Yeast-secreted Recombinant Extracellular Domain of Human CD105 Antigen is Able to Bind TGF-beta Type II Receptor In Vitro

Human CD105 antigen, a type I integral membrane glycoprotein, is expressed as homodimer and oligomer by human endothelial cells, and forms a heteromeric association with TGF-β signaling receptors I and II. Several mutations of CD105 antigen gene are involved in a vascular disorder known as hereditary hemorrhagic telangiectasia type 1. The proposed mechanism by which CD105 is involved in said disorder is haploinsufficiency. We report expression and characterization of human CD105 antigen extracellular domain in yeast Saccharomyces cerevisiae. Different strategies to influence the release of heterologous proteins in the medium, such as alteration of cell wall integrity or coexpression of protein disulfide isomerase, were addressed. Purified extracellular domain of human CD105 antigen retains capacity to bind human TGF-β receptor II in vitro.     

Tetramerization and Cooperativity in Plasmodium falciparum Glutathione S-Transferase Are Mediated by Atypic Loop 113�C119

Glutathione S-transferase of Plasmodium falciparum (PfGST) displays a peculiar dimer to tetramer transition that causes full enzyme inactivation and loss of its ability to sequester parasitotoxic hemin. Furthermore, binding of hemin is modulated by a cooperative mechanism. Site-directed mutagenesis, steady-state kinetic experiments, and fluorescence anisotropy have been used to verify the possible involvement of loop 113–119 in the tetramerization process and in the cooperative phenomenon. This protein segment is one of the most prominent structural differences between PfGST and other GST isoenzymes. Our results demonstrate that truncation, increased rigidity, or even a simple point mutation of this loop causes a dramatic change in the tetramerization kinetics that becomes at least 100 times slower than in the native enzyme. All of the mutants tested have lost the positive cooperativity for hemin binding, suggesting that the integrity of this peculiar loop is essential for intersubunit communication. Interestingly, the tetramerization process of the native enzyme that occurs rapidly when GSH is removed is prevented not only by GSH but even by oxidized glutathione. This result suggests that protection by PfGST against hemin is independent of the redox status of the parasite cell. Because of the importance of this unique segment in the function/structure of PfGST, it could be a new target for the development of antimalarial drugs.Approximately two million deaths in the world per year are caused by Plasmodium falciparum, the parasite responsible for tropical malaria (1, 2). In the last years, increasing interest has been developing for the peculiar glutathione S-transferase (PfGST)³ expressed by this parasite. Expressed in almost all living organisms, GSTs represent a large superfamily of multifunctional detoxifying enzymes that are able to conjugate GSH to a lot of toxic electrophilic compounds, thus facilitating their excretion. Many other protection roles of GSTs have been described, including the enzymatic reduction of organic peroxides (3–5), the inactivation of the proapoptotic JNK through a GST·JNK complex (6), and the protection of the cell from excess nitric oxide (7). The mammalian cytosolic GSTs are dimeric proteins grouped into eight species-independent classes termed Alpha, Kappa, Mu, Omega, Pi, Sigma, Theta, and Zeta on the basis of sequence similarity, immunological reactivity, and substrate specificity (3, 8–11). PfGST is one of the most abundant proteins expressed by P. falciparum (from 1 to 10%, i.e. from 0.1 to 1 mm) (12), and different from what occurs in many organisms, it is the sole GST isoenzyme expressed by this parasite. Despite its structural similarity to the Mu class GST, this specific isoenzyme cannot be assigned to any known GST class (13). The interest in this enzyme is due to its particular protective role in the parasite. In fact, in addition to the usual GST activity that promotes the conjugation of GSH to electrophilic centers of toxic compounds, this protein efficiently binds hemin, and thus it could protect the parasite (that resides in the erythrocytes) from the parasitotoxic effect of this heme by-product (14). Specific compounds that selectively inhibit its catalytic activity or hemin binding could be promising candidates as antimalarial drugs. In this context, the discovery of structural or mechanistic properties of this enzyme that are not found in other GSTs may be important for designing selective inhibitors that are toxic to the parasite but harmless for the host cells. Two properties never observed in other members of the GST superfamily are of particular interest. The first property is that this enzyme, in the absence of GSH, is inactivated in a short time and loses its ability to bind hemin (15). Recent studies indicated that the inactivation process is related to a dimer to tetramer transition (13, 16, 17). The second property is the strong positive homotropic phenomenon that modulates the affinity of the two subunits for hemin (15). The x-ray crystal structure of PfGST, solved by two different groups (13, 18), provides insights into this effect. From a structural point of view, the most intriguing differences of PfGST when compared with other GSTs are a more solvent-exposed H-site and an atypic extra loop connecting helix α-4 and helix α-5 (residues 113–119; see also Fig. 1) that could be involved in the dimer-dimer interaction. Actually, in the absence of ligands, two biological dimers form a tetramer, and these homodimers are interlocked with each other by loop 113–119 of one homodimer, which occupies an H-site of the other homodimer (13, 18). Upon binding of S-hexylglutathione, loop 113–119 rearranges; residues Asn-114, Leu-115, and Phe-116 form an additional coil in helix α-4; and the side chains of Asn-111, Phe-116, and Tyr-211 flip into the H-site of the same dimer (17, 18). The changed course of residues 113–119 in the liganded enzyme prevents the interlocking of the dimers.Open in a separate windowFIGURE 1.A, structural changes of loop 113–119 occurring in the dimer (light blue model and yellow loop; Protein Data Bank code 2AAW) to tetramer (blue model and orange loop; Protein Data Bank code 1OKT) transition. Red spheres indicate the amino acids replaced in this study to obtain mutants A, B, and C. B, model of hemin·PfGST complex obtained by docking simulation using the crystal structure for Protein Data Bank code 1Q4J (15). Hemin is shown in red, loop 113–119 is in orange, and GSH is shown as yellow sticks.In this paper, by means of site-directed mutagenesis, fluorescence anisotropy, kinetic studies, and size exclusion chromatography, we check the influence of selected mutations of this atypic loop in the tetramerization process and the possible involvement of this protein segment in the cooperative phenomenon that characterizes hemin binding. In addition we describe that the tetramerization process is inhibited not only by GSH but even by GSSG. This finding suggests that hemin binding of PfGST is independent of the redox status of the cell. Finally, we demonstrate that the presence of GSH (or GSSG) in the active site is not essential for hemin binding, but this interaction only requires an active dimeric conformation.     

A comparative molecular modeling study of dydrogesterone with other progestational agents through theoretical calculations and nuclear magnetic resonance spectroscopy

6-Dehydroretroprogesterone (dydrogesterone) and three other natural or synthetic progestins (progesterone, retroprogesterone, and 6-dehydroprogesterone) were submitted to a conformational study through theoretical calculations at the B3LYP/6-31G(*) level and high field NMR spectroscopy. The study allows to define the role of the two structural features which differentiate these steroids, i.e., the C9 and C10 configuration and the C6-C7 unsaturation. The combined effects of the conformational preference of A ring, determined by the configuration at C9 and C10, and the enhanced rigidity due to the C6-C7 double bond, could account both for the higher activity and selectivity of dydrogesterone with respect to the other three steroids.     

Endo‐ and exo‐inulinases: Enzyme‐substrate interaction and rational immobilization

Three‐dimensional models of exoinulinase from Bacillus stearothermophilus and endoinulinase from Aspergillus niger were built up by means of homology modeling. The crystal structure of exoinulinase from Aspergillus awamori was used as a template, which is the sole structure of inulinase resolved so far. Docking and molecular dynamics simulations were performed to investigate the differences between the two inulinases in terms of substrate selectivity. The analysis of the structural differences between the two inulinases provided the basis for the explanation of their different regio‐selectivity and for the understanding of enzyme‐substrate interactions. Surface analysis was performed to point out structural features that can affect the efficiency of enzymes also after immobilization. The computational analysis of the three‐dimensional models proved to be an effective tool for acquiring information and allowed to formulate an optimal immobilized biocatalyst even more active that the native one, thus enabling the full exploitation of the catalytic potential of these enzymes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Differential analysis of "protein corona" profile adsorbed onto different nonviral gene delivery systems

A shotgun proteomics approach was used to characterize and compare the proteins that lead to the formation of a rich “protein corona” adsorbed onto the surfaces of cationic liposomes (CLs), lipoplexes, and lipid/polycation/DNA (LPD) complexes, when they come into contact with plasma. After separation of the nanoparticle–protein complex from plasma, the protein mixture was digested, and peptides were analyzed by nanoliquid chromatography–Orbitrap LTQ-XL mass spectrometry. The number of proteins bound to lipoplexes was double that of those identified in the corona of CLs (208 vs 105), while 77 proteins were common to both coronas. The number of proteins bound to the surface of the LPD complexes (158, 133 of which are common to lipoplexes) is intermediate between those found in the protein corona of both CLs and lipoplexes. About half of them were found in the protein corona of CLs. By overlapping the three formulations, it can be seen that only 12 proteins are peculiar to LPD complexes. These results may help in designing gene delivery systems capable of binding the minimum possible quantity of proteins that influence transfection negatively, binding selectively proteins capable of helping in steering in vivo the vector toward the target, and obtaining more efficient and effective gene therapy.     

Protein deiminases: new players in the developmentally regulated loss of neural regenerative ability

Spinal cord regenerative ability is lost with development, but the mechanisms underlying this loss are still poorly understood. In chick embryos, effective regeneration does not occur after E13, when spinal cord injury induces extensive apoptotic response and tissue damage. As initial experiments showed that treatment with a calcium chelator after spinal cord injury reduced apoptosis and cavitation, we hypothesized that developmentally regulated mediators of calcium-dependent processes in secondary injury response may contribute to loss of regenerative ability. To this purpose we screened for such changes in chick spinal cords at stages of development permissive (E11) and non-permissive (E15) for regeneration. Among the developmentally regulated calcium-dependent proteins identified was PAD3, a member of the peptidylarginine deiminase (PAD) enzyme family that converts protein arginine residues to citrulline, a process known as deimination or citrullination. This post-translational modification has not been previously associated with response to injury. Following injury, PAD3 up-regulation was greater in spinal cords injured at E15 than at E11. Consistent with these differences in gene expression, deimination was more extensive at the non-regenerating stage, E15, both in the gray and white matter. As deimination paralleled the extent of apoptosis, we investigated the effect of blocking PAD activity on cell death and deiminated-histone 3, one of the PAD targets we identified by mass-spectrometry analysis of spinal cord deiminated proteins. Treatment with the PAD inhibitor, Cl-amidine, reduced the abundance of deiminated-histone 3, consistent with inhibition of PAD activity, and significantly reduced apoptosis and tissue loss following injury at E15. Altogether, our findings identify PADs and deimination as developmentally regulated modulators of secondary injury response, and suggest that PADs might be valuable therapeutic targets for spinal cord injury.     

Deletion of Syk in neutrophils prevents immune complex arthritis

The K/BxN serum transfer model of arthritis is critically dependent on FcγR signaling events mediated by spleen tyrosine kinase (Syk). However, the specific cell types in which this signaling is required are not known. We report that deletion of Syk in neutrophils, achieved using syk(f/f) MRP8-cre(+) mice, blocks disease development in serum transfer arthritis. The syk(f/f) MRP8-cre(+) mice display absent joint disease and reduced deposition of pathogenic anti-glucose-6-phosphate isomerase Abs in the joint (with a reciprocal accumulation of these Abs in the peripheral circulation). Additionally, syk(f/f) MRP8-cre(+) mice manifest poor edema formation within 3 h after formation of cutaneous immune complexes (Arthus reaction). Together, this suggests that neutrophil-dependent recognition of immune complexes contributes significantly to changes in vascular permeability during the early phases of immune complex disease. Using mixed chimeric mice, containing both wild-type and syk(f/f) MRP8-cre(+) neutrophils, we find no impairment in recruitment of Syk-deficient neutrophils to the inflamed joint, but they fail to become primed, demonstrating lower cytokine production after removal from the joint. They also display an increased apoptotic rate compared with wild-type cells in the same joint. Mast cell-deficient c-kit(sh/sh) mice developed robust arthritis after serum transfer whereas c-kit(W/Wv) mice did not, suggesting that previous conclusions concerning the central role of mast cells in this model may need to be revised. Basophil-deficient mice also responded normally to K/BxN serum transfer. These results demonstrate that Syk-dependent signaling in neutrophils alone is critically required for arthritis development in the serum transfer model.     

ISCOMATRIX adjuvant combines immune activation with antigen delivery to dendritic cells in vivo leading to effective cross-priming of CD8+ T cells

Cancer vaccines aim to induce CTL responses against tumors. Challenges for vaccine design are targeting Ag to dendritic cells (DCs) in vivo, facilitating cross-presentation, and conditioning the microenvironment for Th1 type immune responses. In this study, we report that ISCOM vaccines, which consist of ISCOMATRIX adjuvant and protein Ag, meet these challenges. Subcutaneous injection of an ISCOM vaccine in mice led to a substantial influx and activation of innate and adaptive immune effector cells in vaccine site-draining lymph nodes (VDLNs) as well as IFN-γ production by NK and NKT cells. Moreover, an ISCOM vaccine containing the model Ag OVA (OVA/ISCOM vaccine) was efficiently taken up by CD8α(+) DCs in VDLNs and induced their maturation and IL-12 production. Adoptive transfer of transgenic OT-I T cells revealed highly efficient cross-presentation of the OVA/ISCOM vaccine in vivo, whereas cross-presentation of soluble OVA was poor even at a 100-fold higher concentration. Cross-presenting activity was restricted to CD8α(+) DCs in VDLNs, whereas Langerin(+) DCs and CD8α(-) DCs were dispensable. Remarkably, compared with other adjuvant systems, the OVA/ISCOM vaccine induced a high frequency of OVA-specific CTLs capable of tumor cell killing in different tumor models. Thus, ISCOM vaccines combine potent immune activation with Ag delivery to CD8α(+) DCs in vivo for efficient induction of CTL responses.