetii had the lowest germination percentage. Root elongation of C. peschiana did not differ with copper concentration, but root elongation of C. cobalticola was higher at the greatest copper concentration (125 µM Cu2+). Conclusions. Even if C. cobalticola presented better growth at highest Cu concentrations, it appeared that C. cobalticola and C. peschiana do not require copper for their early stages of development and could thus be conserved in non-contaminated substrate. Crotalaria cornetii seemed to present a physical seed dormancy

The tRNA synthetase paralog PoxA modifies elongation factor-P with (R)-ß-lysine

The lysyl-tRNA synthetase paralog PoxA modifies elongation factor P (EF-P) with α-lysine at low efficiency. Cell-free extracts contained non-α-lysine substrates of PoxA that modified EF-P by a change in mass consistent with β–lysine, a substrate also predicted by genomic analyses. EF-P was efficiently, functionally, modified with (R)-β-lysine but not (S)-β-lysine or genetically encoded α-amino acids, indicating that PoxA has evolved an activity orthogonal to that of the canonical aminoacyl-tRNA synthetases

Molecular evolution of protein-RNA mimicry as a mechanism for translational control

Elongation factor P (EF-P) is a conserved ribosome-binding protein that structurally mimics tRNA to enable the synthesis of peptides containing motifs that otherwise would induce translational stalling, including polyproline. In many bacteria, EF-P function requires post-translational modification with (R)-β-lysine by the lysyl-tRNA synthetase paralog PoxA. To investigate how recognition of EF-P by PoxA evolved from tRNA recognition by aminoacyl-tRNA synthetases, we compared the roles of EF-P/PoxA polar contacts with analogous interactions in a closely related tRNA/synthetase complex. PoxA was found to recognize EF-P solely via identity elements in the acceptor loop, the domain of the protein that interacts with the ribosome peptidyl transferase center and mimics the 3'-acceptor stem of tRNA. Although the EF-P acceptor loop residues required for PoxA recognition are highly conserved, their conservation was found to be independent of the phylogenetic distribution of PoxA. This suggests EF-P first evolved tRNA mimicry to optimize interactions with the ribosome, with PoxA-catalyzed aminoacylation evolving later as a secondary mechanism to further improve ribosome binding and translation control

Asymmetric Amino Acid Activation by Class II Histidyl-tRNA Synthetase from Escherichia coli*

Aminoacyl-tRNA synthetases (ARSs) join amino acids to their cognate tRNAs to initiate protein synthesis. Class II ARS possess a unique catalytic domain fold, possess active site signature sequences, and are dimers or tetramers. The dimeric class I enzymes, notably TyrRS, exhibit half-of-sites reactivity, but its mechanistic basis is unclear. In class II histidyl-tRNA synthetase (HisRS), amino acid activation occurs at different rates in the two active sites when tRNA is absent, but half-of-sites reactivity has not been observed. To investigate the mechanistic basis of the asymmetry, and explore the relationship between adenylate formation and conformational events in HisRS, a fluorescently labeled version of the enzyme was developed by conjugating 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC) to a cysteine introduced at residue 212, located in the insertion domain. The binding of the substrates histidine, ATP, and 5′-O-[N-(l-histidyl)sulfamoyl]adenosine to MDCC-HisRS produced fluorescence quenches on the order of 6–15%, allowing equilibrium dissociation constants to be measured. The rates of adenylate formation measured by rapid quench and domain closure as measured by stopped-flow fluorescence were similar and asymmetric with respect to the two active sites of the dimer, indicating that conformational change may be rate-limiting for product formation. Fluorescence resonance energy transfer experiments employing differential labeling of the two monomers in the dimer suggested that rigid body rotation of the insertion domain accompanies adenylate formation. The results support an alternating site model for catalysis in HisRS that may prove to be common to other class II aminoacyl-tRNA synthetases