A heme tag for in vivo synthesis of artificial cytochromes

A genetic approach is described here that enables the specific covalent attachment of heme via a short C-terminal peptide tag to an otherwise non-heme-binding protein. Covalent attachment of heme to the apo-protein is catalysed by the cytochrome c maturation system of Escherichia coli. While its original enzymatic activity is retained, the resulting heme-tagged protein is red, has peroxidase activity and is redox active. The presence or absence of a C-terminal histidine tag results in low-spin heme iron with six- or high-spin heme iron with five coordinate ligands, respectively. The heme tag can be used as a tool for the rational design of artificial c-type cytochromes and metalloenzymes, thereby overcoming previous limitations set by chemical approaches. Moreover, the tag allows direct visualisation of the red fusion protein during purification.     

Solution structure of the Ca2+-Binding EGF3-4 pair from vitamin K-dependent protein S: identification of an unusual fold in EGF3

Vitamin K-dependent protein S is a cofactor of activated protein C, a serine protease that regulates blood coagulation. Deficiency of protein S can cause venous thrombosis. Protein S has four EGF domains in tandem; domains 2-4 bind calcium with high affinity whereas domains 1-2 mediate interaction with activated protein C. We have now solved the solution structure of the EGF3-4 fragment of protein S. The linker between the two domains is similar to what has been observed in other calcium-binding EGF domains where it provides an extended conformation. Interestingly, a disagreement between NOE and RDC data revealed a conformational heterogeneity within EGF3 due to a hinge-like motion around Glu186 in the Cys-Glu-Cys sequence, the only point in the domain where flexibility is allowed. The dominant, bent conformation of EGF3 in the pair has no precedent among calcium-binding EGF domains. It is characterized by a change in the psi angle of Glu186 from 160 degrees +/- 40 degrees , as seen in ten other EGF domains, to approximately 0 degrees +/- 15 degrees . NOESY data suggest that Tyr193, a residue not conserved in other calcium-binding EGF domains (except in the homologue Gas6), induces the unique fold of EGF3. However, SAXS data, obtained on EGF1-4 and EGF2-4, showed a dominant, extended conformation in these fragments. This may be due to a counterproductive domain-domain interaction between EGF2 and EGF4 if EGF3 is in a bent conformation. We speculate that the ability of EGF3 to adopt different conformations may be of functional significance in protein-protein interactions involving protein S.     

Probing the influence of stereoelectronic effects on the biophysical properties of oligonucleotides: comprehensive analysis of the RNA affinity, nuclease resistance, and crystal structure of ten 2'-O-ribonucleic acid modifications

The syntheses of 10 new RNA 2'-O-modifications, their incorporation into oligonucleotides, and an evaluation of their properties such as RNA affinity and nuclease resistance relevant to antisense activity are presented. All modifications combined with the natural phosphate backbone lead to significant gains in terms of the stability of hybridization to RNA relative to the first-generation DNA phosphorothioates (PS-DNA). The nuclease resistance afforded in particular by the 2'-O-modifications carrying a positive charge surpasses that of PS-DNA. However, small electronegative 2'-O-substituents, while enhancing the RNA affinity, do not sufficiently protect against degradation by nucleases. Similarly, oligonucleotides containing 3'-terminal residues modified with the relatively large 2'-O-[2-(benzyloxy)ethyl] substituent are rapidly degraded by exonucleases, proving wrong the assumption that steric bulk will generally improve protection against nuclease digestion. To analyze the factors that contribute to the enhanced RNA affinity and nuclease resistance we determined crystal structures of self-complementary A-form DNA decamer duplexes containing single 2'-O-modified thymidines per strand. Conformational preorganization of substituents, favorable electrostatic interactions between substituent and sugar-phosphate backbone, and a stable water structure in the vicinity of the 2'-O-modification all appear to contribute to the improved RNA affinity. Close association of positively charged substituents and phosphate groups was observed in the structures with modifications that protect most effectively against nucleases. The promising properties exhibited by some of the analyzed 2'-O-modifications may warrant a more detailed evaluation of their potential for in vivo antisense applications. Chemical modification of RNA can also be expected to significantly improve the efficacy of small interfering RNAs (siRNA). Therefore, the 2'-O-modifications introduced here may benefit the development of RNAi therapeutics.     

Role of host plant alternation in the precocity and intensity of parasitoid activity on of cereal aphids

Integrated pest management programs tend to reduce the chemical input by enhancing the development of biological control. Cereal aphids cause important damages to winter wheat in Europe but are currently under the pressure of several parasitoid species (Braconidae: Aphidiinae). Previous ecological studies have reported the existence of an asynchrony between aphid and parasitoid populations in early spring in cereal cultures. Here, we tested the presence of rose bushes (Rosa rugosa) as a host plant for alternative aphid-host. Aphid and parasitoid densities were recorded for two years using the plant cutting sampling method. The main results were: (i) rose bushes constitute a potential reservoir of alternative aphid hosts species for a number of parasitoid and predator species, (ii) rose aphids appear earlier in spring and were more abundant than the aphids on wheat, (iii) parasitism activity in rose bushes is synchronized with cereal fields, however (iv) rose bushes management did not induce a decrease of cereal aphid population.     

Activation of the Ca(2+)/calcineurin/NFAT2 pathway controls smooth muscle cell differentiation

Cellular mechanisms controlling smooth muscle cells (SMCs) phenotypic modulation are largely unknown. Intracellular Ca2+ movements are essential to ensure SMC functions; one of the roles of Ca2+ is to regulate calcineurin, which in turn induces nuclear localization of the nuclear factor of activated T-cell (NFAT). In order to investigate, during phenotypic differentiation of SMCs, the effect of calcineurin inhibition on NFAT2 nuclear translocation, we used a culture model of SMC differentiation in serum-free conditions. We show that the treatment of cultured SMC with the calcineurin inhibitor cyclosporine A induced their dedifferentiation while preventing their differentiation. These findings suggest that nuclear translocation of NFAT2 is dependent of calcineurin activity during the in vitro SMC differentiation kinetic and that the nuclear presence of NFAT2 is critical in the acquisition and maintenance of SMC differentiation.     

Expression, refolding, and purification of recombinant human granzyme B

Granzyme B (GrB) is a member of a family of serine proteases involved in cytotoxic T-lymphocyte-mediated killing of potentially harmful cells, where GrB induces apoptosis by cleavage of a limited number of substrates. To investigate the suitability of GrB as an enzyme for specific fusion protein cleavage, two derivatives of human GrB, one dependent on blood coagulation factor Xa (FXa) cleavage for activation and one engineered to be self-activating, were recombinantly expressed in Escherichia coli. Both derivatives contain a hexa-histidine affinity tag fused to the C-terminus and expressed as inclusion bodies. These were isolated and solubilized in guanidiniumHCl, immobilized on a Ni2+-NTA agarose column, and refolded by application of a cyclic refolding protocol. The refolded pro-rGrB-H6 could be converted to a fully active form by cleavage with FXa or, for pro(IEPD)-rGrB-H6, by autocatalytic processing during the final purification step. A self-activating derivative in which the unpaired cysteine of human GrB was substituted with phenylalanine was also prepared. Both rGrB-H6 and the C228F mutant were found to be highly specific and efficient processing enzymes for the cleavage of fusion proteins, as demonstrated by cleavage of fusion proteins containing the IEPD recognition sequence of GrB.     

Oligomerization of the fifth transmembrane domain from the adenosine A2A receptor

The human adenosine A_2A receptor (A_2AR) belongs to one of the largest family of membrane proteins, the G-protein coupled receptors (GPCRs), characterized by seven transmembrane (TM) helices. Little is known about the determinants of their structures, folding, assembly, activation mechanisms, and oligomeric states. Previous studies in our group showed that peptides corresponding to all seven TM domains form stable helical structures in detergent micelles and lipid vesicles. However, the peptides behave differently; TM5 is the only peptide to have a ratio [θ]₂₂₂/[θ]₂₀₈ obtained by circular dichroism (CD) spectroscopy>1. This finding suggested to us that TM5 might self-associate. In the present study, we investigate the unique properties of the TM5 domain. We performed detailed analyses of TM5 peptide behavior in membrane-mimetic environments using CD spectroscopy, fluorescence spectroscopy and Förster resonance energy transfer, and gel electrophoresis. We find that TM5 peptide has the ability to self-associate to form oligomeric structures in various hydrophobic milieus and that these oligomers are highly resistant to temperature and chemical denaturation. We also find that mutation of the full-length A_2AR at position M193, which is located in the fifth TM domain, noticeably alters A_2AR monomer: dimer ratio as observed on SDS-PAGE. Our results suggest that parallel association of TM5 dimers may play a role in the known adenosine A_2A receptor dimerization. This study represents the first evidence of an individual GPCR transmembrane domain self-association.