Domain-swapped structure of a mutant of cyanovirin-N

Cyanovirin-N (CV-N) is a potent 11 kDa HIV-inactivating protein that binds with high affinity to the HIV surface envelope protein gp120. A double mutant P51S/S52P of CV-N was engineered by swapping two critical hinge-region residues Pro51 and Ser52. This mutant has biochemical and biophysical characteristics equivalent to the wild-type CV-N and its structure resembles that of wild-type CV-N. However, the mutant shows a different orientation in the hinge region that connects two domains of the protein. The observation that this double mutant crystallizes under a wide variety of conditions challenges some of the current hypotheses on domain swapping and on the role of hinge-region proline residues in domain orientation. The current structure contributes to the understanding of domain swapping in cyanovirins, permitting rational design of domain-swapped CV-N mutants.     

Identification and characterization of a novel human DNA glycosylase for repair of cytosine-derived lesions

Two candidate human orthologs of Escherichia coli MutM/Nei were recently identified in the human genome database, and one of these, NEH1, was characterized earlier (Hazra, T. K., Izumi, T., Boldogh, I., Imhoff, B., Kow, Y. W., Jaruga, P., and Dizdaroglu, M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 3523-3528). Here we report characterization of the second protein, originally named NEH2 and now renamed NEIL2 (Nei-like). The 37-kDa wild-type NEIL2 expressed in and purified from E. coli has DNA glycosylase/AP lyase activity, primarily for excising oxidative products of cytosine, with highest activity for 5-hydroxyuracil, one of the most abundant and mutagenic lesions induced by reactive oxygen species, and with lower activity for 5,6-dihydrouracil and 5-hydroxycytosine. It has negligible or undetectable activity with 8-oxoguanine, thymine glycol, 2-hydroxyadenine, hypoxanthine, and xanthine. NEIL2 is similar to NEIL1 in having N-terminal Pro as the active site. However, unlike NEIL1, its expression was independent of the cell cycle stage in fibroblasts, and its highest expression was observed in the testes and skeletal muscle. Despite the absence of a putative nuclear localization signal, NEIL2 was predominantly localized in the nucleus. These results suggest that NEIL2 is involved in global genome repair mainly for removing oxidative products of cytosine.     

Activation of capsaicin-sensitive primary sensory neurones induces anandamide production and release

The inhibitory cannabinoid 1 receptor and the excitatory vanilloid receptor 1, both of which are responsive to the endogenous ligand anandamide, are co-expressed on a subpopulation of primary sensory neurones. We report that activation of the cannabinoid 1 receptor/vanilloid receptor 1-co-expressing primary sensory neurones induces the production and release of anandamide. Application of capsaicin (3 nm-1 micro m) to cultured primary sensory neurones evoked calcitonin gene-related peptide release, which was significantly increased by the selective cannabinoid 1 receptor antagonist, SR141716A (200 nm). Mass spectrometric analyses of the extracellular solution revealed that exposure to capsaicin (10 nm or 100 nm) enhanced the anandamide concentration of the medium from less then 0.05 pmol/ micro L to more then 2 pmol/ micro L. Depolarization of the neurones with 50 mm KCl also enhanced the anandamide content of the buffer. Both the capsaicin- and KCl-induced anandamide release depended on extracellular Ca2+. Prolonged treatment of the cultures with capsaicin (10 micro m) reduced both the capsaicin- and KCl-induced anandamide release. These findings indicate that activation of capsaicin-sensitive primary sensory neurones evokes anandamide production and release, and that anandamide might be a key endogenous regulator of the excitability of these neurones.     

Protection against late-onset AIDS in macaques prophylactically immunized with a live simian HIV vaccine was dependent on persistence of the vaccine virus

This is a 5-year follow-up study on 12 macaques that were immunized orally with two live SHIV vaccines, six with V1 and six with V2. All 12 macaques became persistently infected after transient replication of the vaccine viruses; all were challenged vaginally 6 mo later with homologous pathogenic SHIV(KU-1). Two of the V1 group developed full-blown AIDS without evidence of vaccine virus DNA in tissues. The data on the 10 vaccinated survivors showed that all 10 became infected with SHIV(KU-1) and that DNA of both vaccine and SHIV(KU-1) viruses were present 6 mo postchallenge, with minimal replication of SHIV(KU-1). During the following 5 years, these animals remained persistently infected, but with only one of the two viruses. Six animals eliminated their vaccine virus after variable periods of time and four of these succumbed to reactivation of the challenge virus and AIDS. Five years after challenge, four latently infected animals, two with V2 and two with SHIV(KU-1), were reinoculated with SHIV(KU-1.) This resulted in transient superinfection and the animals promptly returned to their prechallenge status. Immunosuppression of the four animals 1 year later with Abs to CD8+ lymphocytes resulted in transiently productive replication of their respective latent viruses, and upon recovery of CD8+ lymphocytes, they reverted to their latent virus status. The major finding was that of eight animals that eliminated the vaccine virus, six developed AIDS. The two others harboring SHIV(KU-1) remain at risk for developing late-onset disease. The primary correlate against AIDS was persistence of the vaccine virus.     

Towards synthetic vaccines built on carbohydrate cores

Lipophilic polyfunctional carbohydrate core/templates havebeen designed and developed for drug/vaccine delivery. Three carbohydrate-based templates containing four protectedN-terminal arms were synthesised from glucose and galactose. Methyl -D-glucopyranoside was converted to two derivativesbearing a carboxylic acid handle for attachment to solidsupports, spacer arms of differing hydrophilicity, andphthaloyl-protected amino groups suitable for peptide chainextension. -D-Galactopyranosyl azide was converted to atemplate bearing a carboxylic acid handle and fourBOC-protected amines. All the templates were found to besuitable for attachment to solid supports and subsequentcleavage from resins, using either BOC- or FMOC-methodologies.     

Induced Phenotypic Resistance to Valine in Mycobacterium pellegrino

Valine coordinately increases the levels of three of the enzymes participating in the biosynthesis of isoleucine and valine in Mycobacterium pellegrino. The amount of valine required for end-product induction depends on the condition of the cells. Isoleucine inhibits the effect of valine. Acetohydroxy acid synthetase, the enzyme catalyzing the first common step in the biosynthesis of valine and isoleucine, is inhibited by valine. The induction effect of valine appears to be due to its ability to inhibit the activity of this enzyme, thus causing isoleucine deficiency, which in turn leads to derepression. This conclusion is supported by the fact that valine, under certain conditions, inhibits growth.     

Hemoglobin-degrading plasmepsin II is active as a monomer

A family of aspartic proteases called plasmepsins is important for hemoglobin degradation in intraerythrocytic Plasmodium parasites. Plasmepsin II (PM II) is the best studied member of this family. PM II and its close orthologs and paralogs form homodimers with extensive interfaces in all known crystal structures. This raised the question whether the homodimer is the functional subunit of plasmepsins in solution. We have used gel filtration chromatography, site-directed mutagenesis, and analytical ultracentrifugation to study the oligomeric status of PM II in solution. Our results reveal that PM II exists mainly as a monomer in solution and that the monomer is fully functional for catalysis. A hydrophobic loop at the PM II monomer surface, which would be buried in a PM II dimer, is shown to be essential for the hemoglobin degradation capability of PM II.     

Lipidation and glycosylation of a T cell antigen receptor (TCR) transmembrane hydrophobic peptide dramatically enhances in vitro and in vivo function

A T cell antigen receptor (TCR) transmembrane sequence derived peptide (CP) has been shown to inhibit T cell activation both in vitro and in vivo at the membrane level of the receptor signal transduction. To examine the effect of sugar or lipid conjugations on CP function, we linked CP to 1-aminoglucosesuccinate (GS), N-myristate (MYR), mono-di-tripalmitate (LP1, LP2, or LP3), and a lipoamino acid (LA) and examined the effects of these compounds on T cell activation in vitro and by using a rat model of adjuvant-induced arthritis, in vivo. In vitro, antigen presentation results demonstrated that lipid conjugation enhanced CP's ability to lower IL-2 production from 56.99%+/-15.69 S.D. observed with CP, to 12.08%+/-3.34 S.D. observed with LA. The sugar conjugate GS resulted in only a mild loss of in vitro activity compared to CP (82.95%+/-14.96 S.D.). In vivo, lipid conjugation retarded the progression of adjuvant-induced arthritis by approximately 50%, whereas the sugar conjugated CP, GS, almost completely inhibited the progression of arthritis. This study demonstrates that hydrophobic peptide activity is markedly enhanced in vitro and in vivo by conjugation to lipids or sugars. This may have practical applications in drug delivery and bioavailability of hydrophobic peptides.     

FKBP12 Modulation of the Binding of the Skeletal Ryanodine Receptor onto the II-III Loop of the Dihydropyridine Receptor

In skeletal muscle, excitation-contraction coupling involves a functional interaction between the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR). The domain corresponding to Thr⁶⁷¹-Leu⁶⁹⁰ of the II-III loop of the skeletal DHPR α₁-subunit is able to regulate RyR properties and calcium release from sarcoplasmic reticulum, whereas the domain corresponding to Glu⁷²⁴-Pro⁷⁶⁰ antagonizes this effect. Two peptides, covering these sequences (peptide A_Sk and C_Sk, respectively) were immobilized on polystyrene beads. We demonstrate that peptide A_Sk binds to the skeletal isoform of RyR (RyR1) whereas peptide C_Sk does not. Using surface plasmon resonance detection, we show that 1) domain Thr⁶⁷¹-Leu⁶⁹⁰ is the only sequence of the II-III loop binding with RyR1 and 2) the interaction of peptide A_Sk with RyR1 is not modulated by Ca²⁺ (pCa 9-2) nor by Mg²⁺ (up to 10 mM). In contrast, this interaction is strongly potentiated by the immunophilin FKBP12 (EC₅₀ = 10 nM) and inhibited by both rapamycin (IC₅₀ = 5 nM) and FK506. Peptide A_Sk induces a 300% increase of the opening probability of the RyR1 incorporated in lipid bilayer. Removal of FKBP12 from RyR1 completely abolishes this effect of domain A_Sk on RyR1 channel behavior. These results demonstrate a direct interaction of the RyR1 with the discrete domain of skeletal DHPR α₁-subunit corresponding to Thr⁶⁷¹-Leu⁶⁹⁰ and show that the association of FKBP12 with RyR1 specifically modulates this interaction.     

Discovery of novel steroidal histamine H3 receptor antagonists/inverse agonists

Emerging from an HTS campaign, novel steroid-based histamine H₃ receptor antagonists were identified and characterized. Structural moieties of the hit compounds were combined to improve binding affinities which resulted in compound 4 as lead molecule. During the lead optimization due to the versatile modifications of diamino steroid derivatives, several in vitro potent compounds with subnanomolar binding affinities to histamine H₃ receptors were found. The unfavorable binding to rat muscarinic receptors was successfully reduced by tuning the basicity. Compound 20 showed significant in vivo activity in the rat dipsogenia model and could serve as a pharmacological tool in the future.