Structural properties of the protein SV-IV.

We have investigated the molecular mechanisms that produce different structural and functional behavior in the monomeric and trimeric forms of seminal vesicle protein no. 4, a protein with immunomodulatory, anti-inflammatory, and procoagulant activity secreted from the rat seminal vesicle epithelium. The monomeric and trimeric forms were characterized in solution by CD. Details of the self-association process and structural changes that accompany aggregation were investigated by different experimental approaches: trypsin proteolysis, sequence analysis, chemical modification, and computer modeling. The self-association process induces conformational change mainly in the 1-70 region, which appears to be without secondary structure in the monomer but contains alpha-helix in the trimer. In vivo, proteolysis of seminal vesicle protein no. 4 generates active peptides and this is affected by the monomer/trimer state, which is regulated by the concentration of the protein. The information obtained shows how conformational changes between the monomeric and trimeric forms represent a crucial aspect of activity modulation.     

Transglutaminase activity is involved in polyamine-induced programmed cell death.

Natural polyamines, i.e., putrescine, spermidine, and spermine, are ubiquitous molecules essential for cell proliferation and differentiation. In the present study, the effect of polyamines on primary cultures of bovine aortic endothelial cells (BAECs), rat aortic smooth muscle cells (RASMCs), and a human melanoma cell line was examined. While in the absence of fetal calf serum (FCS) polyamines had no effect on viability, in the presence of FCS spermidine and spermine, at concentrations close to physiologic levels, induced a dose-dependent cell death, whereas putrescine was ineffective. RASMCs were significantly more sensitive than other cells. FACS analysis, oligo-nucleosome ELISA, Hoechst nuclear staining, and Annexin V-FITC quantification showed that cell death was likely due to apoptosis. Cells exposed to spermidine showed a marked increase of intracellular transglutaminase (TGase) activity ( approximately 30-fold over control). Inhibitors of polyamine oxidation or inhibitors of TGase activity prevented polyamine-induced apoptosis. Moreover, tissue TGase overexpression significantly increased cell sensitivity to polyamine, suggesting that this effect is likely related to enhanced intracellular TGase activity. These data indicate that polyamines may modulate cell viability through a novel TGase-dependent process.     

Prediction of the protein structural class by specific peptide frequencies

We evaluated the i-peptides occurrence frequency in the protein sequences belonging to the two datasets which include proteins with a sequence similarity lower than 25% and 40%, respectively. We worked out a new structural class prediction algorithm using the most frequent i-peptides (with i=2, 3, 4), which characterize the four structural classes. Using the tri-peptides, much more able to gain structural information from sequences compared to the di-peptides, the best results were obtained. Compared to the other methods, similarly founded on peptide occurrence frequencies, our method achieves the best prediction accuracy. We compared it also with methods founded on more sophisticated computational approaches.     

Assessment of the conformational features of vasoactive intestinal peptide in solution by limited proteolysis experiments

The structural features of vasoactive intestinal peptide (VIP) and of its Gln16-diaminopropane derivative (VIP-DAP) in solution were investigated by limited proteolysis experiments with trypsin and thermolysin. The proteolysis of the native peptide by both proteinases takes place near the residues in positions 12 and 21/22, suggesting that these amino acids are embedded in segments more flexible than the rest of the molecule. VIP-DAP appears to be more resistant to the proteolytic attack of trypsin, indicating that the derivatization in position 16 is able to stabilize the structure of the peptide. Moreover, the analysis of the mass spectra of the proteolytic mixtures supports the evidence that the derivatization is also able to protect Met17 against oxidation. From these data it can be concluded that VIP in solution under physiological conditions is characterized by the presence of segments with secondary structure, linked together by "hinge" regions that confer flexibility to the peptide, whereas VIP-DAP is embedded in a more rigid conformation, more suitable to receptor interaction.     

Modeling the 3D structure of wheat subtilisin/chymotrypsin inhibitor (WSCI). Probing the reactive site with two susceptible proteinases by time-course analysis and molecular dynamics simulations

Comparative modeling and time-course hydrolysis experiments have been applied to investigate two enzyme-inhibitor complexes formed between the wheat subtilisin-chymotrypsin inhibitor (WSCI) and two susceptible proteinases. WSCI represents the first case of a wheat protein inhibitor active against animal chymotrypsins and bacterial subtilisins. The model was created using as template structure that of the CI-2A inhibitor from barley (PDB code: 2CI2), which shares 87% sequence identity with WSCI. Under these conditions of high similarity, the comparative modeling approach can be successfully applied. We predicted the WSCI 3D model and used it to investigate enzyme-inhibitor complex systems. Experimental observations indicated that chymotrypsin, but not subtilisin, in addition to cleavage at the primary reactive site Met48-Glu49, is able to hydrolyze a second peptide bond between Phe58 and Val59. Here, we report on cleavage of the peptide bond at the inhibitor's reactive site (Met48-Glu49) determined using time-course hydrolysis experiments; the same event was investigated for both subtilisin/WSCI and chymotrypsin/WSCI complexes using molecular dynamics simulations. The molecular details of the initial inhibitor-enzyme interactions, as well as of the changes observed during the simulations, allow us to speculate on the different fates of the two WSCI-proteinase complexes.     

Glycated fibroblast growth factor-2 is quickly produced in vitro upon low-millimolar glucose treatment and detected in vivo in diabetic mice

Angiogenesis impairment in hyperglycemic patients represents a leading cause of severe vascular complications of both type-1 and -2 diabetes mellitus (DM). Angiogenesis dysfunction in DM is related to glycemic control; however, molecular mechanisms involved are still unclear. Fibroblast growth factor-2 (FGF-2) is a potent angiogenic factor and, according to previous evidence, may represent a key target of molecular modifications triggered by high-sugar exposure. Therefore, the purpose of this study was to investigate whether short incubation with hyperglycemic levels of glucose affected FGF-2 and whether glucose-modified FGF-2 was detectable in vivo. Biochemical analyses carried out with SDS-PAGE, fluorescence emission, mass-spectrometry, immunoblot, and competitive ELISA experiments demonstrated that human FGF-2 undergoes a rapid and specific glycation upon 12.5-50 mm glucose exposure. In addition, FGF-2 exposed for 30 min to 12.5 mm glucose lost mitogenic and chemotactic activity in a time- and dose-dependent manner. Under similar conditions, binding affinity to FGF receptor 1 was dramatically reduced by 20-fold, as well as FGF receptor 1 and ERK-1/2 phosphorylation, and FGF-2 lost about 45% of angiogenic activity in two different in vivo angiogenic (Matrigel and chorioallantoic-membrane) assays. Such glucose-induced modification was specific, because other angiogenic growth factors, namely platelet-derived growth factor BB and placental-derived growth factor were not significantly or markedly less modified. Finally, for the first time, glycated-FGF-2 was detected in vivo, in tissues from hyperglycemic nonobese diabetic mice, in significantly higher amounts than in normoglycemic mice. In conclusion, hyperglycemic levels of glucose may strongly affect FGF-2 structure and impair its angiogenic features, and endogenous glycated-FGF-2 is present in diabetic mice, indicating a novel pathogenetic mechanism underlying angiogenesis defects in DM.     

The CD8alpha from sea bass (Dicentrarchus labrax L.): Cloning, expression and 3D modelling

In this paper we describe the cloning, expression and structural study by modelling techniques of the CD8alpha from sea bass (Dicentrarchus labrax L.). The sea bass CD8alpha cDNA is comprised of 1490 bp and is translated in one reading frame to give a protein of 217 amino acids, with a predicted 26 amino acids signal peptide, a 88 bp 5'-UTR and a 748 bp 3'-UTR. A multiple alignment of CD8alpha from sea bass with other known CD8alpha sequences shows the conservation of most amino acid residues involved in the peculiar structural domains found within CD8alpha's. Cysteine residues that are involved in disulfide bonding to form the V domain are conserved. In contrast, an extra cysteine residue found in most mammals in this region is not present in sea bass. The transmembrane and cytoplasmic regions are the most conserved regions within the molecule in the alignment analysis. However, the motif (CXCP) that is thought to be responsible for binding p56lck is missing in the sea bass sequence. Phylogenetic analysis conducted using amino acid sequences showed that sea bass CD8alpha grouped with other known teleost sequences and that three different clusters were formed by the mammalian, avian and fish CD8alpha sequences. The thymus was the tissue with the highest CD8alpha expression, followed by gut, gills, peripheral blood leukocytes and spleen. Lower CD8alpha mRNA levels were found in head kidney, liver and brain. It was possible to create a partial 3D model using the human and mouse structures as template. The CD8alpha 11-120 amino acid region was taken into consideration and the best obtained 3D model shows the presence of ten beta-strands, involving about 50% of the sequence. The global structure was defined as an immunoglobulin-like beta-sandwich made of two anti-parallel sheets. Two cysteines were present in this region and they were at a suitable distance to form an S-S bond as seen in the template human and mouse structures.