Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol mono-oxygenase

Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol mono-oxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure-function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a C-terminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidine motif, only mutation of Glu137 to alanine led to an 18-fold increase in the Michaelis-Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase.     

Cycloheximide as a tool to investigate protein import in peroxisomes: a case study of the subcellular localization of isoprenoid biosynthetic enzymes

Cytosolic background fluorescence is often observed when native low-abundance peroxisomal proteins carrying a weak peroxisomal targeting sequence are expressed as fluorescent fusion protein using a strong constitutive promoter in transiently transformed plant cells. This cytosolic fluorescence usually comes from the strong expression of the low-abundance proteins exceeding the peroxisome import efficiency. This often results in a misinterpretation of the protein subcellular localization, as there is doubt as to whether proteins are dually targeted to the cytosol and peroxisome or are exclusively localized to peroxisomes. To circumvent this experimental difficulty, the protein peroxisome import study can be optimized by de novo protein synthesis inhibition in transiently transformed cells using the translation inhibitor cycloheximide. This approach was used here successfully for the study of the subcellular localization of distinct plant isoprenoid biosynthetic enzymes, allowing us to clearly demonstrate that 5-phosphomevalonate kinase, mevalonate 5-diphosphate decarboxylase and a short isoform of farnesyl diphosphate synthase from Catharanthus roseus are exclusively localized to peroxisomes.     

Kruppel-like factor 1 (KLF1), KLF2, and Myc control a regulatory network essential for embryonic erythropoiesis

The Krüppel-like factor 1 (KLF1) and KLF2 positively regulate embryonic β-globin expression and have additional overlapping roles in embryonic (primitive) erythropoiesis. KLF1(-/-) KLF2(-/-) double knockout mice are anemic at embryonic day 10.5 (E10.5) and die by E11.5, in contrast to single knockouts. To investigate the combined roles of KLF1 and KLF2 in primitive erythropoiesis, expression profiling of E9.5 erythroid cells was performed. A limited number of genes had a significantly decreasing trend of expression in wild-type, KLF1(-/-), and KLF1(-/-) KLF2(-/-) mice. Among these, the gene for Myc (c-Myc) emerged as a central node in the most significant gene network. The expression of the Myc gene is synergistically regulated by KLF1 and KLF2, and both factors bind the Myc promoters. To characterize the role of Myc in primitive erythropoiesis, ablation was performed specifically in mouse embryonic proerythroblast cells. After E9.5, these embryos exhibit an arrest in the normal expansion of circulating red cells and develop anemia, analogous to KLF1(-/-) KLF2(-/-) embryos. In the absence of Myc, circulating erythroid cells do not show the normal increase in α- and β-like globin gene expression but, interestingly, have accelerated erythroid cell maturation between E9.5 and E11.5. This study reveals a novel regulatory network by which KLF1 and KLF2 regulate Myc to control the primitive erythropoietic program.     

Randomness + determinism = progresses: Why random processes could be favored by evolution

Biologists are somehow pioneers on the idea that progress can be driven by randomness: randomness is one of the main engine of evolution; small variations induced by randomness coupled with natural selection allows the species to self-adapt to their moving environment. Studies from the last 40 years in computer science suggest that randomness is in fact able of doing much more and revealed unexpected possibilities which might appear impossible at first. Furthermore, it turns out that these discoveries are faster, cheaper and above all exponentially thriftier than their deterministic alternatives. This means that random explorations would almost surely generate a stochastic process way before any equivalent deterministic counterpart is found. It follows that most likely these processes are favored by evolution and should thus be known to anyone dealing with systems (alive or not) having access to random sources. This article presents some of these counter-intuitive results as a possible source of inspiration for studying systems fed with randomness.     

Co-expression of the small heat shock protein, Lo18, with β-glucosidase in Escherichia coli improves solubilization and reveals various associations with overproduced heterologous protein, GroEL/ES

We developed a new system to improve the overproduction of soluble proteins in E. coli based on a plasmid encoding the small heat-shock protein, Lo18, derived from the lactic acid bacterium Oenococcus oeni. The efficiency of this system was compared with that of another system based on production of the E. coli universal chaperone GroEL/ES. A compatible plasmid encoding β-glucosidase was constructed for the overproduction and aggregation of this enzyme. Co-expression with Lo18 resulted in an increase in soluble β-glucosidase levels similar to that obtained in the GroEL/ES co-expression system. Lo18 was found preferentially in the insoluble fraction, associated with aggregated enzyme. By contrast, GroEL/ES was more abundant in the soluble fraction.     

Biological traits of European pond macroinvertebrates

Whilst biological traits of river macroinvertebrates show unimodal responses to geographic changes in habitat conditions in Europe, we still do not know whether spatial turnover of species result in distinct combinations of biological traits for pond macroinvertebrates. Here, we used data on the occurrence of 204 macroinvertebrate taxa in 120 ponds from four biogeographic regions of Europe, to compare their biological traits. The Mediterranean, Atlantic, Alpine, and Continental regions have specific climate, vegetation and geology. Only two taxa were exclusively found in the Alpine and Continental regions, while 28 and 34 taxa were exclusively recorded in the Atlantic and Mediterranean regions, respectively. Invertebrates in the Mediterranean region allocated much energy to reproduction and resistance forms. Most Mediterranean invertebrate species had narrow thermal ranges. In Continental areas, invertebrates allocated lesser energy to reproduction and dispersal, and organisms were short lived with high diversity of feeding groups. These characteristics suggest higher resilience. The main difference between ponds in the Alpine and Atlantic regions was their elevation. Alpine conditions necessitate specific adaptations related to rapid temperature fluctuations, and low nutrient concentrations. Even if our samples did not cover the full range of pond conditions across Europe, our analyses suggest that changes in community composition have important impacts on pond ecosystem functions. Consistent information on a larger set of ponds across Europe would be much needed, but their low accessibility (unpublished data and/or not disclosed by authors) remains problematic. There is still, therefore, a pressing need for the incorporation of high quality data sets into a standardized database so that they can be further analyzed in an integrated European-wide manner.     

Flagellar hook flexibility is essential for bundle formation in swimming Escherichia coli cells

Swimming Escherichia coli cells are propelled by the rotary motion of their flagellar filaments. In the normal swimming pattern, filaments positioned randomly over the cell form a bundle at the posterior pole. It has long been assumed that the hook functions as a universal joint, transmitting rotation on the motor axis through up to ～90° to the filament in the bundle. Structural models of the hook have revealed how its flexibility is expected to arise from dynamic changes in the distance between monomers in the helical lattice. In particular, each of the 11 protofilaments that comprise the hook is predicted to cycle between short and long forms, corresponding to the inside and outside of the curved hook, once each revolution of the motor when the hook is acting as a universal joint. To test this, we genetically modified the hook so that it could be stiffened by binding streptavidin to biotinylated monomers, impeding their motion relative to each other. We found that impeding the action of the universal joint resulted in atypical swimming behavior as a consequence of disrupted bundle formation, in agreement with the universal joint model.