Abnormal splicing of ABCA1 pre-mRNA in Tangier disease due to a IVS2 +5G>C mutation in ABCA1 gene

Two point mutations of ABCA1 gene were found in a patient with Tangier disease (TD): i) G>C in intron 2 (IVS2 +5G>C) and ii) c.844 C>T in exon 9 (R282X). The IVS2 +5G>C mutation was also found in the brother of another deceased TD patient, but not in 78 controls and 33 subjects with low HDL. The IVS2 +5G>C mutation disrupts ABCA1 pre-mRNA splicing in fibroblasts, leading to three abnormal mRNAs: devoid of exon 2 (Ex2-/mRNA), exon 4 (Ex4-/mRNA), or both these exons (Ex2-/Ex4-/mRNA), each containing a translation initiation site. These mRNAs are expected either not to be translated or generate short peptides. To investigate the in vitro effect of IVS2 +5G>C mutation, we constructed two ABCA1 minigenes encompassing Ex1-Ex3 region, one with wild-type (WTgene) and the other with mutant (MTgene) intron 2. These minigenes were transfected into COS1 and NIH3T3, two cell lines with a different ABCA1 gene expression. In COS1 cells, WTgene pre-mRNA was spliced correctly, while the splicing of MTgene pre-mRNA resulted in Ex2-/mRNA. In NIH3T3, no splicing of MTgene pre-mRNA was observed, whereas WTgene pre-mRNA was spliced correctly. These results stress the complexity of ABCA1 pre-mRNA splicing in the presence of splice site mutations.     

Effective genetic vaccination with a widely shared endogenous retroviral tumor antigen requires CD40 stimulation during tumor rejection phase

Endogenous retrovirus (ERV) products are recognized by T lymphocytes in mice and humans. As these Ags are preferentially expressed by neoplastic tissues, they might represent an ideal target for active immunization by genetic vaccination. However, i.m. inoculation of plasmid DNA encoding mouse gp70 or p15E, two products of the env gene of an endogenous murine leukemia virus, elicited a weak Ag-specific T lymphocyte response and resulted in partial protection from challenge with mouse tumors possessing these Ags. Depletion experiments showed that CD8(+), but not CD4(+), T lymphocytes were crucial for the antitumor activity of the vaccines. Systemic administration of agonistic anti-CD40 mAb increased the therapeutic potential of genetic vaccination, but only when given during the tumor rejection phase and not at the time of immunization. This effect correlated with a dramatic increase in the number of ERV-specific CD8(+) T lymphocytes. Adjuvant activity of CD40 agonists thus seems to be relevant to enhance the CD8(+) T cell-dependent response in tumor-bearing hosts, suggesting that sustaining tumor-specific T lymphocyte survival in subjects undergoing vaccination might be a key event in the successful vaccination with weak tumor Ags.     

Molecular interaction and enzymatic activity of macrophage migration inhibitory factor with immunorelevant peptides

Disulfide reduction is an important step in antigen processing for HLA class II restricted T cell responses. Migration inhibitory factor (MIF) is a member of the thioredoxin family and has been classically defined as a cytokine. Using enzyme-linked immunosorbent assay and CD analysis, here we describe the binding to MIF of two peptides, hepatitis B surface antigen (HBsAg) and insulin B (InsB) with high affinity for HLA class II allo-types, HLA-DP2 and HLA-DQ8, respectively. At neutral pH, cysteinylated InsB was a substrate for MIF thiol reductase activity, as assessed by mass spectroscopy/electrospray analysis. Finally, a biologically active form of MIF co-immunopurified with mature forms of HLA DP2/15, and a peptide derived from the HLA-DP beta1 helix could be used for affinity purification of MIF. The possibility that MIF participates in class II antigen presentation and/or as a chaperone is discussed.     

Unfolding and refolding of porcine odorant binding protein in guanidinium hydrochloride: equilibrium studies at neutral pH

Unfolding and refolding studies on porcine odorant binding protein (pOBP) have been performed at pH 7 in the presence of guanidinium hydrochloride (GdnHCl). Unfolding, monitored by following changes of protein fluorescence and circular dichroism (CD), was found to be a reversible process, in terms of recovered structure and function. The equilibrium transition data were fitted by a simple two-state sigmoidal function of denaturant concentration and the thermodynamic folding parameters, derived from the two techniques, were very similar (average values: C(1/2) approximately 2.4 M, m approximately 2 kcal mol(-1) M(-1), DeltaG(unf,w)(0) approximately 4.7 kcal mol(-1)). The transition was independent of protein concentration, indicating that only monomeric species are involved. Only a minor protective effect by the fluorescent ligand 1-amino-anthracene (AMA) against protein unfolding was detected, whereas dihydromyrcenol (DHM) stabilised the protein to a larger extent (DeltaC(1/2) approximately 0.5 M). Refolding was complete, when the protein, denatured with GdnHCl, was diluted with buffer. On the other hand, refolding by dialysis was largely prevented by concomitant aggregation. The present results on pOBP are compared with those on bovine OBP (bOBP) [Biochim. Biophys. Acta 1599 (2002) 90], where subunit folding is accompanied by domain swapping. We finally suggest that the generally observed two-state folding of many lipocalins is probably favoured by their beta-barrel topology.     

Unfolding and pH studies on manganese peroxidase: role of heme and calcium on secondary structure stability

The present study characterizes the unfolding and folding processes of recombinant manganese peroxidase. This enzyme contains five disulfide bonds, two calcium ions, and one heme prosthetic group. Circular dichroism in the far UV was used to monitor global changes of the protein secondary structure, whereas UV-visible spectroscopy of the Soret band provided information about local changes in the heme cavity. The effects of reducing agents, oxidizing agents, and denaturants on this process were investigated. In addition to affecting the secondary structure content, these factors also affect the binding of the heme and the calcium ions, both of which have a significant effect on the folding process. Our results also show that denaturants induce irreversible changes, which are most likely due to the inability of the denatured protein to rebind either calcium or the heme. Breaking of disulfide bonds by 30 mM dithiothreitol causes complete unfolding of recombinant manganese peroxidase. The unfolding process was also studied at low and high pH, where the protein reaches the final unfolded state through two different intermediate states. The data also indicate that only the acidic folding-unfolding process is reversible. Our results indicate a complex synergistic relationship between the secondary structure content, the tertiary structure arrangement, and the binding of the heme and the calcium ions and disulfide bridge formation.     

Study of the cytotoxic effect of a peptidic inhibitor of the p53-hdm2 interaction in tumor cells

Inhibitors of the p53-hdm2 interaction are attractive molecules for stimulating the p53 pathway in tumors. In this report, an inhibitor of the p53-hdm2 interaction, the AP peptide, is used to activate p53 in tumor cells expressing various levels of hdm2 protein. It induces apoptosis only in cells expressing high endogenous levels of hdm2 protein. The absence of apoptosis in tumor cells with low hdm2 levels is due not to alterations in the p53-dependent apoptotic pathway but to a different regulation of this pathway. The peptide is also less toxic for non-tumor cells than for tumor cells overexpressing the hdm2 protein.     

Solution structure of a monoheme ferrocytochrome c from Shewanella putrefaciens and structural analysis of sequence-similar proteins: functional implications

Within the frame of the characterization of the structure and function of cytochromes c, an 81-amino acid cytochrome c was identified in the genome of Shewanella putrefaciens. Because of the scarce information about bacterial cytochromes of this type and the large variability in sequences and possibly function, we decided to proceed to its structural characterization. This protein was expressed in Escherichia coli and purified. The oxidized species is largely high spin, with a detached methionine, whereas the reduced species has the classical His/Met axial ligation to iron. The NMR solution structure of the reduced form was determined on a (15)N-labeled sample, for which 99% of all non-proline backbone (1)H and (15)N resonances have been assigned. One thousand three hundred two meaningful NOEs, out of 1775 NOEs, together with 66 dihedral angles provide a structure with rmsd values from the mean of 0.50 and 0.96 A for backbone and all heavy atoms, respectively. A search of gene banks allowed us to locate 10 different cytochromes c, the sequences of which are more than 30% identical to that of the S. putrefacienscytochrome. For two of them, the structures are known. The structures of the others have been modeled by using the available templates and internally validated. Structural similarities in terms of surface properties account for their biophysical features and provide hints about the function.     

HIV protease inhibitors are potent anti-angiogenic molecules and promote regression of Kaposi sarcoma

Treatment with HIV-1 protease inhibitors (PI) is associated with a reduced incidence or regression of Kaposi sarcoma (KS). Here we show that systemic administration of the PIs indinavir or saquinavir to nude mice blocks the development and induces regression of angioproliferative KS-like lesions promoted by primary human KS cells, basic fibroblast growth factor (bFGF), or bFGF and vascular endothelial growth factor (VEGF) combined. These PIs also block bFGF or VEGF-induced angiogenesis in the chorioallantoic membrane assay with a potency similar to paclitaxel (Taxol). These effects are mediated by the inhibition of endothelial- and KS-cell invasion and of matrix metalloproteinase-2 proteolytic activation by PIs at concentrations present in plasma of treated individuals. As PIs also inhibit the in vivo growth and invasion of an angiogenic tumor-cell line, these data indicate that PIs are potent anti-angiogenic and anti-tumor molecules that might be used in treating non-HIV KS and in other HIV-associated tumors.     

Periods of organogenesis in shoots of Nothofagus dombeyi (Mirb.) oersted (Nothofagaceae)

The organogenetic cycle of main-branch shoots of Nothofagus dombeyi (Nothofagaceae) was studied. Twelve samples of 52-59 parent shoots were collected from a roadside population between September 1999 and October 2000. Variations over time in the number of nodes of terminal and axillary buds, and the length, diameter and number of leaves of shoots derived from these buds (sibling shoots) were analysed. The number of nodes of buds developed by parent shoots was compared with the number of nodes of buds developed, I year later, by sibling shoots. The length, diameter and number of leaves of sibling shoots increased from October 1999 to February 2000 in those shoots with a terminal bud. However, extension of most sibling shoots, including the first five most distal leaf primordia, ceased before February due to abscission of the shoot apex. Axillary buds located most distally on a shoot had more nodes than both terminal buds and more proximal axillary buds. The longest shoots included a preformed part and a neoformed part. The organogenetic event which initiated the neoformed organs continued until early autumn, giving rise to the following year's preformation. The absence of cataphylls in terminal buds could indicate a low intensity of shoot rest. The naked terminal bud of Nothofagus spp. could be interpreted as a structure less specialized than the scaled bud found in genera of Fagaceae and Betulaceae.     

Probing the hirudin-thrombin interaction by incorporation of noncoded amino acids and molecular dynamics simulation

Thrombin is a primary target for the development of novel anticoagulants, since it plays two important and opposite roles in hemostasis: procoagulant and anticoagulant. All thrombin functions are influenced by Na+ binding, which triggers the transition of this enzyme from an anticoagulant (slow) form to a procoagulant (fast) form. In previous studies, we have conveniently produced by chemical synthesis analogues of the N-terminal fragment 1-47 of hirudin HM2 containing noncoded amino acids and displaying up to approximately 2700-fold more potent antithrombin activity, comparable to that of full-length hirudin. In the work presented here, we have exploited the versatility of chemical synthesis to probe the structural and energetic properties of the S3 site of thrombin through perturbations introduced in the structure of hirudin fragment 1-47. In particular, we have investigated the effects of systematic replacement of Tyr3 with noncoded amino acids retaining the aromatic nucleus of Tyr, as well as similar hydrophobic and steric properties, but possessing different electronic (e.g., p-fluoro-, p-iodo-, or p-nitro-Phe), charge (p-aminomethyl-Phe), or conformational (homo-Phe) properties. Our results indicate that the affinity of fragment 1-47 for thrombin is proportional to the desolvation free energy change upon complex formation, and is inversely related to the electric dipole moment of the amino acid side chain at position 3 of hirudin. In this study, we have also identified the key features that are responsible for the preferential binding of hirudin to the procoagulant (fast) form of thrombin. Strikingly, shaving at position 3, by Tyr --> Ala exchange, abolishes the differences in the affinity for thrombin allosteric forms, whereas a bulkier side chain (e.g., beta-naphthylalanine) improves binding preferentially to the fast form. These results provide strong, albeit indirect, evidence that the procoagulant (fast) form of thrombin is in a more open and accessible conformation with respect to the less forgiving structure it acquires in the slow form. This view is also supported by the results of molecular dynamics simulations conducted for 18 ns on free thrombin in full explicit water, showing that after approximately 5 ns thrombin undergoes a significant conformational transition, from a more open conformation (which we propose can be related to the fast form) to a more compact and closed one (which we propose can be related to the slow form). This transition mainly involves the Trp148 and Trp60D loop, the S3 site, and the fibrinogen binding site, whereas the S1 site, the Na+-binding site, and the catalytic pocket remain essentially unchanged. In particular, our data indicate that the S3 site of the enzyme is less accessible to water in the putative slow form. This structural picture provides a reasonable molecular explanation for the fact that physiological substrates related to the procoagulant activity of thrombin (fibrinogen, thrombin receptor 1, and factor XIII) orient a bulky side chain into the S3 site of the enzyme. Taken together, our results can have important implications for the design of novel thrombin inhibitors, of practical utility in the treatment of coagulative disorders.