A specific and unusual nuclear localization signal in the DNA binding domain of the Rev-erb orphan receptors.

The orphan receptors Rev-erbalpha and Rev-erbbeta are members of the nuclear receptors superfamily and act as transcriptional repressors. Rev-erbalpha is expressed with a robust circadian rhythm and is involved in liver metabolism through repression of the ApoA1 gene, but no role has been yet defined for Rev-erbbeta. To gain better understanding of their function and mode of action, we characterized the proteins encoded by these two genes. Both Rev-erbalpha and Rev-erbbeta proteins were nuclear when transiently transfected in COS-1 cells. The major nuclear location signal (NLS) of Rev-erbalpha is in the amino-terminal region of the protein. Fusion of green fluorescent protein (GFP) to the amino terminus of Rev-erbalpha deletion mutants showed that the NLS is located within a 53 amino acid segment of the DNA binding domain (DBD). The homologous region of Rev-erbbeta fused to GFP also targeted the fusion protein to the nucleus, suggesting that the location of this NLS is conserved among all the Rev-erb group members. Interestingly, members of the phylogenetically closest nuclear orphan receptor group (ROR), which exhibit 58% amino acid identity with Rev-erb in the DBD, do not have their NLS located within the DBD. GFP/DBD. RORalpha or GFP/DBD.RORbeta remained cytoplasmic, in contrast to GFP/DBD. Rev-erb fusion proteins. Alignment of human Rev-erb and ROR DBD amino acid sequences predicted that the two basic residues, K167 and R168, located just upstream from the second zinc finger, could play a critical part in the nuclear localization of Rev-erb proteins. Substitution of these two residues with those found in ROR, in the GFP/DBD. Rev-erb context, resulted in cytoplasmic proteins. In contrast, the reverse mutation of the GFP/DBD. RORalpha towards the Rev-erbalpha residues targeted the fusion protein to the nucleus. Our data demonstrate that Rev-erb proteins contain a functional NLS in the DBD. Its location is unusual within the nuclear receptor superfamily and suggests that Rev-erb orphan receptors control their intracellular localization via a mechanism different from that of other nuclear receptors.

Identification of amino acid residues in the ETS transcription factor Erg that mediate Erg-Jun/Fos-DNA ternary complex formation.

Jun, Fos, and Ets proteins belong to distinct families of transcription factors that target specific DNA elements often found jointly in gene promoters. Physical and functional interactions between these families play important roles in modulating gene expression. Previous studies have demonstrated a direct interaction between the DNA-binding domains of the two partners. However, the molecular details of the interactions have not been investigated so far. Here we used the known three-dimensional structures of the ETS DNA-binding domain and Jun/Fos heterodimer to model an ETS-Jun/Fos-DNA ternary complex. Docking procedures suggested that certain ETS domain residues in the DNA recognition helix alpha3 interact with the N-terminal basic domain of Jun. To support the model, different Erg ETS domain mutants were obtained by deletion or by single amino acid substitutions and were tested for their ability to mediate DNA binding, Erg-Jun/Fos complex formation, and transcriptional activation. We identified point mutations that affect both the DNA binding properties of Erg and its physical interaction with Jun (R367K), as well as mutations that essentially prevent transcriptional synergy with the Jun/Fos heterodimer (Y371V). These results provide a framework of the ETS/bZIP interaction linked to the manifestation of functional activity in gene regulation.

2- and 3-substituted 1,4-naphthoquinone derivatives as subversive substrates of trypanothione reductase and lipoamide dehydrogenase from Trypanosoma cruzi: synthesis and correlation between redox cycling activities and in vitro cytotoxicity.

Trypanothione reductase (TR) is both a valid and an attractive target for the design of new trypanocidal drugs. Starting from menadione, plumbagin, and juglone, three distinct series of 1,4-naphthoquinones (NQ) were synthesized as potential inhibitors of TR from Trypanosoma cruzi (TcTR). The three parent molecules were functionalized at carbons 2 and/or 3 by various polyamine chains. Optimization of TcTR inhibition and TcTR specificity versus human disulfide reductases was achieved with the 3,3'-[polyaminobis(carbonylalkyl)]bis(1,4-NQ) series 19-20, in which an optimum chain length was determined for inhibition of the trypanothione disulfide reduction. The most active derivatives against trypanosomes in cultures were also studied as subversive substrates of TcTR and lipoamide dehydrogenase (TcLipDH). The activities were measured by following NAD(P)H oxidation as well as coupling the reactions to the reduction of cytochrome c which permits the detection of one-electron transfer. For TcTR, 20(4-c) proved to be a potent subversive substrate and an effective uncompetitive inhibitor versus trypanothione disulfide and NADPH. Molecular modeling studies based on the known X-ray structures of TcTR and hGR were conducted in order to compare the structural features, dimensions, and accessibility of the cavity at the dimer interface of TcTR with that of hGR, as one of the putative NQ binding sites. TcLipDH reduced the plumbagin derivatives by an order of magnitude faster than the corresponding menadione derivatives. Such differences were not observed with the pig heart enzyme. The most efficient and specific subversive substrates of TcTR and TcLipDH exhibited potent antitrypanosomal activity in in vitro T. brucei and T. cruzi cultures. The results obtained here confirm that reduction of NQs by parasitic flavoenzymes is a promising strategy for the development of new trypanocidal drugs.

Contribution of cation-pi interactions to the stability of protein-DNA complexes.

Cation-pi interactions between an aromatic ring and a positive charge located above it have proven to be important in protein structures and biomolecule associations. Here, the role of these interactions at the interface of protein-DNA complexes is investigated, by means of ab initio quantum mechanics energy calculations and X-ray structure analyses. Ab initio energy calculations indicate that Na ions and DNA bases can form stable cation-pi complexes, whose binding strength strongly depends on the type of base, on the position of the Na ion, and whether the base is isolated or included in a double-stranded B-DNA. A survey of protein-DNA complex structures using appropriate geometrical criteria revealed cation-pi interactions in 71% of the complexes. More than half of the cation-pi pairs involve arginine residues, about one-third asparagine or glutamine residues that only carry a partial charge, and one-seventh lysine residues. The most frequently observed pair, which is also the most stable as monitored by ab initio energy calculations, is arginine- guanine. Arginine-adenine interactions are also favorable in general, although to a lesser extent, whereas those with thymine and cytosine are not. Our calculations show that the major contribution to cation-pi interactions with DNA bases is of electrostatic nature. These interactions often occur concomitantly with hydrogen bonds with adjacent bases; their strength is estimated to be from three to four times lower than that of hydrogen bonds. Finally, the role of cation-pi interactions in the stability and specificity of protein-DNA complexes is discussed.

Identification of inhibitors of an 80 kDa protease from Trypanosoma cruzi through the screening of a combinatorial peptide library.

Two orthogonal peptide combinatorial libraries were screened to discover inhibitors of Tc80 protease, a novel target from Trypanosoma cruzi involved in host cell invasion. These libraries were composed of 15,625 structurally diversified tripeptides, partitioned in 125 mixtures. The screening led to a low micromolar inhibitor which was actually an HF cleavage by-product H-Ipe-D-Tic-D-Glu(S-paratolyl)-OH. IC50 values of several analogous molecules of this hit were determined and are discussed. For the best compounds, conformational analysis revealed a high degree of similarity in shape with a potent prolylendopeptidase inhibitor, SUAM-1221.

Synthesis, folding, and structure of the beta-turn mimic modified B1 domain of streptococcal protein G.

The mechanism of beta-sheet formation remains a fundamental issue in our understanding of the protein folding process, but is hampered by the often encountered kinetic competition between folding and aggregation. The role of local versus nonlocal interactions has been probed traditionally by mutagenesis of both turn and strand residues. Recently, rigid organic molecules that impose a correct chain reversal have been introduced in several small peptides to isolate the importance of the long-range interactions. Here, we present the incorporation of a well-studied beta-turn mimic, designated as the dibenzofuran-based (DBF) amino acid, in the B1 domain of streptococcal protein G (B1G), and compare our results with those obtained upon insertion of the same mimic into the N-terminal beta-hairpin of B1G (O Melnyk et al., 1998, Lett Pept Sci 5:147-150). The DBF-B1G domain conserves the structure and the functional and thermodynamical properties of the native protein, whereas the modified peptide does not adopt a native-like conformation. The nature of the DBF flanking residues in the modified B1G domain prevents the beta-turn mimic from acting as a strong beta-sheet nucleator, which reinforces the idea that the native beta-hairpin formation is not driven by the beta-turn formation, but by tertiary interactions.

Characterization of a new family of toxin-like peptides from the venom of the scorpion Leiurus quinquestriatus hebraeus. 1H-NMR structure of leiuropeptide II.

To extend our knowledge about the structural features of short scorpion toxins, the ion-exchange fractions obtained from Leiurus quinquestriatus hebraeus venom were investigated by plasma desorption mass spectrometry in order to select low molecular mass polypeptides. Three toxin-like peptides with molecular mass close to 3 kDa, named leiuropeptides I, II and III, were purified and found devoid of any significant toxicity against mammals and insects. Their amino acid sequences revealed a cysteine pattern analogous to that of short-chain scorpion toxins. The solution structure of leiuropeptide II was determined by 2D 1H-NMR spectroscopy and indicated the presence of a helix accommodating a proline, connected to a two-standard beta-sheet by three disulfide bonds. The overall fold of leiuropeptide II is found to be similar to that of leiurotoxin I, a 31-residue toxin present in the same scorpion venom which acts on K+ channels. In order to rationalize the absence of toxicity, the electrostatic potential of leiuropeptide II was compared to that of leiurotoxin I. The peptide is characterized by a large negative zone around Glu4, Asp5 and Asp8 residues, beginning in the neighbourhood of the beta-turn and extending along the helix. In the same area, leiurotoxin I exhibits a positive surface, around Arg6 and Arg13 basic residues, which are essential for its receptor affinity.

2-Amino diphenylsulfides as inhibitors of trypanothione reductase: modification of the side chain.

A molecular modeling study meant to detect pharmacophore-like patterns in the active site of trypanothione reductase (TR) offered hints about the opportunity of synthesizing and testing diphenylsulfide derivatives with prolonged or branched polyamino side chains as putative TR inhibitors. The inhibition results within the synthesized series confirmed the main working hypothesis inspired by the molecular modeling study. The different compounds were tested in vitro on the enzyme and on Trypanosoma cruzi and Trypanosoma brucei trypomastigotes as well as in vivo in infected mice.

Isolation and characterization of a small putative zinc finger protein from cuttlefish epididymal sperm cells.

At the end of spermiogenesis, sperm chromatin stabilization is ensured by protamine dephosphorylation and, in mammals, by the formation during epididymal transit, of intra- and inter-molecular disulfide bridges between protamines. In cuttlefish, the nuclear protein transition histones-->spermatid-specific protein T-->protamine Sp is very similar to that occurring in mammals during spermiogenesis. However, in cuttlefish, the protamine Sp is devoid of cysteine residues. The protein complement of cuttlefish epididymal sperm nuclei has been investigated. A minor basic protein, called protein E, has been isolated. Its primary structure was established from sequence analysis and mass spectrometry data of the protein and its fragments. Protein E contains a motif -Cys-Xaa2-Cys-Xaa23-His-Cys-Xaa2-Cys- which is likely to adopt a zinc finger conformation. Reduced protein E does fix zinc whereas alkylation of cysteine residues abolishes this ability. The sequence of protein E does not correspond to that of any known protein, but presents some similarities with a part of ZFY protein, a putative human transcription factor specifically expressed in germinal cells and which could be involved in spermatogenesis.

Immunogenicity and antigenicity of synthetic peptides derived from the mite allergen Der p I.

As an immunogen must contain both B- and T-cell epitopes, small peptides are usually reported as non-immunogenic unless coupled to a protein carrier. In this study, the immunogenicity of the Der p I synthetic uncoupled peptides (p52-71, p89-104, p117-133 and p176-187) previously reported as B-cell epitopes, was evaluated. Different schedules of immunization were used. Results indicated that by using the Vaitukaikis' method three injections of the same peptide without protein carrier was sufficient to induce an specific anti-peptide IgG antibody response (evaluated by ELISA). Indeed, the 16-20 amino-acid long peptides p52-71, p117-133 and p89-104 were revealed highly immunogenic in rabbits. Furthermore anti-peptide p52-71 and p117-133 antibodies were shown by Western-blotting or by neutralization assay to recognize the Der p I molecule either in denaturated or native form as well as Der f I (major allergen of Dermatophagoides farinae). Finally, taking into account the location of Der p I-derived peptides in the three-dimensional model of Der p I, the antigenicity and immunogenicity of peptides were discussed.