Potential effects of climate warming on the survivorship of adult Monochamus galloprovincialis

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Data integration uncovers the metabolic bases of phenotypic variation in yeast

The relationship between different levels of integration is a key feature for understanding the genotype-phenotype map. Here, we describe a novel method of integrated data analysis that incorporates protein abundance data into constraint-based modeling to elucidate the biological mechanisms underlying phenotypic variation. Specifically, we studied yeast genetic diversity at three levels of phenotypic complexity in a population of yeast obtained by pairwise crosses of eleven strains belonging to two species, Saccharomyces cerevisiae and S. uvarum. The data included protein abundances, integrated traits (life-history/fermentation) and computational estimates of metabolic fluxes. Results highlighted that the negative correlation between production traits such as population carrying capacity (K) and traits associated with growth and fermentation rates (J_max) is explained by a differential usage of energy production pathways: a high K was associated with high TCA fluxes, while a high J_max was associated with high glycolytic fluxes. Enrichment analysis of protein sets confirmed our results.This powerful approach allowed us to identify the molecular and metabolic bases of integrated trait variation, and therefore has a broad applicability domain.     

Metabolic engineering provides insight into the regulation of thiamin biosynthesis in plants

Thiamin (or thiamine) is a water-soluble B-vitamin (B1), which is required, in the form of thiamin pyrophosphate, as an essential cofactor in crucial carbon metabolism reactions in all forms of life. To ensure adequate metabolic functioning, humans rely on a sufficient dietary supply of thiamin. Increasing thiamin levels in plants via metabolic engineering is a powerful strategy to alleviate vitamin B1 malnutrition and thus improve global human health. These engineering strategies rely on comprehensive knowledge of plant thiamin metabolism and its regulation. Here, multiple metabolic engineering strategies were examined in the model plant Arabidopsis thaliana. This was achieved by constitutive overexpression of the three biosynthesis genes responsible for B1 synthesis, HMP-P synthase (THIC), HET-P synthase (THI1), and HMP-P kinase/TMP pyrophosphorylase (TH1), either separate or in combination. By monitoring the levels of thiamin, its phosphorylated entities, and its biosynthetic intermediates, we gained insight into the effect of either strategy on thiamin biosynthesis. Moreover, expression analysis of thiamin biosynthesis genes showed the plant’s intriguing ability to respond to alterations in the pathway. Overall, we revealed the necessity to balance the pyrimidine and thiazole branches of thiamin biosynthesis and assessed its biosynthetic intermediates. Furthermore, the accumulation of nonphosphorylated intermediates demonstrated the inefficiency of endogenous thiamin salvage mechanisms. These results serve as guidelines in the development of novel thiamin metabolic engineering strategies.     

Calmodulin and calcium differentially regulate the neuronal Nav1.1 voltage-dependent sodium channel

Mutations in the neuronal Nav1.1 voltage-gated sodium channel are responsible for mild to severe epileptic syndromes. The ubiquitous calcium sensor calmodulin (CaM) bound to rat brain Nav1.1 and to the human Nav1.1 channel expressed by a stably transfected HEK-293 cell line. The C-terminal region of the channel, as a fusion protein or in the yeast two-hybrid system, interacted with CaM via a consensus C-terminal motif, the IQ domain. Patch clamp experiments on HEK1.1 cells showed that CaM overexpression increased peak current in a calcium-dependent way. CaM had no effect on the voltage-dependence of fast inactivation, and accelerated the inactivation kinetics. Elevating Ca⁺⁺ depolarized the voltage-dependence of fast inactivation and slowed down the fast inactivation kinetics, and for high concentrations this effect competed with the acceleration induced by CaM alone. Similarly, the depolarizing action of calcium antagonized the hyperpolarizing shift of the voltage-dependence of activation due to CaM overexpression. Fluorescence spectroscopy measurements suggested that Ca⁺⁺ could bind the Nav1.1 C-terminal region with micromolar affinity.     

The enzymes of the transsulfuration pathways: active-site characterizations

The diversity of reactions catalyzed by enzymes reliant on pyridoxal 5'-phosphate (PLP) demonstrates the catalytic versatility of this cofactor and the plasticity of the protein scaffolds of the major fold types of PLP-dependent enzymes. The enzymes of the transsulfuration (cystathionine γ-synthase and cystathionine β-lyase) and reverse transsulfuration (cystathionine β-synthase and cystathionine γ-lyase) pathways interconvert l-cysteine and l-homocysteine, the immediate precursor of l-methionine, in plants/bacteria and yeast/animals, respectively. These enzymes provide a useful model system for investigation of the mechanisms of substrate and reaction specificity in PLP-dependent enzymes as they catalyze distinct side chain rearrangements of similar amino acid substrates. Exploration of the underlying factors that enable enzymes to control the substrate and reaction specificity of this cofactor will enable the engineering of these properties and the development of therapeutics and antimicrobial compounds. Recent studies probing the role of active-site residues, of the enzymes of the transsulfuration pathways, as determinants of substrate and reaction specificity are the subject of this review. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.     

Hidden in plain sight: subtle effects of the 8-oxoguanine lesion on the structure, dynamics, and thermodynamics of a 15-base pair oligodeoxynucleotide duplex

The base lesion 8-oxoguanine is formed readily by oxidation of DNA, potentially leading to G → T transversion mutations. Despite the apparent similarity of 8-oxoguanine-cytosine base pairs to normal guanine-cytosine base pairs, cellular base excision repair systems effectively recognize the lesion base. Here we apply several techniques to examine a single 8-oxoguanine lesion at the center of a nonpalindromic 15-mer duplex oligonucleotide in an effort to determine what, if anything, distinguishes an 8-oxoguanine-cytosine (8oxoG-C) base pair from a normal base pair. The lesion duplex is globally almost indistinguishable from the unmodified parent duplex using circular dichroism spectroscopy and ultraviolet melting thermodynamics. The DNA mismatch-detecting photocleavage agent Rh(bpy)(2)chrysi(3+) cleaves only weakly and nonspecifically, revealing that the 8oxoG-C pair is locally stable at the level of the individual base pairs. Nuclear magnetic resonance spectra are also consistent with a well-conserved B-form duplex structure. In the two-dimensional nuclear Overhauser effect spectra, base-sugar and imino-imino cross-peaks are strikingly similar between parent and lesion duplexes. Changes in chemical shift due to the 8oxoG lesion are localized to its complementary cytosine and to the 2-3 bp immediately flanking the lesion on the lesion strand. Residues further removed from the lesion are shown to be unperturbed by its presence. Notably, imino exchange experiments indicate that the 8-oxoguanine-cytosine pair is strong and stable, with an apparent equilibrium constant for opening equal to that of other internal guanine-cytosine base pairs, on the order of 10(-6). This collection of experiments shows that the 8-oxoguanine-cytosine base pair is incredibly stable and similar to the native pair.     

Solid character of membrane ceramides: a surface rheology study of their mixtures with sphingomyelin

The compression and shear viscoelasticities of egg-ceramide and its mixtures with sphingomyelin were investigated using oscillatory surface rheology performed on Langmuir monolayers. We found high values for the compression and shear moduli for ceramide, compatible with a solid-state membrane, and extremely high surface viscosities when compared to typical fluid lipids. A fluidlike rheological behavior was found for sphingomyelin. Lateral mobilities, measured from particle tracking experiments, were correlated with the monolayer viscosities through the usual hydrodynamic relationships. In conclusion, ceramide increases the solid character of sphingomyelin-based membranes and decreases their fluidity, thus drastically decreasing the lateral mobilities of embedded objects. This mechanical behavior may involve important physiological consequences in biological membranes containing ceramides.